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Characterization of dolichol kinase from bovine thyroid microsomes
Int. J. Biochem. Vol. 19, No. 5, pp. 419-426, 1987
0020-711X/87 $3.00+0.00
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Copyright © 1987 Pergamon Journals Ltd
CHARACTERIZATION OF DOLICHOL KINASE FROM BOVINE THYROID MICROSOMES L u c STEENI, GUIDO VAN DESSEL2'*, MARC DE WOLF l, ALBERT LAGROUl, HERWIG HILDERSON1 and WILFRIED DIERICKL2 tRUCA-Laboratory for Human Biochemistry, University of Antwerp, B2020 Antwerp, Belgium 2UIA-Laboratory for Pathological Biochemistry, University of Antwerp, B2610 Antwerp, Belgium (Received 31 July 1986) Abstract--1. Bovine thyroid microsomes are able to phosphorylate exogenous [l-3H]dolichol as well as
endogenous dolichol. 2. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [l-3H]dolichol to [1-3H]DMP as well as the formation of [32p]DMP from endogenous dolichol and [7-32p]CTP. 3. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. 4. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. 5. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca 2+- and Mn2+-ions. 6. In accordance, EDTA was strongly inhibitory. 7. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. 8. The apparent K,,-value for exogenous dolichol amounted to 4 #M. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [l-3H]dolichol depending on the source of CTP. 9. With endogenous dolichol Kin-values for CTP of 27.8 and 6.1 #M were calculated in respectively the absence and presence of 5 mM VO] . 10. Triton X-100 (0.15%) was necessary in the [l-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [7-32p]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). ll. The levels of the enzymatic activity could be doubled by the inclusion of 18-21 m M N a F ([1-3H]dolichol kinase) as phosphatase inhibitor; VOW- had practically no effect. 12. In contrast with [7-32p]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO43- while F - resulted into no appreciable effect. 13. In both approaches sulphydryl groups were essential for enzymatic activity. 14. Bacitracin inhibited dolichol kinase. 15. Propylthiouracyl, cAMP, TSH and several other agonists were without effect on the system in vitro. 16. VOS, PO 3-, P2074- and several phosphomonoesters had no influence on the enzymatic activity. [l-3H]dolichol kinase activity could readily be solubilized (~70%).
INTRODUCTION
W o r k from several laboratories has suggested that D M P functions as a coenzyme in the assembly of asparagine-linked oligosaccharides of glycoproteins in eucaryotes (Elbein, 1979; Hemming, 1974; Parodi and Leloir, 1979). A growing body of experimental evidence indicates that the concentration of dolichyl monophosphate may be a rate limiting factor in the glycosylation process. The bulk of the dolichol in bovine thyroid (Van Dessel et al., 1979) and also in other organs (Dallner and Hemming, 1981) is present in non-phosphorylated form. D M P concentration levels seem to be regulated by altering the relative activities of dolichol kinase and D M P phosphatase. In this context data have been reported showing some correlation between the dolichol kinase activity and the glycoprotein synthesis (De *To whom correspondence should be addressed. Abbreviations: DMP: dolichylmonophosphate; DPP: dol-
ichylpyrophosphate; sphingomyelin.
PA: phosphatidic acid;
SPH: 419
B.C. 19/5--B
Rosa and Lucas, 1982). A number of reports have appeared regarding the occurrence and properties of dolichol kinase from several sources (Rip and Carroll, 1980; Gandhi and Keenan, 1983) using endogenous and exogenous dolichol as phosphoryl acceptor. This paper reports on the dolichol kinase activity in bovine thyroid microsomal membranes studied with both approaches. Some aspects of this work have been presented in a preliminary form (Steen et aL, 1982, 1985). MATERIALS AND METHODS
Chemicals
Dolichol was isolated from bovine thyroid as described earlier (Steen et al., 1984). Dolichol and DMP standards (pig liver), phospholipids, calmodulin, bacitracin, tunicamycin, glucose-6-phosphate and ATP were purchased from Sigma. Acetate kinase and almost all nucleotides were supplied by Boehringer Mannheim Corp. CTP was also obtained from Calbiochem-Behring. [32p]H3PO 4 (carrier free) was acquired from New England Nuclear. p-Nitrophenylphosphate, di-Na-phenylphosphate, fl-glycerophosphate and vanadium oxide sulphate were from Merck.
420
Luc STEENet aL
Sodium orthovanadate was obtained from BDH. Hormones were purchased from Pharmachemic. TSH was from Armour Pharmaceutical Co. All other reagents and chemicals used in these experiments were purchased from standard commercial sources. Chromatographic methods Thin-layer chromatography was performed on precoated silica gel 60F-254 plates (Merck) using benzene/ethylacetate (9:1; v/v) (eluant A) chloroform/methanol/water (60:25 :4; v/v) (eluant B), chloroform/methanol/25% (w/v) NH 3 (65:35:5; v/v) (eluant C) or diisobutylketone/HAc/water (60:45:6; v/v) (eluant D) and on PEI cellulose plates (Merck) using 0.5 M KH2PO 4 (pH 3.4) (eluant E). Column chromatography was done on AG1-X2 (2 g; Bio-Rad Lab) converted to the acetate form by treating the resin with I M NH4OOCCH 3 in chloroform/methanol (2:1; v/v) until C I free followed by equilibration of the resin in chloroform/methanol (1 : 1; v/v). Preparation of substrates [l-3H]dolichol was obtained from bovine thyroid dolichol using the labelling method described by Keenan and Kruczek (1975). D,-32p]CTP was prepared enzymatically by the procedure of Bauer and Varady (1978): CDP and [7)zP] CTP were eluted from DEAE-cellulose respectively by 53 and 90 mM NH4HCO 3 (Bauer and Varady, 1978). Enzyme preparation Fresh bovine thyroid glands were obtained from a local slaughterhouse. Further manipulations were performed at 4°C. The glands were trimmed free of surrounding tissue and freed from blood by washing several times with 0.25 M sucrose 5 mM Tris-HCl (pH 7.4). The thyroid tissue was cut into small cubes with scissors and minced in a meat mill. After homogenization in a Waring Blendor homogenisator (30sec high speed and 30sec low speed with a 30sec intermittent cooling on ice) and filtration through a double layer of cheese cloth, nuclei and cell debris were pelleted (1200g, 10 rain). The postnuclear supernatant was centrifuged at 73,300 g for 15 min. The resultant supernatant was further centrifuged at 144,700g for 60min (Voets et al., 1979). The pellet was suspended in 0.25 M sucrose-5 mM Tris-HC1 (pH 7.4) (60-80mg protein/ml) and divided in 0.5 ml aliquots which were frozen at -20°C until use. This fraction was designated as the microsomal membrane fraction. All experiments have been performed with this enzyme preparation unless stated otherwise. Kinase assays [1-3H]dolichol kinase (procedure I). Activity was monitored by measuring the formation of [I-3H]DMP from [l-3H]dolichol. 1,250,000 dpm [1-3H]dolichol (4 Ci/mmol) dissolved in chloroform/methanol (2:1; v/v) was mixed intensively with sufficient Triton X-100 to give a final concentration of 0.15 % (w/v) in the incubation mixture. The organic solvent was evaporated under N 2. The reaction was carried out in a total volume of 200/~1 containing 200 mM Tris-maleate (pH 7.0), 24 mM MgCI:, 20 mM NaF, 18 mM CTP (pH 7) and up to 1.5 mg protein prepared as described. The mixture was shaken in a Vortex and incubated at 37°C after the addition of the enzyme fraction. In routine experiments the incubation time was 3 min. The reaction was terminated by the addition of 4 ml chloroform/methanol (2:1; v/v) and this mixture was washed with 0.6 ml water. Two clear phases and the denatured protein interface were separated by low speed centrifugation. The lower phase was applied to a AG1-X2 column prepared as described in chromatographic methods. [l-3H]dolichol was eluted with chloroform/methanol (1:1; v/v) (16ml) and [I-3H]DMP with 0.25 M NH4OOCCH3 in chloroform/methanol (1:1; v/v) (10 ml). The [1-3H]DMP fraction was collected directly in a scintillation vial and dried under a stream of air. After
addition of 10 ml lipoluma (Lumac) the amount of radioactivity was determined in a Beckman LS 8100 liquid scintillation counter. 90-95% of the radioactivity in the 0.25 M NH4OOCCH 3 chloroform/methanol (1:t; v/v) fraction migrated on TLC with authentic DMP in eluent B, C and D. The remaining radioactivity was localized at the origin of the thin-layer plate and at the level of [l-3H]dolichol. [7-seP]CTP dolichol kinase (procedure lI). The phosphorylation of endogenous dolichol was determined by estimating the amount of [~ZP]DMP formed. The 200/~1 assay, containing 200mM Tris-maleate (pH7), 20mM MgC12, 5mMVO~L 4#M [y-32p]CTP (~4pCi) and enzyme protein (~ t.5 mg) was incubated at 37°C for 3 min. Further manipulations were carried out as described for [l-3H]dolichol kinase. All enzyme assays were performed in duplicate and always agreed within 10% Solubilization of bovine thyroid dolichol kinas~ activit~ To solubilize dolichol kinase activity microsomes (8-16 mg protein/ml) were treated with 0.25% (w/v) Triton X-100, After mixing, the detergent suspension was shaken for 15 rain at 4°C. Approximately 70% of the dolichol kinase activity was recovered in the supernatant fluid after centrifugation at 144,700 g for 60 rain. Analytical methods" Protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS Kinetics With both incubation systems (l, lI) D M P is formed as a result of a non-linear time-dependent phosphorylation of dolichol [Fig. l(a) and (b)]. On the other hand, the product formation is directly proportional to membrane protein concentration up to 3 rag/incubation in both systems [Fig. l(c) and (d)]. At higher protein concentration, a sharp decrease in the amount of reaction product is noted in system 1. Dolichol kinase exhibits a p H - o p t i m u m centered at around pH 7. In system II the pH-activity curve displays a broad profile in contrast to system I where a sharp p H optimum is obtained. Comparison of the effect of different divalent cations demonstrated that in both systems Mg2+-ions are much better than Ca 2~- or Mn2+-ions (Table 1). The results with Ca 2+and Mn2+-ions are reversed in system I and II; system 1: Ca2+-ions>Mn2+-ions; system II: Mn2+-ions >>Ca2+-ions. the divalent cation requirement for full enzymatic activity is in accordance with the strong inhibition by E D T A (Table 1). The enzyme preparation itself probably contains divalent cations because E D T A addition is necessary to abolish totally the dolichol kinase activity. Detergent Dolichol kinases are generally membrane bound and their substrate is water insoluble. Therefore, especially in system I, detergent administration is needed for enzymatic activity. Several detergents were tested [Fig. 2(a)]. The highest activity is obtained with 0.15% (w/v) Triton X-100. At higher concentrations a decrease in the amount of [1-3H]DMP formed is noted. In the absence of Triton X-100 only 3% of the enzymatic activity registered at the optim u m detergent concentration 0.15% (w/v) is found. Dependent on the Triton X-100 concentration a
Bovine thyroid dolichol kinase 15
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421
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Fig. 1. (a) [l-3H]dolichol kinase activity in function of incubation time. IS]: without NaF; i : in the presence of 21 mM NaF (CTP, Boehringer). (b) b, J2p]dolichol kinase activity in function of incubation time. 0 : without VO]- and NaF; 2: in the presence of 5 mM NaF; O: in the presence of 5 mM VOW-. (c) [l-3H]dolichol kinase in function of protein concentration (CTP, Boehringer). (d) [7-32P]dolicholkinase in function of protein concentration. different course of the enzymatic activity vs CTP concentration is observed. The Km and Vmaxvalues for CTP as calculated from the Lineweaver-Burke plots are tabulated in Table 2. The influence of different detergents on the enzymatic activity in system II is
presented in Fig. 2(b). Generally at low concentrations, detergents activate; at higher concentrations inhibition occurs. The highest stimulation ( ~ 30%) is also found with Triton X-100, but at 0.1% (w/v) concentration and higher a strong inhibition is noted.
Phospholipids Table 1. Influence of divalent cations and EDTA Concentration (pmol product formed) Divalent cation System I Mg#÷ ( + 12 mM EDTA) Ca 2÷ Mg 2+ System II without VO]Mg 2+ Ca 2+ Mn :+ with 5 mM VO~Mg 2+ ( + 1 2 m M EDTA) Ca :+ Mn 2+
Control 5 m M 10mM
20raM
0.34
1.01 0.90 0.65
2.15 1.95 0.54
4.44(1.62)* 0.58 0.39
0.83
0.243 0.069 0.215
0.270 0.078 0.269
0.334 0.087 0.345
1.04
0.965 0.204 0.348
1.166 0.208 0.385
1.44(0.620)* 0.216 0.491
*The values in parentheses are obtained in the presence of 12 mM EDTA.
The effect of different phospholipids on dolichol phosphorylation process was examined. Low concentrations (1-100/~M) have only little influence ( ~ 30% reduction) on the enzymatic activity, 1 mM concentration results into a strong inhibition (50-90%) in system I. Only with SPH an enhancement of [I-3H]DMP formation (40%) is obtained. Addition of phospholipids in system II provokes minor effects. Merely, the presence of PA (1 mM) leads to a decrease of the phosphorylating activity with 69%.
Phosphatase inhibitors The phosphorylation of [l-3H]dolichol can be stimulated 1.7-fold by the addition of 18-21 mM N a F in system I. When adding VOW-, alone or together with N a F (1 mM), to the incubation mixture the amount of reaction product increases (30-40%) at low V043-
422
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Fig. 2. Dolichol kinase activity in function of detergent concentration (%, w/v final concentration). (a) [13H]doliehol kinase (CTP, Boehringer). I-q: control (without detergent); m: Triton X-100; U: sodium taurocholate; I1: sodium deoxycholate; I]11]: cetyltrimethyl a m m o n i u m bromide. (b) [y32P]dolichol kinase. I"1: control (without detergent); I1: Triton X-100; Ill: sodium taurocholate; I : sodium deoxycholate; II: Nonidet P-40.
Fig. 3. (a) Influence of the source ol CTP on the [1-3H]dolichol kinase activity in function of VO3 concentration. CTP, Calbiochem. 0 : control without NaF; 0: control with 21 mM NaF; Fq: incubation without NaF; n : incubation with 21 mM NaF. CTP-Boehringer. A: control with 21 mM NaF; 0 : incubation with 21 mM NaF, C': mM VO3-. (b) Influence of VO43- on the [y3ZP]dolicho] kinase activity. I--l: control with ATP; i : VO3- without ATP; O: VO]- without ATP; O: V O ] in the presence of 20mMATP; z% VOS without ATP; A: VOS in the presence of 20 mM ATP. C': mM VO]- or VOS,
concentrations (0.02-0.1 mM); at VO43- concentrations above 0.5 m M an inhibition (CTP, Calbiochem) is observed [Fig. 3(a)]. Different effects are noted with C T P Boehringer as cosubstrate. In system II, F --ions cause little influence on the kinase activity ( 5 r a M N a F : 10% stimulation). At higher concen-
trations there is even inhibition. Trinucleotides (10mM), on the other hand, increase the kinase activity (24 times for CTP, 2-3 times for the others). Purine dinucleotides are activating while U D P has no effect and C D P reacts inhibitory. In system II different VO43- effects are observed whether A T P is
o 0.05
0.01
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0.02
Detergent
0.05 (%,
0.~0
0.50
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Table 2. Kinetic constants Dolichol
CTP
o~ (wl,vi Enzyme [1)H]dolichol kinase
K,, 4.10 -6 M
Vm~ x
11.69
Source Calbiochem Boehringer
['f32P]CTP dolichol kinase
*V~x is expressed as pmol product formedlmin/mg protein.
K~, 3.88.10-
Vma~ 3M
10.75.10 3 M 27.8-10-6 M (without VOW-) 5.1.10-6M (+5raM VOW-)
Triton X-IO0
1.0~
015
0.689 2.020 1.250 1.400
0.25 IL15 0.25
423
Bovine thyroid dolichol kinase Table 3. Influence of ionic strength and sulphydryl reagent % vs control Concentration Added substance (mM) System I System II NaCI
50 100 200
94 84 69
95 98 97
50 100 200
97 87 68
98 100 101
Dithiothreitol
5 10
103 113
101 112
fl-Mercaptoethanol
5 10
101 95
100 104
(NH4)2SO4
p -Chloromercuribenzoate HgC12
0.1
3-
2-
24
100
1
1
26
I
0.1
6
22
- O.4
I
2
5
Table 4. Influence of some phosphatase inhibitors* % vs control Concentration Added substance (mM) System I System II VOS 0.1 116"I" 115 1 1021" 117 KH2PO4-K2HPO4(pH =7) 5 107 100 Na4 P207 5 104 55 Glucose-6-phosphate 5 102 100 fl-Glycerophosphate 5 102 89 *Incubations were performed in the absence of NaF (system I) and VO43- (system II). ~lncubation with VOS was performed with CTP, Calbiochem.
present or not. In the absence o f A T P a strong activation is f o u n d while in the presence of A T P no effect is seen [Fig. 3(b)]. VOS, P O 3-, P20~ - a n d substrates for some p h o s p h a t a s e in 5 m M concentration are w i t h o u t effect o n the enzymatic activity in b o t h systems except for P20~- with [7-32p]cTP dolichol kinase (Table 4).
CTP specificity F r o m all nucleotides tested, only C T P increases the basal level o f dolichol kinase in b o t h systems (Table 5). This is further s u b s t a n t i a t e d by the inhibitory effect o f CDP. In system I, optimal enzymatic activity is observed at 1 6 - 2 0 m M CTP. A t higher c o n c e n t r a t i o n (substrate ?) inhibition occurs. Moreover, w h e n studying a n u m b e r o f parameters different results are o b t a i n e d regarding the C T P source. A t optimal Mg2+-ions conditions the dolichol kinase activity is higher with C T P from Boehringer t h a n m e a s u r e d with C T P from Calbiochem. The stimulatory effect of VOW- (0.02-0.1 m M ) is only
--0.2
I/CTP
I 0.4
0.2
0
I 0.6
( r a M ) -1
Fig. 4. [1-3H]dolichol kinase in function of CTP concentration. Incubations were performed with two sources of CTP (O: CTP, Calbiochem; A: CTP, Boehringer) and in the presence of 0.15% (w/v) Triton X-100. Reaction rate (v) is the amount of [1-3H]DMP formed (pmol/min/mg protein). observed when C T P from C a l b i o c h e m is used as p h o s p h o r y l donor. The a p p a r e n t Kin-values for C T P from C a l b i o c h e m a n d C T P from Boehringer a m o u n t to respectively 3.88 a n d 10.75 m M (Table 2; Fig. 4). If this p h e n o m e n o n is due to c o n t a m i n a t i o n , it suggests t h a t C T P from C a l b i o c h e m contains an inhibitor from the mixed type (partial n o n competitive; partial anti-competitive with ~ = 0.36 a n d fl =0.52). Such a n inversion point ( 1 2 m M MgE+-ions) is also found when checking the effect o f Mg2+-ions for b o t h C T P sources. S u p p l e m e n t a r y addition of C T P after declination of the enzymatic activity in function of time has only a m i n o r influence (5%). The a p p a r e n t K m for C T P in system II is 27.8 # M in the absence o f V O 3- a n d decreases 4-fold when 5 m M VOW- is a d d e d (Table 2). T h e a p p a r e n t Km for dolichol in system I a m o u n t s to 4 # M .
12-
"o E
9
Q. ,.-, 6 T I ,.--.
Table 5. Nucleotide specificity and influence of nucleoside monoand diphosphates % vs control Added Concentration substance (mM) System I System II ATP 20 1 248 UTP 20 1 182 GTP 20 1 I 14 CTP 20 100 2402 ADP 5 109 241 UDP 5 92 104 GDP 5 130 160 CDP 5 69 8 CMP 5 115 92
o.
//
I -4
I -3
[ -2
I --1
tog
I 0
I I
[ 2
I 3
C'
Fig. 5. Influence of DMP on [1-3H]dolichol kinase activity. &: control without NaF; I1: control in the presence of 21 mM NaF; A: incubation without NaF; r-I: incubation in the presence of 2 1 m M N a F . C': /~M DMP (CTP, Boehringer).
424
LtJc S'~r~ et al.
Table 6. Influence of D M P and dolichol on the [~,-32P]CTP dolichol kinase activity* Concentration (# M)
Added substance
DMP
% vs control
3.5 103.5 2.2 7.2 36.2 180.9
Dolichol
are involved in the enzymatic function (Table 3). Only at 200 mM (NH4)~SO4 or NaC1 (system I) the enzymatic activity is affected (Table 3). Contrary, in system II the ionic strength is without any influence (Table 3).
185 f55 101 1t3 123 t 36
*The added substances were suspended in 0.01% (w/v) Triton X-100 final concentration.
Effect of exogenous DMP and dolichol Addition of exogenous DMP in a concentration range of 1-30/tM stimulates the formation of [1-3H]DMP (Fig. 5) as well as [32p]DMP (Table 6). At higher amounts ( > 1 0 0 # M ) an inhibition of the amount of [1-3H]DMPformed is observed. In system I, adding NaF together with DMP has no influence on the profile of the curve. Simultaneous addition of DMP, NaF and VOW- gives rise to a limited but no absolute additivity (data not shown). Addition of exogenous dolichol in system II enhances the DMP production (36%) (Table 6). Stability, sulphydryl reagents and ionic strength Dolichol kinase is relative stable against repeated freezing and thawing but the activity is lost by prolonged storage at 4°C (Table 7). Sulphydryl groups Table 7. Stability of [l-3H]dolichol kinase and [~,-32P]CTP dolichol kinase
Bio-effectors Some bio-effectors were studied with the purpose to check if there is any direct effect on the kinase activity in vitro. From Table 7 it is obvious that most bio-effectors are without effect. The antibiotic bacitracin shows to be most effective (40% inhibition). Calmodulin is without appreciable influence. Characterization of the reaction product The reaction product is identified as DMP in different ways. The product cochromatographed with standard pig liver DMP in three different thin layer chromatographic systems (eluant B, C and D). Furthermore, upon anion exchange chromatography on AG1-X2, the product co-eluted with DMP in the 0.25 M ammonium acetate C/M (1:1; v/v) eluate. The reaction product was stable against mild alkaline and acid hydrolysis. Finally the reaction product could enhance the glucosyltransferase activity in a microsomal enzyme preparation (unpublished results). Solubilization Dolichol kinase (system I) can easy be solubilized [up to 70% with 0.25% (w/v) Triton X-100]. The properties of the solubilized enzyme are very similar to those of system I (data hot shownj.
% vs control Procedure
Times
Freezing/thawing
hr
System I
System II
DISCUSSION
24 48 96
89 73 71 65 56 50
85 80 79 74 65 6
The presence of an enzymatic activity catatysing the phosphorylation of dolichol in bovine thyroid microsomes has been demonstrated using both exogenous [l-3H]dolichol/CTP and endogenous dolichol/[7-32p]CTP as respective substrate pairs. A
1 2 3
Preservation at 4"C
Table 8. Influence of bio-effectors on the dolichol kinase activity in a (M + L)-fraction* % vs control Effector
Concentration't
[l-~H]dolichol kinase
[~)2P]CTP dolichol kinase
lsoproterenol Eserinesulphate Carbachol Norepinephrine Epinephrine Phenylephrine Pilocarpine Chloropromazine cAMP
1# M 1# M 1# M 1# M 1# M 1/z M 1# M 1/z M 0.04 mM 0.4 mM 0.015% (w/v) 0.15% (w/v) 216#g/ml 0.5 gg/ml 5 lag/ml 6.25 gg/ml 31.25 #g/ml 125 g g/ml 0.15 mM 1.5 mM
103 98 89 102 87 102 96 105 I 11 ( 109):~ I I 0 (107):~ 117 107 106 110 115 92 95 87 73 59
105 109 104 99 100 I0 t 103 109 106 96 99 t)~ 103
PTU TSH Tunicamycin Calmodulin§
Baeitracin
lII
*The protein concentration of the (M + L)-fraction/incubation amounts to 1 mg. tFinal concentration incubation mixture. ~/Values on parentheses are those for AMP. §The incubation with calmodulin was carried out with Ca ~÷- instead of Mg2+-ions. The concentration used in the incubation mixture was 12.5raM. Calmolulin was dissolved in 0.25 M sucrose/ 5 mM Tris-HCI pH 7.4.
Bovine thyroid dolichol kinase number of data are concording between the two incubation systems but also some striking differences are noted. System I and II both require specifically CTP as phosphoryl donor as well as the presence of divalent cations. The CTP specificity was confirmed by the inhibitory effect of CDP. All eukaryotic dolichol kinases so far characterized show this same cosubstrate specificity in contrast to bacterial polyprenyl kinases which are primarily ATP dependent. In bovine thyroid dolichol kinase (both approaches) shows a preference for Mg2+-ions, which is confirmed by the strong inhibition by EDTA. Previously, dolichol kinases have been reported to be dependent on either Mg 2+- (Rip and Carroll, 1980), Ca 2+- (Keller et aL, 1982) or Mn2+-ions (Burton et aL, 1981). Calcium activation seems to be calmodulin mediated (Gandhi and Keenan, 1983) while magnesium stimulates through another mechanism (Rip and Carroll, 1980). The apparent Kin-value for CTP in system I is much higher than in system II. Similar pronounced differences have also been reported in the literature: Km-value for [y-32p]CTP (endogenous dolichol) is always in the ~M range (Rip and Carroll, 1980); K,,-value for cold CTP (exogenous dolichol) in the mM range (Gandhi and Keenan, 1983). On the other hand, the apparent Vmax-valuesare of the same order of magnitude. This is also in agreement with the literature (Gandhi and Keenan, 1983). Up to now, there is no unequivocal explanation for the great difference between the apparent Kin-values for CTP between system I and system II at hand. Some possible explanations are: (i) A different degree of DMP-Pase and CTP-Pase inhibition in both systems. (ii) The enzyme-substrate complex must be formed in a totally different physico-chemical situation (system I: 0.15% Triton X-100; system II: no detergent). Consequently the accessibility and/or affinity of the enzyme for CTP may be different (charge effects, conformational changes). (iii) The difference in apparent Kin-values according to the source of CTP could be explained by the presence of varying amounts of contaminating phosphatase inhibitors in the respective CTP preparations. The apparent K~ for exogenous dolichol is 4 # M. Generally, higher apparent Kin-values are found in the literature (factor 10 to 102) (Rossignol et al., 1983). Different results are obtained for various phosphatase inhibitors tested in system I and system II. The sequence for the kinase stimulation in system I is F - > ATP > VOW-; in system II VOW- ~ ATP >>F - . The lack of VO]- effect in system I when using CTP from Boehringer could be due to contamination of the trinucleotide by VOW- in analogy with some ATP preparations (Sigma A6144). Since the effects of ATP and VO43- in system II are not additive it seems likely that both are operating through an analogous mechanism, possibly at the level of the degradation of CTP and DMP. The enhanced kinase activity in the presence of 5mM VO 3- without addition of ATP points to a supplementary effect from this anion that is blocked by ATP. In system II, the behaviour of F - is comparable with results mentioned in the literature (Keller et al., 1982). It is clear that the stimulatory effect may not exclusively be ascribed to phosphatase
425
inhibition. Indeed, phosphate, the most general phosphatase inhibitor, does not influence the kinase activity at 5 mM concentration while DMP-Pase clearly becomes inhibited (Steen et aL, 1986). The same holds for VOS. In agreement with some reports (Rip and Carroll, 1980; Burton et al., 1979), but opposite to others (Allan et al., 1978; Keller et al., 1982), the time activity course of thyroidal dolichol kinase is not linear,cvcn at early times. The absence of a lag period in this curve is supporting evidence for the absence of a C T P derived phosphoryl donor, meaning that C T P itselfmust act as direct phosphoryl donor (Allan et al., 1978). The declination at longer incubation time can partially bc ascribed to a restricteddenaturation of the enzyme; indeed the enzymatic activity decreases rapidly upon storage at 4°C. The linear relationship of the enzymatic activity in function of protein concentration is in agreement with the literature (Allan et al., 1978; Keller et al., 1982). Plotting the enzymatic activity in function of protein x time (data not given) shows that prolonging the incubation time acts more inhibitory than increasing the enzyme amount. All kinasc assays wcrc performed at optimal conditions, i.e. 4.5 m i n x m g protein. The neutral pH-optimum for bovine thyroid kinasc is in agreement with data in the literature (Burton et al., 1979). U p to now there is no clcarcut explanation for the difference in pH-profilc for system I and If. W h e n studying the detergent effect it should be stressed that systems I and II do represent two totally different physico-chcmical situations. Using exogenous [l-3H]dolichol, addition of detergent is necessary to disperse the water insoluble substratc and to increase the accessibilityof enzyme and substratc to each other. In system II, dolichol and the kinasc arc both present together in the same membranes, tbercfore 0.02% (w/v) detergent is already sufficient for optimal activity.The low Triton X-100 requirement for system II was also noted in the literature(Burton et al., 1979). In our hands the maximal stimulation is found at 0.15% (w/v) Triton X-100 in system I whereas other reports (Allan et al., 1978) refer to 0.5% (w/v) Triton X-100 for optimal stimulation. This finding may reflectthe higher dolichol concentration used by tbesc authors. Finally, it should bc mentioned that the optimal detergent concentration also depends on the protein concentration in the incubation mixture (Keller et al., 1983). At higher protein concentration the detergent optima shift towards higher values. The enzyme in system II is evidently not saturated with lipid substratc; indeed addition of exogenous dolichol increase the amount of D M P formed. This suggests that the level of endogenous dolichol is rate limiting in this in vitro system. The increase of dolichol kinasc upon addition of extra dolichol with bovine thyroid is much less pronounced than reported in the literature(22% vs 300%) (Burton et al., 1979). A possible explanation for this phenomenon is the relativehigh amount of dolichol in bovine thyroid (Van Dcssel et al., 1979). In contrast to our findings, Rip and Carroll (1980) did not find an increase in the amount of labelled D M P when cold D M P was added in a concentration range of 3-30#M. Such an elevation can bc ascribed to inhibition of DMP-Pasc
426
Luc STEENet al.
of non-radioactive DMP. The decrease of D M P synthesis at higher concentration of D M P is perhaps the consequence of product inhibition as proposed by Rossignol et aL (1981) in sea urchin embryos. The limited additive character of DMP, N a F and VOJ indicates that these effectors operate at different loci. The influence of phospholipids in system I can be due to (i) changes of the physico-chemical situation and (ii) dilution effects at the level of the dolichol substrate. In the different physico-chemical condition of system II phospholipids appear to have practically no influence except for PA. Although bacitracin has been shown to be an inhibitor of the undecaprenylpyrophosphate dephosphorylation in bacteria and of DPP (not DMP) dephosphorylation in eukaryotic systems (Appelkvist et al., 1981), the antibiotic, at 1 m M concentration, also inhibits the kinase activity in system 1. The fact that, after in situ synthesis from endogenous dolichol and [7-32p]CTP, endogenous [32P]DMP can be hydrolysed suggests that both dolichol kinase and D M P phosphohydrolase are membrane bound and that the enzymes are closely positioned in the membrane. Its membrane association is further sustained by the observation that the product formed by system II can be used as substrate for glycosyltransferases located at the cytoplasmic site of the membrane. Preliminary experimental results concerning the topology of dolichol kinase suggest that the active centre of thyroidal dolichol kinase is facing the cytoplasmic surface of the membrane as already postulated by Adair and Cafmeyer (1981) for rat liver. Dolichol kinase can be easily solubilized, an indication for a peripheral localization. As a conclusion, the results presented demonstrate that bovine thyroid possesses an enzyme capable of phosphorylating dolichol. The enzyme differs in some aspects from the other dolichol kinases reported, although there is also some conformity. Operating in coordination with the D M P phosphatase, dolichol kinase could play a role in the regulation of the cellular level of the active lipid carrier. Some components (VO43-, VOS, NaF, PO 3- and dolichol) may be significant in the regulation of the D M P level in view of their different effects on, respectively, dolichol kinase and DMP-Passe. Further experiments are in progress in order to elicit the subcellular distribution and possible regulatory mechanisms in vitro. Acknowledgements--The authors are indebted to Mrs L.
Naessens for technical assistance. We are grateful to Mr P. Van Dyck and Mr J. Van Sonhoven for expert typing and artwork. This work was partly supported by a grant No. 3.0002.83 from the Nationaal Fonds voor Wetenschappelijk Onderzoek. REFERENCES
Adair W. L. Jr and Cafmeyer N. (1983) Topography of dolichyl phosphate synthesis in rat liver microsomes. Transbilayer arrangement of doliehol kinase and longchain prenyl transferase. Biochim. biophys. Acta 751; 21-26. Allan C. M., Kalin J. R., Sack J. and Verizzo D. (1978) CTP-dependent dolichol phosphorylation by mammalian cell homogenates. Biochemistry 17, 5026-5026. Appelkvist E. L., Chojnacki T. and Dallner G. (1981) Dolichol (C55) mono- and pyrophosphatases of the rat liver. Biosci. Rep. 1, 619-626.
Bauer P. I. and Varady C. (1978) A simple method for synthesizing y)2p nucleoside triphosphates using [32p]acetylphosphate and acetate kinase. Analyt. Biochem. 91, 613-617. Burton W. A., Lucas J. J. and Waechter C. J. (1981) Enhanced chick oviduct dolichol kinase activity during estrogen-induced differentiation. J. biol. Chem. 256, 632-635. Burton W. A., Scher M. G and Waechter C. J. 11979) Enzymatic phosphorylation of dolichol in central nervous tissue. J. biol. Chem. 254, 7129-7136. Datlner G. and Hemming F. W. (1981) Lipid carriers in microsomal membranes. In Mitochondria and Microsomes (Edited by Lee C. P., Schatz G. and Dallner G.L pp. 655-681. Addison Wesley, New York. De Rosa P. A. and Lucas J. J. (1982) Estrogen-induced changes in chick oviduct membrane glycoproteins. J. bioL Chem. 257, 1017 1024. Elbein A. D. (1979) The role of lipid-linked saccharides in the biosynthesis of complex carbohydrates. A. Rev. Plant Physiol. 30, 239-272. Gandhi C. and Keenan R. W. (1983) The role ofcalmodulin in the regulation of dolichol kinase. J hiol. Chem, 258, 7639 7643. Hemming F. W. (1974) Formation of sugar derivatives of dolichyl phosphate in mammalian systems. In Biochemistry of the Lipids (Edited by Goodwin T. W), pp. 39-97. University Park Press, Baltimore, Md. Keenan R. W. and Kruczek M. (1975) The preparation of tritiated betulaprenols and dolichols. Analyt. Biochem. 69, 504 509. Keller R. K., Rottler G. D., Cafmeyer N. and Adair W. L. Jr (1982) Separation of dolichols and polyprenols by straight-phase high performance liquid chromatography. Biochim. biophys. Acta 719, 1t8 125. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. I. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. Parodi A. J. and Leloir L. F. (1979) The role of lipid intermediates in the glycosylation of proteins in the eukaryotic cell. Biochim. biophys. Acta 559, 1-37. Rip J. W. and Carroll K. K. (1980) Properties of a dolichol phosphokinase activity associated with rat liver microsomes. Can. J. Biochem. 58, 1051-1056. Rossignol D. P., Lennarz W. J. and Waechter C. J. (1981) Induction of phosphorylation of dolichol during embryonic development of the sea urchin. J. biol. Chem. 256, 10538-10542. Rossignol D. P., Scher M., Waechter C. J. and Lennarz W. J. (1983) Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo. J. biol. Chem. 258, 9122-9127. Steen L., Van Dessel G., De Wolf M., Hilderson H. J., Lagrou A. and Dierick W. (1982) A CTP-dependent dolichol phosphokinase in bovine thyroid. Archs int. Physiol. Biochim. 90, BI56-BI51. Steen L., Van Dessel G., De Wolf M., Lagrou A., Hilderson H. J. and Dierick W. (1985) Further studies on bovine thyroid microsomal dolichol phosphokinase using [7-32p]CTP. Archs int. Physiol, Biochim 93, B54-B55. Steen L., Van Dessel G., De Wolf M., Lagrou A., Hilderson H. J., De Keukeleire D., Pinkse F. A., Fokkens R. H. and Dierick W. (1984) Identification and characterization of dolichyl dolichoate, a novel isoprenoic derivative in bovine thyroid. Biochim. biophys. Acta 796, 294-303. Van Dessel G., Lagrou A,, Hilderson H, J., Dommisse R,, Esmans E. and Dierick W. (1979) Isolation and identification of polyprenols from bovine thyroid gland. Biochim. biophys. Acta 573, 296--300. Voets R., Lagrou A., Hilderson H. J., Van Dessel G. and Dieriek W. (1979) RNA synthesis in isolated nuclei and nucleoli from bovine thyroid. Hoppe-Seyler's Z. physiol. Chem. 360, 1271-1283.
0020-711X/87 $3.00+0.00
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CHARACTERIZATION OF DOLICHOL KINASE FROM BOVINE THYROID MICROSOMES L u c STEENI, GUIDO VAN DESSEL2'*, MARC DE WOLF l, ALBERT LAGROUl, HERWIG HILDERSON1 and WILFRIED DIERICKL2 tRUCA-Laboratory for Human Biochemistry, University of Antwerp, B2020 Antwerp, Belgium 2UIA-Laboratory for Pathological Biochemistry, University of Antwerp, B2610 Antwerp, Belgium (Received 31 July 1986) Abstract--1. Bovine thyroid microsomes are able to phosphorylate exogenous [l-3H]dolichol as well as
endogenous dolichol. 2. The properties and specificity of the dolichol kinase activity have been studied by following the phosphorylation of [l-3H]dolichol to [1-3H]DMP as well as the formation of [32p]DMP from endogenous dolichol and [7-32p]CTP. 3. The dolichol kinase activity was not linear with respect to time and exhibited a neutral pH-optimum. 4. Product formation was directly proportional to microsomal protein concentration up to 2.5 mg protein/incubation. 5. The enzyme was found to depend on divalent cations for activity: Mg2+-ions being much more effective than Ca 2+- and Mn2+-ions. 6. In accordance, EDTA was strongly inhibitory. 7. The enzyme exhibited specificity for CTP as phosphoryl donor and was found to be inhibited by the reaction product CDP. 8. The apparent K,,-value for exogenous dolichol amounted to 4 #M. Those for CTP were estimated to be 3.88 and 10.75 mM with exogenous [l-3H]dolichol depending on the source of CTP. 9. With endogenous dolichol Kin-values for CTP of 27.8 and 6.1 #M were calculated in respectively the absence and presence of 5 mM VO] . 10. Triton X-100 (0.15%) was necessary in the [l-3H]dolichol kinase assay (only 3% of enzymatic activity in the absence of detergent), while with [7-32p]CTP dolichol kinase detergent was only of minor influence (30% stimulation at 0.02% Triton X-100). ll. The levels of the enzymatic activity could be doubled by the inclusion of 18-21 m M N a F ([1-3H]dolichol kinase) as phosphatase inhibitor; VOW- had practically no effect. 12. In contrast with [7-32p]CTP dolichol kinase, the enzymatic activity could be enhanced 4-fold by addition of 5 mM VO43- while F - resulted into no appreciable effect. 13. In both approaches sulphydryl groups were essential for enzymatic activity. 14. Bacitracin inhibited dolichol kinase. 15. Propylthiouracyl, cAMP, TSH and several other agonists were without effect on the system in vitro. 16. VOS, PO 3-, P2074- and several phosphomonoesters had no influence on the enzymatic activity. [l-3H]dolichol kinase activity could readily be solubilized (~70%).
INTRODUCTION
W o r k from several laboratories has suggested that D M P functions as a coenzyme in the assembly of asparagine-linked oligosaccharides of glycoproteins in eucaryotes (Elbein, 1979; Hemming, 1974; Parodi and Leloir, 1979). A growing body of experimental evidence indicates that the concentration of dolichyl monophosphate may be a rate limiting factor in the glycosylation process. The bulk of the dolichol in bovine thyroid (Van Dessel et al., 1979) and also in other organs (Dallner and Hemming, 1981) is present in non-phosphorylated form. D M P concentration levels seem to be regulated by altering the relative activities of dolichol kinase and D M P phosphatase. In this context data have been reported showing some correlation between the dolichol kinase activity and the glycoprotein synthesis (De *To whom correspondence should be addressed. Abbreviations: DMP: dolichylmonophosphate; DPP: dol-
ichylpyrophosphate; sphingomyelin.
PA: phosphatidic acid;
SPH: 419
B.C. 19/5--B
Rosa and Lucas, 1982). A number of reports have appeared regarding the occurrence and properties of dolichol kinase from several sources (Rip and Carroll, 1980; Gandhi and Keenan, 1983) using endogenous and exogenous dolichol as phosphoryl acceptor. This paper reports on the dolichol kinase activity in bovine thyroid microsomal membranes studied with both approaches. Some aspects of this work have been presented in a preliminary form (Steen et aL, 1982, 1985). MATERIALS AND METHODS
Chemicals
Dolichol was isolated from bovine thyroid as described earlier (Steen et al., 1984). Dolichol and DMP standards (pig liver), phospholipids, calmodulin, bacitracin, tunicamycin, glucose-6-phosphate and ATP were purchased from Sigma. Acetate kinase and almost all nucleotides were supplied by Boehringer Mannheim Corp. CTP was also obtained from Calbiochem-Behring. [32p]H3PO 4 (carrier free) was acquired from New England Nuclear. p-Nitrophenylphosphate, di-Na-phenylphosphate, fl-glycerophosphate and vanadium oxide sulphate were from Merck.
420
Luc STEENet aL
Sodium orthovanadate was obtained from BDH. Hormones were purchased from Pharmachemic. TSH was from Armour Pharmaceutical Co. All other reagents and chemicals used in these experiments were purchased from standard commercial sources. Chromatographic methods Thin-layer chromatography was performed on precoated silica gel 60F-254 plates (Merck) using benzene/ethylacetate (9:1; v/v) (eluant A) chloroform/methanol/water (60:25 :4; v/v) (eluant B), chloroform/methanol/25% (w/v) NH 3 (65:35:5; v/v) (eluant C) or diisobutylketone/HAc/water (60:45:6; v/v) (eluant D) and on PEI cellulose plates (Merck) using 0.5 M KH2PO 4 (pH 3.4) (eluant E). Column chromatography was done on AG1-X2 (2 g; Bio-Rad Lab) converted to the acetate form by treating the resin with I M NH4OOCCH 3 in chloroform/methanol (2:1; v/v) until C I free followed by equilibration of the resin in chloroform/methanol (1 : 1; v/v). Preparation of substrates [l-3H]dolichol was obtained from bovine thyroid dolichol using the labelling method described by Keenan and Kruczek (1975). D,-32p]CTP was prepared enzymatically by the procedure of Bauer and Varady (1978): CDP and [7)zP] CTP were eluted from DEAE-cellulose respectively by 53 and 90 mM NH4HCO 3 (Bauer and Varady, 1978). Enzyme preparation Fresh bovine thyroid glands were obtained from a local slaughterhouse. Further manipulations were performed at 4°C. The glands were trimmed free of surrounding tissue and freed from blood by washing several times with 0.25 M sucrose 5 mM Tris-HCl (pH 7.4). The thyroid tissue was cut into small cubes with scissors and minced in a meat mill. After homogenization in a Waring Blendor homogenisator (30sec high speed and 30sec low speed with a 30sec intermittent cooling on ice) and filtration through a double layer of cheese cloth, nuclei and cell debris were pelleted (1200g, 10 rain). The postnuclear supernatant was centrifuged at 73,300 g for 15 min. The resultant supernatant was further centrifuged at 144,700g for 60min (Voets et al., 1979). The pellet was suspended in 0.25 M sucrose-5 mM Tris-HC1 (pH 7.4) (60-80mg protein/ml) and divided in 0.5 ml aliquots which were frozen at -20°C until use. This fraction was designated as the microsomal membrane fraction. All experiments have been performed with this enzyme preparation unless stated otherwise. Kinase assays [1-3H]dolichol kinase (procedure I). Activity was monitored by measuring the formation of [I-3H]DMP from [l-3H]dolichol. 1,250,000 dpm [1-3H]dolichol (4 Ci/mmol) dissolved in chloroform/methanol (2:1; v/v) was mixed intensively with sufficient Triton X-100 to give a final concentration of 0.15 % (w/v) in the incubation mixture. The organic solvent was evaporated under N 2. The reaction was carried out in a total volume of 200/~1 containing 200 mM Tris-maleate (pH 7.0), 24 mM MgCI:, 20 mM NaF, 18 mM CTP (pH 7) and up to 1.5 mg protein prepared as described. The mixture was shaken in a Vortex and incubated at 37°C after the addition of the enzyme fraction. In routine experiments the incubation time was 3 min. The reaction was terminated by the addition of 4 ml chloroform/methanol (2:1; v/v) and this mixture was washed with 0.6 ml water. Two clear phases and the denatured protein interface were separated by low speed centrifugation. The lower phase was applied to a AG1-X2 column prepared as described in chromatographic methods. [l-3H]dolichol was eluted with chloroform/methanol (1:1; v/v) (16ml) and [I-3H]DMP with 0.25 M NH4OOCCH3 in chloroform/methanol (1:1; v/v) (10 ml). The [1-3H]DMP fraction was collected directly in a scintillation vial and dried under a stream of air. After
addition of 10 ml lipoluma (Lumac) the amount of radioactivity was determined in a Beckman LS 8100 liquid scintillation counter. 90-95% of the radioactivity in the 0.25 M NH4OOCCH 3 chloroform/methanol (1:t; v/v) fraction migrated on TLC with authentic DMP in eluent B, C and D. The remaining radioactivity was localized at the origin of the thin-layer plate and at the level of [l-3H]dolichol. [7-seP]CTP dolichol kinase (procedure lI). The phosphorylation of endogenous dolichol was determined by estimating the amount of [~ZP]DMP formed. The 200/~1 assay, containing 200mM Tris-maleate (pH7), 20mM MgC12, 5mMVO~L 4#M [y-32p]CTP (~4pCi) and enzyme protein (~ t.5 mg) was incubated at 37°C for 3 min. Further manipulations were carried out as described for [l-3H]dolichol kinase. All enzyme assays were performed in duplicate and always agreed within 10% Solubilization of bovine thyroid dolichol kinas~ activit~ To solubilize dolichol kinase activity microsomes (8-16 mg protein/ml) were treated with 0.25% (w/v) Triton X-100, After mixing, the detergent suspension was shaken for 15 rain at 4°C. Approximately 70% of the dolichol kinase activity was recovered in the supernatant fluid after centrifugation at 144,700 g for 60 rain. Analytical methods" Protein was determined according to the method of Lowry et al. (1951) using bovine serum albumin as standard. RESULTS Kinetics With both incubation systems (l, lI) D M P is formed as a result of a non-linear time-dependent phosphorylation of dolichol [Fig. l(a) and (b)]. On the other hand, the product formation is directly proportional to membrane protein concentration up to 3 rag/incubation in both systems [Fig. l(c) and (d)]. At higher protein concentration, a sharp decrease in the amount of reaction product is noted in system 1. Dolichol kinase exhibits a p H - o p t i m u m centered at around pH 7. In system II the pH-activity curve displays a broad profile in contrast to system I where a sharp p H optimum is obtained. Comparison of the effect of different divalent cations demonstrated that in both systems Mg2+-ions are much better than Ca 2~- or Mn2+-ions (Table 1). The results with Ca 2+and Mn2+-ions are reversed in system I and II; system 1: Ca2+-ions>Mn2+-ions; system II: Mn2+-ions >>Ca2+-ions. the divalent cation requirement for full enzymatic activity is in accordance with the strong inhibition by E D T A (Table 1). The enzyme preparation itself probably contains divalent cations because E D T A addition is necessary to abolish totally the dolichol kinase activity. Detergent Dolichol kinases are generally membrane bound and their substrate is water insoluble. Therefore, especially in system I, detergent administration is needed for enzymatic activity. Several detergents were tested [Fig. 2(a)]. The highest activity is obtained with 0.15% (w/v) Triton X-100. At higher concentrations a decrease in the amount of [1-3H]DMP formed is noted. In the absence of Triton X-100 only 3% of the enzymatic activity registered at the optim u m detergent concentration 0.15% (w/v) is found. Dependent on the Triton X-100 concentration a
Bovine thyroid dolichol kinase 15
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Fig. 1. (a) [l-3H]dolichol kinase activity in function of incubation time. IS]: without NaF; i : in the presence of 21 mM NaF (CTP, Boehringer). (b) b, J2p]dolichol kinase activity in function of incubation time. 0 : without VO]- and NaF; 2: in the presence of 5 mM NaF; O: in the presence of 5 mM VOW-. (c) [l-3H]dolichol kinase in function of protein concentration (CTP, Boehringer). (d) [7-32P]dolicholkinase in function of protein concentration. different course of the enzymatic activity vs CTP concentration is observed. The Km and Vmaxvalues for CTP as calculated from the Lineweaver-Burke plots are tabulated in Table 2. The influence of different detergents on the enzymatic activity in system II is
presented in Fig. 2(b). Generally at low concentrations, detergents activate; at higher concentrations inhibition occurs. The highest stimulation ( ~ 30%) is also found with Triton X-100, but at 0.1% (w/v) concentration and higher a strong inhibition is noted.
Phospholipids Table 1. Influence of divalent cations and EDTA Concentration (pmol product formed) Divalent cation System I Mg#÷ ( + 12 mM EDTA) Ca 2÷ Mg 2+ System II without VO]Mg 2+ Ca 2+ Mn :+ with 5 mM VO~Mg 2+ ( + 1 2 m M EDTA) Ca :+ Mn 2+
Control 5 m M 10mM
20raM
0.34
1.01 0.90 0.65
2.15 1.95 0.54
4.44(1.62)* 0.58 0.39
0.83
0.243 0.069 0.215
0.270 0.078 0.269
0.334 0.087 0.345
1.04
0.965 0.204 0.348
1.166 0.208 0.385
1.44(0.620)* 0.216 0.491
*The values in parentheses are obtained in the presence of 12 mM EDTA.
The effect of different phospholipids on dolichol phosphorylation process was examined. Low concentrations (1-100/~M) have only little influence ( ~ 30% reduction) on the enzymatic activity, 1 mM concentration results into a strong inhibition (50-90%) in system I. Only with SPH an enhancement of [I-3H]DMP formation (40%) is obtained. Addition of phospholipids in system II provokes minor effects. Merely, the presence of PA (1 mM) leads to a decrease of the phosphorylating activity with 69%.
Phosphatase inhibitors The phosphorylation of [l-3H]dolichol can be stimulated 1.7-fold by the addition of 18-21 mM N a F in system I. When adding VOW-, alone or together with N a F (1 mM), to the incubation mixture the amount of reaction product increases (30-40%) at low V043-
422
L u c STEEN et al. t5
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tog C
Fig. 2. Dolichol kinase activity in function of detergent concentration (%, w/v final concentration). (a) [13H]doliehol kinase (CTP, Boehringer). I-q: control (without detergent); m: Triton X-100; U: sodium taurocholate; I1: sodium deoxycholate; I]11]: cetyltrimethyl a m m o n i u m bromide. (b) [y32P]dolichol kinase. I"1: control (without detergent); I1: Triton X-100; Ill: sodium taurocholate; I : sodium deoxycholate; II: Nonidet P-40.
Fig. 3. (a) Influence of the source ol CTP on the [1-3H]dolichol kinase activity in function of VO3 concentration. CTP, Calbiochem. 0 : control without NaF; 0: control with 21 mM NaF; Fq: incubation without NaF; n : incubation with 21 mM NaF. CTP-Boehringer. A: control with 21 mM NaF; 0 : incubation with 21 mM NaF, C': mM VO3-. (b) Influence of VO43- on the [y3ZP]dolicho] kinase activity. I--l: control with ATP; i : VO3- without ATP; O: VO]- without ATP; O: V O ] in the presence of 20mMATP; z% VOS without ATP; A: VOS in the presence of 20 mM ATP. C': mM VO]- or VOS,
concentrations (0.02-0.1 mM); at VO43- concentrations above 0.5 m M an inhibition (CTP, Calbiochem) is observed [Fig. 3(a)]. Different effects are noted with C T P Boehringer as cosubstrate. In system II, F --ions cause little influence on the kinase activity ( 5 r a M N a F : 10% stimulation). At higher concen-
trations there is even inhibition. Trinucleotides (10mM), on the other hand, increase the kinase activity (24 times for CTP, 2-3 times for the others). Purine dinucleotides are activating while U D P has no effect and C D P reacts inhibitory. In system II different VO43- effects are observed whether A T P is
o 0.05
0.01
%
0.02
Detergent
0.05 (%,
0.~0
0.50
w/v)
Table 2. Kinetic constants Dolichol
CTP
o~ (wl,vi Enzyme [1)H]dolichol kinase
K,, 4.10 -6 M
Vm~ x
11.69
Source Calbiochem Boehringer
['f32P]CTP dolichol kinase
*V~x is expressed as pmol product formedlmin/mg protein.
K~, 3.88.10-
Vma~ 3M
10.75.10 3 M 27.8-10-6 M (without VOW-) 5.1.10-6M (+5raM VOW-)
Triton X-IO0
1.0~
015
0.689 2.020 1.250 1.400
0.25 IL15 0.25
423
Bovine thyroid dolichol kinase Table 3. Influence of ionic strength and sulphydryl reagent % vs control Concentration Added substance (mM) System I System II NaCI
50 100 200
94 84 69
95 98 97
50 100 200
97 87 68
98 100 101
Dithiothreitol
5 10
103 113
101 112
fl-Mercaptoethanol
5 10
101 95
100 104
(NH4)2SO4
p -Chloromercuribenzoate HgC12
0.1
3-
2-
24
100
1
1
26
I
0.1
6
22
- O.4
I
2
5
Table 4. Influence of some phosphatase inhibitors* % vs control Concentration Added substance (mM) System I System II VOS 0.1 116"I" 115 1 1021" 117 KH2PO4-K2HPO4(pH =7) 5 107 100 Na4 P207 5 104 55 Glucose-6-phosphate 5 102 100 fl-Glycerophosphate 5 102 89 *Incubations were performed in the absence of NaF (system I) and VO43- (system II). ~lncubation with VOS was performed with CTP, Calbiochem.
present or not. In the absence o f A T P a strong activation is f o u n d while in the presence of A T P no effect is seen [Fig. 3(b)]. VOS, P O 3-, P20~ - a n d substrates for some p h o s p h a t a s e in 5 m M concentration are w i t h o u t effect o n the enzymatic activity in b o t h systems except for P20~- with [7-32p]cTP dolichol kinase (Table 4).
CTP specificity F r o m all nucleotides tested, only C T P increases the basal level o f dolichol kinase in b o t h systems (Table 5). This is further s u b s t a n t i a t e d by the inhibitory effect o f CDP. In system I, optimal enzymatic activity is observed at 1 6 - 2 0 m M CTP. A t higher c o n c e n t r a t i o n (substrate ?) inhibition occurs. Moreover, w h e n studying a n u m b e r o f parameters different results are o b t a i n e d regarding the C T P source. A t optimal Mg2+-ions conditions the dolichol kinase activity is higher with C T P from Boehringer t h a n m e a s u r e d with C T P from Calbiochem. The stimulatory effect of VOW- (0.02-0.1 m M ) is only
--0.2
I/CTP
I 0.4
0.2
0
I 0.6
( r a M ) -1
Fig. 4. [1-3H]dolichol kinase in function of CTP concentration. Incubations were performed with two sources of CTP (O: CTP, Calbiochem; A: CTP, Boehringer) and in the presence of 0.15% (w/v) Triton X-100. Reaction rate (v) is the amount of [1-3H]DMP formed (pmol/min/mg protein). observed when C T P from C a l b i o c h e m is used as p h o s p h o r y l donor. The a p p a r e n t Kin-values for C T P from C a l b i o c h e m a n d C T P from Boehringer a m o u n t to respectively 3.88 a n d 10.75 m M (Table 2; Fig. 4). If this p h e n o m e n o n is due to c o n t a m i n a t i o n , it suggests t h a t C T P from C a l b i o c h e m contains an inhibitor from the mixed type (partial n o n competitive; partial anti-competitive with ~ = 0.36 a n d fl =0.52). Such a n inversion point ( 1 2 m M MgE+-ions) is also found when checking the effect o f Mg2+-ions for b o t h C T P sources. S u p p l e m e n t a r y addition of C T P after declination of the enzymatic activity in function of time has only a m i n o r influence (5%). The a p p a r e n t K m for C T P in system II is 27.8 # M in the absence o f V O 3- a n d decreases 4-fold when 5 m M VOW- is a d d e d (Table 2). T h e a p p a r e n t Km for dolichol in system I a m o u n t s to 4 # M .
12-
"o E
9
Q. ,.-, 6 T I ,.--.
Table 5. Nucleotide specificity and influence of nucleoside monoand diphosphates % vs control Added Concentration substance (mM) System I System II ATP 20 1 248 UTP 20 1 182 GTP 20 1 I 14 CTP 20 100 2402 ADP 5 109 241 UDP 5 92 104 GDP 5 130 160 CDP 5 69 8 CMP 5 115 92
o.
//
I -4
I -3
[ -2
I --1
tog
I 0
I I
[ 2
I 3
C'
Fig. 5. Influence of DMP on [1-3H]dolichol kinase activity. &: control without NaF; I1: control in the presence of 21 mM NaF; A: incubation without NaF; r-I: incubation in the presence of 2 1 m M N a F . C': /~M DMP (CTP, Boehringer).
424
LtJc S'~r~ et al.
Table 6. Influence of D M P and dolichol on the [~,-32P]CTP dolichol kinase activity* Concentration (# M)
Added substance
DMP
% vs control
3.5 103.5 2.2 7.2 36.2 180.9
Dolichol
are involved in the enzymatic function (Table 3). Only at 200 mM (NH4)~SO4 or NaC1 (system I) the enzymatic activity is affected (Table 3). Contrary, in system II the ionic strength is without any influence (Table 3).
185 f55 101 1t3 123 t 36
*The added substances were suspended in 0.01% (w/v) Triton X-100 final concentration.
Effect of exogenous DMP and dolichol Addition of exogenous DMP in a concentration range of 1-30/tM stimulates the formation of [1-3H]DMP (Fig. 5) as well as [32p]DMP (Table 6). At higher amounts ( > 1 0 0 # M ) an inhibition of the amount of [1-3H]DMPformed is observed. In system I, adding NaF together with DMP has no influence on the profile of the curve. Simultaneous addition of DMP, NaF and VOW- gives rise to a limited but no absolute additivity (data not shown). Addition of exogenous dolichol in system II enhances the DMP production (36%) (Table 6). Stability, sulphydryl reagents and ionic strength Dolichol kinase is relative stable against repeated freezing and thawing but the activity is lost by prolonged storage at 4°C (Table 7). Sulphydryl groups Table 7. Stability of [l-3H]dolichol kinase and [~,-32P]CTP dolichol kinase
Bio-effectors Some bio-effectors were studied with the purpose to check if there is any direct effect on the kinase activity in vitro. From Table 7 it is obvious that most bio-effectors are without effect. The antibiotic bacitracin shows to be most effective (40% inhibition). Calmodulin is without appreciable influence. Characterization of the reaction product The reaction product is identified as DMP in different ways. The product cochromatographed with standard pig liver DMP in three different thin layer chromatographic systems (eluant B, C and D). Furthermore, upon anion exchange chromatography on AG1-X2, the product co-eluted with DMP in the 0.25 M ammonium acetate C/M (1:1; v/v) eluate. The reaction product was stable against mild alkaline and acid hydrolysis. Finally the reaction product could enhance the glucosyltransferase activity in a microsomal enzyme preparation (unpublished results). Solubilization Dolichol kinase (system I) can easy be solubilized [up to 70% with 0.25% (w/v) Triton X-100]. The properties of the solubilized enzyme are very similar to those of system I (data hot shownj.
% vs control Procedure
Times
Freezing/thawing
hr
System I
System II
DISCUSSION
24 48 96
89 73 71 65 56 50
85 80 79 74 65 6
The presence of an enzymatic activity catatysing the phosphorylation of dolichol in bovine thyroid microsomes has been demonstrated using both exogenous [l-3H]dolichol/CTP and endogenous dolichol/[7-32p]CTP as respective substrate pairs. A
1 2 3
Preservation at 4"C
Table 8. Influence of bio-effectors on the dolichol kinase activity in a (M + L)-fraction* % vs control Effector
Concentration't
[l-~H]dolichol kinase
[~)2P]CTP dolichol kinase
lsoproterenol Eserinesulphate Carbachol Norepinephrine Epinephrine Phenylephrine Pilocarpine Chloropromazine cAMP
1# M 1# M 1# M 1# M 1# M 1/z M 1# M 1/z M 0.04 mM 0.4 mM 0.015% (w/v) 0.15% (w/v) 216#g/ml 0.5 gg/ml 5 lag/ml 6.25 gg/ml 31.25 #g/ml 125 g g/ml 0.15 mM 1.5 mM
103 98 89 102 87 102 96 105 I 11 ( 109):~ I I 0 (107):~ 117 107 106 110 115 92 95 87 73 59
105 109 104 99 100 I0 t 103 109 106 96 99 t)~ 103
PTU TSH Tunicamycin Calmodulin§
Baeitracin
lII
*The protein concentration of the (M + L)-fraction/incubation amounts to 1 mg. tFinal concentration incubation mixture. ~/Values on parentheses are those for AMP. §The incubation with calmodulin was carried out with Ca ~÷- instead of Mg2+-ions. The concentration used in the incubation mixture was 12.5raM. Calmolulin was dissolved in 0.25 M sucrose/ 5 mM Tris-HCI pH 7.4.
Bovine thyroid dolichol kinase number of data are concording between the two incubation systems but also some striking differences are noted. System I and II both require specifically CTP as phosphoryl donor as well as the presence of divalent cations. The CTP specificity was confirmed by the inhibitory effect of CDP. All eukaryotic dolichol kinases so far characterized show this same cosubstrate specificity in contrast to bacterial polyprenyl kinases which are primarily ATP dependent. In bovine thyroid dolichol kinase (both approaches) shows a preference for Mg2+-ions, which is confirmed by the strong inhibition by EDTA. Previously, dolichol kinases have been reported to be dependent on either Mg 2+- (Rip and Carroll, 1980), Ca 2+- (Keller et aL, 1982) or Mn2+-ions (Burton et aL, 1981). Calcium activation seems to be calmodulin mediated (Gandhi and Keenan, 1983) while magnesium stimulates through another mechanism (Rip and Carroll, 1980). The apparent Kin-value for CTP in system I is much higher than in system II. Similar pronounced differences have also been reported in the literature: Km-value for [y-32p]CTP (endogenous dolichol) is always in the ~M range (Rip and Carroll, 1980); K,,-value for cold CTP (exogenous dolichol) in the mM range (Gandhi and Keenan, 1983). On the other hand, the apparent Vmax-valuesare of the same order of magnitude. This is also in agreement with the literature (Gandhi and Keenan, 1983). Up to now, there is no unequivocal explanation for the great difference between the apparent Kin-values for CTP between system I and system II at hand. Some possible explanations are: (i) A different degree of DMP-Pase and CTP-Pase inhibition in both systems. (ii) The enzyme-substrate complex must be formed in a totally different physico-chemical situation (system I: 0.15% Triton X-100; system II: no detergent). Consequently the accessibility and/or affinity of the enzyme for CTP may be different (charge effects, conformational changes). (iii) The difference in apparent Kin-values according to the source of CTP could be explained by the presence of varying amounts of contaminating phosphatase inhibitors in the respective CTP preparations. The apparent K~ for exogenous dolichol is 4 # M. Generally, higher apparent Kin-values are found in the literature (factor 10 to 102) (Rossignol et al., 1983). Different results are obtained for various phosphatase inhibitors tested in system I and system II. The sequence for the kinase stimulation in system I is F - > ATP > VOW-; in system II VOW- ~ ATP >>F - . The lack of VO]- effect in system I when using CTP from Boehringer could be due to contamination of the trinucleotide by VOW- in analogy with some ATP preparations (Sigma A6144). Since the effects of ATP and VO43- in system II are not additive it seems likely that both are operating through an analogous mechanism, possibly at the level of the degradation of CTP and DMP. The enhanced kinase activity in the presence of 5mM VO 3- without addition of ATP points to a supplementary effect from this anion that is blocked by ATP. In system II, the behaviour of F - is comparable with results mentioned in the literature (Keller et al., 1982). It is clear that the stimulatory effect may not exclusively be ascribed to phosphatase
425
inhibition. Indeed, phosphate, the most general phosphatase inhibitor, does not influence the kinase activity at 5 mM concentration while DMP-Pase clearly becomes inhibited (Steen et aL, 1986). The same holds for VOS. In agreement with some reports (Rip and Carroll, 1980; Burton et al., 1979), but opposite to others (Allan et al., 1978; Keller et al., 1982), the time activity course of thyroidal dolichol kinase is not linear,cvcn at early times. The absence of a lag period in this curve is supporting evidence for the absence of a C T P derived phosphoryl donor, meaning that C T P itselfmust act as direct phosphoryl donor (Allan et al., 1978). The declination at longer incubation time can partially bc ascribed to a restricteddenaturation of the enzyme; indeed the enzymatic activity decreases rapidly upon storage at 4°C. The linear relationship of the enzymatic activity in function of protein concentration is in agreement with the literature (Allan et al., 1978; Keller et al., 1982). Plotting the enzymatic activity in function of protein x time (data not given) shows that prolonging the incubation time acts more inhibitory than increasing the enzyme amount. All kinasc assays wcrc performed at optimal conditions, i.e. 4.5 m i n x m g protein. The neutral pH-optimum for bovine thyroid kinasc is in agreement with data in the literature (Burton et al., 1979). U p to now there is no clcarcut explanation for the difference in pH-profilc for system I and If. W h e n studying the detergent effect it should be stressed that systems I and II do represent two totally different physico-chcmical situations. Using exogenous [l-3H]dolichol, addition of detergent is necessary to disperse the water insoluble substratc and to increase the accessibilityof enzyme and substratc to each other. In system II, dolichol and the kinasc arc both present together in the same membranes, tbercfore 0.02% (w/v) detergent is already sufficient for optimal activity.The low Triton X-100 requirement for system II was also noted in the literature(Burton et al., 1979). In our hands the maximal stimulation is found at 0.15% (w/v) Triton X-100 in system I whereas other reports (Allan et al., 1978) refer to 0.5% (w/v) Triton X-100 for optimal stimulation. This finding may reflectthe higher dolichol concentration used by tbesc authors. Finally, it should bc mentioned that the optimal detergent concentration also depends on the protein concentration in the incubation mixture (Keller et al., 1983). At higher protein concentration the detergent optima shift towards higher values. The enzyme in system II is evidently not saturated with lipid substratc; indeed addition of exogenous dolichol increase the amount of D M P formed. This suggests that the level of endogenous dolichol is rate limiting in this in vitro system. The increase of dolichol kinasc upon addition of extra dolichol with bovine thyroid is much less pronounced than reported in the literature(22% vs 300%) (Burton et al., 1979). A possible explanation for this phenomenon is the relativehigh amount of dolichol in bovine thyroid (Van Dcssel et al., 1979). In contrast to our findings, Rip and Carroll (1980) did not find an increase in the amount of labelled D M P when cold D M P was added in a concentration range of 3-30#M. Such an elevation can bc ascribed to inhibition of DMP-Pasc
426
Luc STEENet al.
of non-radioactive DMP. The decrease of D M P synthesis at higher concentration of D M P is perhaps the consequence of product inhibition as proposed by Rossignol et aL (1981) in sea urchin embryos. The limited additive character of DMP, N a F and VOJ indicates that these effectors operate at different loci. The influence of phospholipids in system I can be due to (i) changes of the physico-chemical situation and (ii) dilution effects at the level of the dolichol substrate. In the different physico-chemical condition of system II phospholipids appear to have practically no influence except for PA. Although bacitracin has been shown to be an inhibitor of the undecaprenylpyrophosphate dephosphorylation in bacteria and of DPP (not DMP) dephosphorylation in eukaryotic systems (Appelkvist et al., 1981), the antibiotic, at 1 m M concentration, also inhibits the kinase activity in system 1. The fact that, after in situ synthesis from endogenous dolichol and [7-32p]CTP, endogenous [32P]DMP can be hydrolysed suggests that both dolichol kinase and D M P phosphohydrolase are membrane bound and that the enzymes are closely positioned in the membrane. Its membrane association is further sustained by the observation that the product formed by system II can be used as substrate for glycosyltransferases located at the cytoplasmic site of the membrane. Preliminary experimental results concerning the topology of dolichol kinase suggest that the active centre of thyroidal dolichol kinase is facing the cytoplasmic surface of the membrane as already postulated by Adair and Cafmeyer (1981) for rat liver. Dolichol kinase can be easily solubilized, an indication for a peripheral localization. As a conclusion, the results presented demonstrate that bovine thyroid possesses an enzyme capable of phosphorylating dolichol. The enzyme differs in some aspects from the other dolichol kinases reported, although there is also some conformity. Operating in coordination with the D M P phosphatase, dolichol kinase could play a role in the regulation of the cellular level of the active lipid carrier. Some components (VO43-, VOS, NaF, PO 3- and dolichol) may be significant in the regulation of the D M P level in view of their different effects on, respectively, dolichol kinase and DMP-Passe. Further experiments are in progress in order to elicit the subcellular distribution and possible regulatory mechanisms in vitro. Acknowledgements--The authors are indebted to Mrs L.
Naessens for technical assistance. We are grateful to Mr P. Van Dyck and Mr J. Van Sonhoven for expert typing and artwork. This work was partly supported by a grant No. 3.0002.83 from the Nationaal Fonds voor Wetenschappelijk Onderzoek. REFERENCES
Adair W. L. Jr and Cafmeyer N. (1983) Topography of dolichyl phosphate synthesis in rat liver microsomes. Transbilayer arrangement of doliehol kinase and longchain prenyl transferase. Biochim. biophys. Acta 751; 21-26. Allan C. M., Kalin J. R., Sack J. and Verizzo D. (1978) CTP-dependent dolichol phosphorylation by mammalian cell homogenates. Biochemistry 17, 5026-5026. Appelkvist E. L., Chojnacki T. and Dallner G. (1981) Dolichol (C55) mono- and pyrophosphatases of the rat liver. Biosci. Rep. 1, 619-626.
Bauer P. I. and Varady C. (1978) A simple method for synthesizing y)2p nucleoside triphosphates using [32p]acetylphosphate and acetate kinase. Analyt. Biochem. 91, 613-617. Burton W. A., Lucas J. J. and Waechter C. J. (1981) Enhanced chick oviduct dolichol kinase activity during estrogen-induced differentiation. J. biol. Chem. 256, 632-635. Burton W. A., Scher M. G and Waechter C. J. 11979) Enzymatic phosphorylation of dolichol in central nervous tissue. J. biol. Chem. 254, 7129-7136. Datlner G. and Hemming F. W. (1981) Lipid carriers in microsomal membranes. In Mitochondria and Microsomes (Edited by Lee C. P., Schatz G. and Dallner G.L pp. 655-681. Addison Wesley, New York. De Rosa P. A. and Lucas J. J. (1982) Estrogen-induced changes in chick oviduct membrane glycoproteins. J. bioL Chem. 257, 1017 1024. Elbein A. D. (1979) The role of lipid-linked saccharides in the biosynthesis of complex carbohydrates. A. Rev. Plant Physiol. 30, 239-272. Gandhi C. and Keenan R. W. (1983) The role ofcalmodulin in the regulation of dolichol kinase. J hiol. Chem, 258, 7639 7643. Hemming F. W. (1974) Formation of sugar derivatives of dolichyl phosphate in mammalian systems. In Biochemistry of the Lipids (Edited by Goodwin T. W), pp. 39-97. University Park Press, Baltimore, Md. Keenan R. W. and Kruczek M. (1975) The preparation of tritiated betulaprenols and dolichols. Analyt. Biochem. 69, 504 509. Keller R. K., Rottler G. D., Cafmeyer N. and Adair W. L. Jr (1982) Separation of dolichols and polyprenols by straight-phase high performance liquid chromatography. Biochim. biophys. Acta 719, 1t8 125. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. I. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. Parodi A. J. and Leloir L. F. (1979) The role of lipid intermediates in the glycosylation of proteins in the eukaryotic cell. Biochim. biophys. Acta 559, 1-37. Rip J. W. and Carroll K. K. (1980) Properties of a dolichol phosphokinase activity associated with rat liver microsomes. Can. J. Biochem. 58, 1051-1056. Rossignol D. P., Lennarz W. J. and Waechter C. J. (1981) Induction of phosphorylation of dolichol during embryonic development of the sea urchin. J. biol. Chem. 256, 10538-10542. Rossignol D. P., Scher M., Waechter C. J. and Lennarz W. J. (1983) Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo. J. biol. Chem. 258, 9122-9127. Steen L., Van Dessel G., De Wolf M., Hilderson H. J., Lagrou A. and Dierick W. (1982) A CTP-dependent dolichol phosphokinase in bovine thyroid. Archs int. Physiol. Biochim. 90, BI56-BI51. Steen L., Van Dessel G., De Wolf M., Lagrou A., Hilderson H. J. and Dierick W. (1985) Further studies on bovine thyroid microsomal dolichol phosphokinase using [7-32p]CTP. Archs int. Physiol, Biochim 93, B54-B55. Steen L., Van Dessel G., De Wolf M., Lagrou A., Hilderson H. J., De Keukeleire D., Pinkse F. A., Fokkens R. H. and Dierick W. (1984) Identification and characterization of dolichyl dolichoate, a novel isoprenoic derivative in bovine thyroid. Biochim. biophys. Acta 796, 294-303. Van Dessel G., Lagrou A,, Hilderson H, J., Dommisse R,, Esmans E. and Dierick W. (1979) Isolation and identification of polyprenols from bovine thyroid gland. Biochim. biophys. Acta 573, 296--300. Voets R., Lagrou A., Hilderson H. J., Van Dessel G. and Dieriek W. (1979) RNA synthesis in isolated nuclei and nucleoli from bovine thyroid. Hoppe-Seyler's Z. physiol. Chem. 360, 1271-1283.