Properties of uridine diphosphoglucose dehydrogenase from pollen of Lilium longiflorum

ARCHIVES

OF

BIOCHEMISTRY

Properties

AND

of Uridine

of Lilium

D. DAVIES2

Department

of Horticulture, Received

5341

(1972)

Diphosphoglucose

Pollen MICHAEL

162,

BIOPHYSICS

March

Dehydrogenase

from

longiflorum’ DAVID

AND

B. DICKINSON

University of Illiwis, 15, 1972; accepted

Urbana, May

Illinois

61801

30, 1972

UDP-glucose dehydrogenase was partially purified from germinating lily pollen (Lilium 1ongijZorum). The enzyme was associated with the particulate fraction in extracts of nongerminated pollen but could be solubilized when this fraction was treated with the detergent Tween 80. The enzyme was inhibited by UDP-galacturonic acid, UDP-glucuronic acid, and UDP-xylose. The latter was a powerful inhibitor when concentration of the substrate UDP-glucose was low and caused the normally hyperbolic UDP-glucose saturation curve to become sigmoid. These data were interpreted as indicating cooperative effects, UDP-glucose dehydrogenase being viewed as a regulatory enzyme.

UDP-glucose dehydrogenase (UDP-(Y-Dglucose: NAD oxidoreductase, EC 1.1.1.22) is the first step of a branched pathway leading to plant cell-wall polysaccharides which contain glucuronic and galacturonic acids and the pentoses xylose, arabinose, and apiose (1, 2). This enzyme is also thought to be important in the synthesis of glucuronides in animals (3) and capsular polysaccharides of certain yeasts (4) and bacteria (5). UDP-glucose dehydrogenase from several different tissues is inhibited in vitro by the reaction product UDP-n-glucuronic acid and by UDP-D-xy10sc (4, 6-S). Hence the enzyme may be an important metabolic control point, being feedback-inhibited in viva by sugar nucleotides which are immediate precursors of the polysaccharides. Such a mechanism would resemble those found for other metabolic pathways, particularly the branched pathways concerned with amino acid, purine, and pyrimidine i This research was supported in part by Na.tional Science Foundation Grant GB-8764 and the Illinois Agricultural Experiment Station. 2 Present address: Instituto de Biologia, Escola Superior de Agricultura, Universidade Federal de Vicosa, Vicosa, Minas Gerais, Brazil.

biosynthesis (9-11). Allosteric properties were ascribed to UDP-glucose dehydrogenase since both homotropic and heterotropic cooperative effects were observed (4, 6, 7). Kinetic studies of the enzyme from higher plants were done with UDP-glucose dehydrogenase isolated from seedsof germinating peas (6, la), and there is little information about the enzyme from actively growing plant tissue. The present study is concerned with purification and regulatory properties of UDPglucose dehydrogenase from a rapidly growing plant tissue-germinating lily pollen (13, 14). Information was also gained concerning intracellular distribution of the enzyme and whether its activity changed during germination. Our earlier report (15) described a rapid radiochemical assay for the pollen UDPglucose dehydrogenase. The study included enzyme product characterization and measurement of stoichiometry. One UDPglucuronate and two NADH molecules appeared for each molecule of UDPglucose which disappeared. The same stoichiometry was reported for the enzyme from other organisms (3, 12).

53 Copyright All rights

@ 1972 by Academic Press, of reproduction in any form

Inc. reserved.

54

DAVIES MATERIALS

AND

AND

METHODS

Reagents Unlabeled sugar nucleotides and protamine sulfate (Grade I) were obtained from Sigma Chemical Company. UDP-n-[U-14C]glucose, 273 mCi/mmole, and UDP-n-[U-14C]xylose, 200 mCi/ mmole, were obtained from Amersham-Searle; UDP-n-[U-“C]glucuronic acid, 61.6 mCi/mmole, was from ICN. The identity and radiochemical purity of these isotopes were established with paper chromatography. The identity of unlabeled sugar nucleotides was also confirmed. Ammonium sulfate was enzyme grade from Mann. Tween 80 (polyoxyethylene sorbitan mono-oleate) was from Nutritional Biochemicals Company. Phosphodiesterase (EC 3.1.4.1) from Crotalus damanteus venom (0.32 units/mg protein) was purchased from Sigma Chemical Company, and E. coli alkaline phosphatase (EC 3.1.3.1, 10 units/mg protein) was from Worthington Biochemical Corporation. Glass-distilled water was used for all solutions.

Chromatography

was handled and germinated as reported earlier (21). Anthers were taken from freshly opened lily flowers, allowed to dry under a microbiological hood for about 10 hr, and stored at 2°C for a month or less. Pollen was scraped from the anthers and used directly as a source of enzyme or else it was germinated 2 hr in the standard germina.tion medium, pH 5.2, containing0.29 M pentaerythritol, 1.27 mM Ca(NOt)z, 0.16 mM H3B03, 0.99 mM KNOI, and 10 pg tetracycline/ml. Germination was carried out in 125.ml Erlenmeyer flasks containing 100 mg pollen and 15 ml culture medium and incubated on a rotary shaker at 30°C. Percentage of germination (generally 6570%) was determined by removing 0.05.ml aliquots and counting germinated grains under 20 X magnification. Germinated pollen was transferred to chilled centrifuge tubes, recovered by a 10.min centrifugstion at 5009, and carefully drained of culture medium by filtering under a slight vacuum on Whatman No. 1 paper discs. This constituted the germinated preparation used for enzyme isolation.

and filectrophoresis

Enzyme substrates and products were separated and identified by paper chromatography and paper electrophoresis. Paper chromatograms were run on strips of Whatman No. 1 or 3 MM paper in the following solvents : 95% ethanol : 0.1 M ammonium acetate6602 M EDTA, 7:3 (16) and ethyl acetate: pyridine:water, 8:2:1 (17). Ion-exchange paper chromatograms were run on strips of DEAE-cellulose paper (Whatman DE-81) in the following solvents: water and 0.1 M LiCI. The use of anionexchange paper was adapted from the work of Verachtert, et al. (18) in which nucleotides were separated on paper impregnated with polyethyleneimine. Paper electrophoresis was carried out on Whatman No. 1 paper in 0.02 M borate, pH 8.6 with a potential of 7 V/cm (19). St,andard sugars and sugar acids were chromatographed together with radioactive enzyme substrates or products. Radioactive spots were located with a Packard radiochromatogram scanner, and standards were visualized with Tollen’s reagent spra.y or with aniline :diphenylamine-phosphoric acid, 5: 5: 1 (20). Radioactive portions of the chromatograms (4-5 cm) were cut out and placed in vials containing 15 ml of scintillation fluid. The scintillation fluid contained 5 g of 2,5-diphenyloxazole (PPO) and 0.5 g of 1,4-his (2-[5-phenyloxazolyl])-benzene (POPOP) per liter of toluene. The samples were counted at 68% efficiency in a Packard Tri-Carb liquid scintillation counter, model 3375.

Plant Material Bulbs purchased

DICKINSON

of L&urn longijlorum, variety from George J. Ball, Inc.

Ace were The pollen

Enzyme Isolation Nongerminated or germinated pollen (prepared as described above) was ground several minutes in a mortar with isolation medium (100 mg pollen: 1 ml isolation medium). The isolation medium was at pH 7.2 and contained 10 mM Hepes (N-X-hydroxyethyl piperazine-N’-2-ethanesulfonic acid) buffer and 1 mM dithiothreitol. Cell breakage was considered complete if less than 5% of the pollen remained intact as determined under low-power magnification. The homogenate was centrifuged (38,OOOg, 30 min, 0%) and the supernatant fluid was the source of crude enzyme. In enzyme-localization studies, the isolation medium was modified slightly to include the detergent Tween 80 at 570, v/v.

PuriJication

of UDP-Glucose

Dehydrogenase

A crude enzyme extract was prepared from germinated pollen as described above. The purification was done a.t O-5%. Protamine sulfate fractionation. Protamine sulfate was added dropwise to the crude extract (0.2 ml of l%, w/v, protamine sulfate/ml crude extract). The solution was kept in ice for 25 min with occasional stirring. The resulting precipitate was removed by centrifugation at 38,OOOg for 20 min. The supernatant fluid was the source of enzyme. Heat Treatment. Sufficient 26rnM NAD was added to the protamine sulfate supernatant fluid to give a final concentration of 0.2 mM. The fluid was placed in a 55°C water bath for 2 min and then rapidly cooled in ice. Precipitated protein was removed by centrifugation at 38,000g for 20 min.

UDP-GLUCOSE

55

DEHYDROGENASE

Ammonium sulfate fractionation. A solution of cold 100% saturated ammonium sulfate was added to the supernatant fluid from the heat step to give a final concentration of 20% saturation. The solution was kept in ice for 25 minutes with occasional stirring. Precipitated protein was removed by centrifugation at 38,OOOg for 20 min. The supernatant fluid was withdrawn and made 40% saturated with ammonium sulfate. The solution was centrifuged and the precipitate dissolved in a volume of isolation medium equal to the volume of the crude extract. This was designated purified enzyme and was used for subsequent characterization.

Enzyme Assays Spectrophotometric assay of UDP-glucose dehydrogenase. This was an adaptation of the published assay (12) and was based on appearance of NADH. Reaction mixtures contained 0.4 rmole UDP-glucase, 0.4 rmole NAD, 0.1 M glycine buffer, pH 8.7, and enzyme in a total volume of 0.2 ml. Reactions were run at 22’C in a Zeiss PMQ II spectrophotometer set at 340 nm and equipped with microcells (l-cm lightpath) and a millivolt recorder. The reaction was initiated by the addition of enzyme. Controls lacking UDP-glucose were run simultaneously and the control rate was subtracted from that of the complete rea.ction mixture. Controls containing crude enzyme exhibited rates as great as one-third the rates seen in complete reaction mixtures while there was no change in controls containing purified enzyme. Isotopic assay of UDP-glucose dehydrogenase. This radioisotopic assay is discussed elsewhere (15). It involves the conversion of UDP-[14C]glucose to UDP-[14C]glucuronate by the pollen enzyme, hydrolysis of the sugar nucleotides with phosphodiesterase and alkaline phosphatase, and separation of labeled glucose from glucuronate on DEAE cellulose paper. Reactions were initiated with enzyme in 75 X lo-mm Kimble culture tubes which contained 0.1 pmole UDP-D-[14C]glucose (3000 cpm/nmole), 0.2pmole NAD, 0.1 M glycine, pH 8.7. Final volume was 50 ~1. Control tubes were included which contained boiled enzyme or lacked NAD. Tubes were incubated 10 min at 3O”C, and the reaction was terminated by a 30-set incubation at 100°C. Subsequent steps involving hydrolysis of sugar nucleotides and separation of [%]glucose from [‘4C]glucuronic acid were done as reported earlier (15). The reaction rate wa,~ linear for 10 min and was proportional to concentration of enzyme up to about 8 nmoles of UDP-glucuronic acid produced in the reaction mixture in 10 min. Assays for contaminating enzymes. The presence or absence of several additional enzymes was verified using the standard isotopic assay with the modifications described below. UDP-n-glucose-4’-

epimerase (EC 5.1.3.2) was assayed by the standard procedure except that the reaction mixture was chromatographed on Whatman No. 1 paper with the ethyl acetate:pyridine:water solvent (36-hr development). Portions of the chromatograms corresponding to galactose and mannose (in case a 2’ epimerase was present) were cut out and counted in scintillation fluid. UDP-L-arabinosel’epimerase (EC 5.1.3.5) was assayed by substitut.ing UDP-n-[14C]xylose (2i,440 cpm/nmole) for UDP-D-[%]glucose in the standard reaction mixture. Formation of UDP-r~-[‘4C]arabinose was detected by the procedure given above for UDP-hexose epimerases. UDP-D-glucuronale4’-epimerase (EC 5.1.3.6) was assayed by substituting UDPn-[14C]glucuronate (4800 cpm/nmole) for UDPn-[“Clglucose in the reaction mixture. After phosphatase treatment, [14C]glucuronate and [*%Igalacturonate were separated by paper electrophoresis for 8 hr in borate (19), and enzyme activit,y was expressed as UDP-n-[14C]galacturonate produced. Standards were located with aniline:diphenylamine:pbosphoric acid spray. UDP-n-glucuronic decarboxylase (EC 4.1.1.35) was assayed according to the standard procedure, using UDPn-[14C]glucuronate (4806 cpm/nmole) instead of UDP-D-[14C]glucose. Decarboxylase activity was indicated by appearance of a neutral compound near the solvent front of the DEAE paper after development with water. Phosphodiesterase (EC 3.1.4.1) was assayed with UDP-n-[14C]glucose in the reaction mixture. The standard procedure was followed except for omission of venom phosphodiesterase. Any [l%]glucose-1-P produced by the pollen enzyme was converted by E. coli alkaline phosphatase to [‘%]glucose which moved near the solvent front of DEAE paper developed with water. Unreacted UDP-[‘4C]glucose remained near the origin since it was not attacked by the alkaline phosphatase. Alcohol dehydrogenaae (EC 1.1.1.1) w&s assayed spectrophotometrically (22).

Kinetics Kinetic constants were calculated from plots of [S]/Y vs. [S] (23,24) from Hill plots (25) in the case of sigmoid saturation curves. Standard isotopic or spectrophotometric assay procedures were followed except that substrate concentrations were varied and inhibitors were present at the concentrations indicated. Inhibitors were added to the reaction mixtures prior to enzyme. Inhibitor constants (Ki) were calculated from Dixon plots (23) except that Hill plots were used when nonhyperbolic inhibitor saturation curves were observed. Data for the saturation curves were expressed as nmoles UDP-glucuronic acid formed/min/ml purified enzyme and estimates of maximal velocities (V,) were expressed in the same units. All K,,,

56

DAVIES

AND

values were expressed in millimolarity of the substrate being varied. Values calculated from Hill plots were SE”, the substrate concentration giving one-half of maximal velocity; Zjo, the concentration of inhibitor causing 50yc inhibition; and n (the slope) which is an estimate of the order of the reaction. RESULTS

Localization

of UDP-Glucose

Dehydrogmase

Initial studies using the spectrophotometric assay failed to detect UDP-glucose dehydrogenase in extracts of nongerminated pollen. After cenkifugation of crude hoTABLE LOCALIZATION

I

OF UDP-GLUCOSE

Treatment

DEHYDROGENASIC

Enzyme Nongerminated pollen

Tween 80 in initial isolation medium” (a) Supernatant fluid (b) Pellet resuspended in original volume of isolation medium, centrifuged, supernatant fluid assayed (c) Pellet resuspended in original volume of isolation medium containing Tween 80, centrifuged, supernatant fluid assayed 2. Tween 80 present in isolation mediumc

activity& Germinated pollen

1. No

0 0

25.7 0

DICKINSON

mogenates, no enzyme activity was observed in the supernatants or in the resuspended pellets. Activity was present in supernatant fluids of extracts from germinated pollen. However, the enzyme was present in nongerminated pollen and could be released from the pellet in an active form with the detergent Tween 80 (Table I). The enzyme was released in an amount equal to that of germinated pollen when the high-speed pellet of a homogenate from nongerminated pollen was treated with detergent (Table I, treatment lc), or when the detergent was included in the initial isolation medium (Table I, treatment 2). No additional enzyme was recovered by detergent treatment of germinated pollen (Table I, treatments lc and 2). Subsequent extraction and purification was done with germinated pollen in the absence of Tween 80. This was done in case the detergent altered possible regulatory properties of the enzyme. Purification

of UDP-Glucose

Dehydroyenase

A purification procedure was devised (Table II) in which the goals were high yield of UDP-glucose dehydrogenase and 26.8

0

TABLE PURIFICATION GLUCOSE

27.6

II

OF POLLEN DEHYDROQENASE~

UDP-

Fraction

Activityb (mu/ml)

Protein (mg/ml)

Specific activity

Purification (-fold)

Crude Protamine sulfate Heat treatment (55”C, 2 min) Ammonium sulfate (20-40%)

22.8 34.6

14.8 7.8

1.5 4.4

1 3

32.5

3.5

9.3

6

26.8

1.5

17.9

12

26.2

Q Activity was measured spectrophotometritally and expressed as nmoles UDP-glucuronic acid/min/ml enzyme extract at 22°C. All pollen samples were replicates, so values in the table represent equivalent numbers of pollen grains. b Samples of nongerminated and germinated pollen were homogenized and centrifuged as described in Materials and Methods. The supernatant fluids were assayed (la) and the pellets were extracted sequentially with isolation medium (lb) and then with isolation medium containing 5y0’,, v/v Tween 80 (1~). c Samples of nongerminated and germinated pollen were homogenized with isolation medium containing 5y0 Tween 80, and the supernatant fluid was assayed after centrifugation.

n Individual steps were those described in Methods. Protein determinations were by the method of Lowry et al. (31). The volume of each fraction was approximately 1.0 ml. The ammonium sulfate fraction was resuspended in 1.0 ml of isolation medium, so measurements of enzyme activity/ml also indicate recovery of total enzyme. b Spectrophotometric assay. 1 mU = 1 nmole of glucuronic acid/min.

UDP-GLUCOSE

DEHYDROGENASE

absence of contaminating enzymes. Good recovery of activity during purification ensured that any regulatory properties observed would be typical of the pollen enzyme. Rapid purification was necessary because the enzyme in the crude extracts was not stable, Activity decreased 50% by 2 hr after isolation when the enzyme was extracted with Tween SO from nongerminated pollen or extracted without detergent from germinated pollen. The enzyme was purified 12-fold (Table II). The purified fraction was free of contaminating enzymes (see below) and showed little loss of activity when stored 24 hr at 0°C or 2 weeks at -12°C. Preliminary experiment,s established that either UDPglucose or NAD protected the enzyme during the heat treatment, but NAD (0.2 mu final concentration) was used in all subsequent work, Without substrate the enzyme was completely inactivated after 2 min at 50°C. Optimum pH UDP-glucose dehydrogenase was assayed at pH 8.7 after preliminary experiments showed that the pH optimum was in the range 8.4-8.8. This is similar to the pea and liver enzymes (3, 12) but differs from the 7.3-7.8 range for the yeast enzyme (4) and the 9.4-9.7 optimum for the Aerobacter aerogenes enzyme (‘26). Contaminating

Enzymes

The purified UDP-glucose dehydrogenase was free of the contaminating enzymes listed under Methods. These enzymes and their activities (nmoles product/ min/ml crude extract) are phosphodiesterase, 94; UDP-D-glucuronate-4’-epimerase, 21; UDPn-glucose-4’-epimerase, 18; UDP-n-glucuronate decarboxylase, 9.0; UDP-L-arabinose4’-epimerase, 6.5. These activities may be underestimates since optimum reaction conditions were not established for each enzyme. 80 UDP-n-mannose was produced from UDP-n-glucose by either the crude or the purified enzyme preparations. Substrate Specificity The pollen enzyme was specific for NAD; NADP was not reduced in t,he presence of

57

UDP-glucose. Reaction mixtures in which UDP-n-glucose was replaced by ADP-Dglucose, GDP-n-glucose, UDP-n-xylose, or UDP-n-mannose did not yield NADH when incubated with purified enzyme. CDP-n-glucose and TDP-n-glucose gave approximately 17 % of the rate observed when UDP-D-glucose was substrate. This may indicate a lack of complete specificity for the pyrimidine base. Similar specificity for sugar and base moieties was observed with crude extracts. With UDP-n-galactose as substrate, the purified enzyme preparation catalyzed NADH reduction at 26 % of the rate observed for UDP-glucose (8.8 vs. 33.6 mu/ml enzyme). Direct evidence for production of UDP-galacturonate was not obtained, but a UDP-glucose contaminant seems unlikely since the UDP-galactose contained no detectable amounts of UDPglucose and the purified pollen enzyme preparation lacked detectable UDP-glucose4’-epimerase. Neither the pea nor the liver UDP-glucose dehydrogenase react. with UDP-galactose (12, 27), although the latter does oxidize both hexodialdose intermediates--UDP-a-n-gluco-dialdose and UDPcY-n-galacto-dialdose (28). Kinetics E$ect of substrate concentration. Substrate saturation curves were obtained using both the spectrophotometric and the radioisotopic assays. There was good agreement between the two procedures. K, values were 0.3 m&f for UDP-glucose and 0.4 mar for NAD. Activity of the purified pollen enzyme was markedly reduced when UDP-glucose exceeded 2 mM. Increasing the UDP-glucose concentration from 2 to 4 m&t in the reaction medium caused a 40 % reduction in rate. Again, similar results were obtained with the spectrophotometric and isotopic assays. Inhibitor studies. A series of compounds was tested for ability to inhibit the purified pollen UDP-glucose dehydrogenase. The following compounds had little or no effect when tested at 2 rn>r in the reaction medium: UDP-n-mannose, UDP-N-acetyl-nglucosaminc, ATP, UTP, ADP, UDP, GDP, CDP, TDP, AMP, CAIP, and Pi. UDP-D-

DAVIES

58

AND DICKINSON I 0

MINUS

UOPGALA

0

0.2

mM

UOPGALA

A

0.5

mM

UDPGALA

A

1.0

mM

UDPGALA

.

2.0

mM

UDPGALA Vmax=

” 38.1

1

I

<;r;ox= 3.9;; ,,*’/’ ,’ Vmox: / /

39.1 /”

(UDPG)mM

Km:%1

-2.0

-1.0

0

0.5

I

I

I.0

1.5

-I 2.0

(UDPG)mM FIG. 1. Relation between UDP-glucose concentration and UDP-glucose dehydrogenase activity in the absence and presence of UDP-galacturonic acid. Saturation curves are upper left and the reciprocal plot is to the right. Activity was measured isotopically.

galactose (1 mM) gave 25 % inhibition, but this effect may be due to the compound acting as a substrate. Three other sugar nucleotides (UDP-D-galacturonate, UDPn-glucuronate, UDP-n-xylose) were inhibitory and were studied in more detail. The effects of UDP-galacturonic acid in the assay medium are shown in Figs. 1 and 2. Inhibition was competitive with UDP-glucose (K; = 0.46 mM) and noncompetitive with NAD (Ki = 4.4 mM). UDP-galacturonic acid has not been shown to inhibit. UDP-glucose dehydrogenase from other organisms. Its effectiveness as an inhibitor of the pollen enzyme paralleled that of the enzyme product UDP-glucuronic acid. UDP-glucuronic acid also inhibited competitively with UDP-glucose (Ki = 0.4 m&I) and noncompetitively with NAD (Ki = 3.2 mM). The yeast, liver, and pea seed (4, 6) enzymes exhibit similar types of inhibition with respect to the two substrates. Since both UDP-glucuronic and UDPgalacturonic acids are inhibitory, the absence of UDP-glucuronic-4’-epimerase activity from the purified UDP-glucose dehydrogenase should be verified. This was done for the pollen enzyme as mentioned earlier. A striking feature of the UDP-xylose

inhibition was the effect on the normally hyperbolic UDP-glucose saturation curve which became sigmoid in the presence of the inhibitor (Fig. 3). This sugar nueleotide was more inhibitory than the uranic acid nucleotides, and 20 I.cm UDP-xylose gave greater than 50 % inhibition when UDPglucose was low. Increasing the substrate overcame the inhibition, and reciprocal plots indicated approximately the same maximal velocity (37 nmoles UDP-glucuronate/min/ml enzyme) for each curve in Fig. 3. The Hill plot (Fig. 3) shows that the inhibitor caused as much as a 3-fold reduction in affinity of enzyme for substrate and that n increased to approximately 2. Such sigmoid curves could be artifacts due to rapid inactivation of the enzyme during incubation with inhibitor at low substrate concentrations, but such was not the case here. The pollen enzyme exhibited a linear time course at a number of different concentrations of UDP-xylose and UDP-glucose. The yeast (4), pea (6), liver (6), and hen oviduct (7) enzymes also exhibited sigmoid UDP-glucose saturation curves in the presence of UDP-xylose as indicated by upward deflections of Lineweaver-Burke plots, but no 8.~ or n values

UDP-GLUCOSE

59

DEHYDROGENASE

0

MINUS

0

0.2

UDPGAL

A

n

05

UDPGAL.

A.

1.0

UDPGAL.A.

a *

2.0

UDPGAL.A.

/

U0PGAL.A.

(NAD)mM

-2.0

-I 0

0

05

I. 0

20

(NAD)mM

FIG. 2. Relation

between NAD sence and presence of UDP-galacturonic upper left and the reciprocal plot

concentration and acid. Activity is to the right.

UDP-glucose was measured

dehydrogenase isotopically.

activity Saturation

in the curves

abare

. 30

/

s:,: il.

A

n=1.7

0

/

.sc

0

.

MINUS

UDPX

A

7.5pM

UDPX

A

20.0,uM

u OPX

0 50.opP.A

LlOPX

/ l3=1.8

/ 1.0

2.0

(UDPGlmM

-4.5

-4.0

-3.5

LOG

FIG. 3. Relation between UDP-glucose the absence and presence of UDP-xylose. was measured isotopically.

concentration The Hill plot

-3.0

(UDPG)

and UDP-glucose dehydrogenase activity in is based on the saturation curves (inset). Activity

60

DAVIES

AND

DICKINSON

of UDP-xylose, and the Ki was 16 PII. The inhibition was competitive with NAD. The highest concentration of UDP-xylose tested, 150 MM, increased the K, for NAD from 0.4 to 2.6 mAI without affecting the maximal velocity. UDP-xylose also gave simple competitive inhibit’ion with NAD in studies of the pea enzyme (6). Different results were apparently obtained with the yeast enzyme because Lineweaver-Burke plots of NAD saturation curves became concave upward in the presence of UDPxylose (4). DISCUSSION

Pollen UDP-glucose dehydrogenase activity did not change during germination. In this respect it resembles the 11 other lily pollen enzymes studied to date (29, 30). It differs from the other pollen enzymes in its association with the particulate fraction and lack of activity prior to extraction with detergent. This property were given for the UDP-glucose saturation curves to show how these kinetic constants has not been reported for other organisms were affected by the presence of UDPin which UDP-glucose dehydrogenase has xylose. The pea stem enzyme was also been considered soluble. A recent report (32) mentions that UDP-glucose dehydrogenase inhibited by UDP-xylose, but no saturation was not detected in extracts from barley curves were presented (8). A complex pattern of inhibition was also and maize seedlings. The particulate nature observed when UDP-xylose inhibitor satura- of the pollen enzyme indicates that UDPtion curves were carried out at several glucose dehydrogenase in other tissues concentrations of UDP-glucose (Fig. 4). might be overlooked if only the soluble The plots of UDP-xylose vs. percentage of fraction is assayed. The transition from an inact’ive, parinhibition were sigmoid when UDP-glucose was low (n approached 2), but approached ticulate enzyme to an active, soluble enzyme first order as UDP-glucose increased. In may be part of the normal pollen germ&ation process. It is also possible that the Fig. 4 the values of V,, (nmoles UDPglucuronate formed/ mini ml enzyme in pollen dehydrogenase is associated with a absence of inhibitor) were 14 for 0.2 rnM cytoplasmic organelle which becomesfragile UDP-glucose, 18 for 0.3 m&r, 20 for 0.4 and easily ruptured after germination begins. However, the possibility that the rnnl, and 27 for 0.75 mnr. pollen dehydrogenase becomes adsorbed Neufeld and Hall (6) also reported that the liver and pea cotyledon enzymes gave to the particulate fraction during homogennonhyperbolic UDP - xylose saturation ization has not yet been ruled out. The results suggest that UDP-glucose curves at several different UDP-glucose concentrations. Hill plots of their data were dehydrogenase is a regulatory enzyme in germinating lily pollen, the inhibition by said to give families of parallel lines with acid and UDP-xylose slopes (n values) of 1.5 for pea and 2.3 for UDP-galacturonic liver; similar results were reported for the being viewed as a feedback mechanism yeast (4) and hen oviduct (7) enzymes. whereby the indirect products of the dehyNo cooperative effects were observed drogenase reaction inhibit the first enzyme with the purified pollen enzyme when NAD in the branched pathway. However, it is was varied in the presence of various levels possible that the only biologically imporFIG. 4. Relation

between UDP-xylose concentration and UDP-glucose dehydrogenase activity at several concentrations of the substrate UDP-glucose. The Hill plot is based on the saturation curves (inset). Activity was measured isotopically.

UDP-GLUCOSE

DEHYDROGENASE

tant inhibitor is UDP-xylose which is by far the most effective in vitro. At low UDPglucose concentrations the level of UDPxylose necessary to give 50% inhibition was 40-fold less than the concentration of UDP-glucuronic or UDP-galacturonic acids required to give equivalent inhibition. These sugar nucleotides have been isolated from plants (33-35), but it is difficult to relate their intracellular concentrations to in vivo enzyme activity when the enzyme may be associated with a subcellular parhicle. The concept that UDP-glucose dehydrogenase is a regulat,ory enzyme is strengthened by the recent report that the bovine cornea enzyme is modulated in viva by externally supplied UDP-xylose (36). The UDP-xylose inhibition was quite different from the inhibition caused by the two UDP-uranic acids. The latter probably acted by binding to the UDP-glucose site on the enzyme since they were competitive and noncompetitive with UDP-glucose with NAD. However, both substrates were competitive with UDP-xylose; sigmoid UDP-xylose saturation curves were noted; and the hyperbolic UDP-glucose saturation curve became sigmoid in the presence of UDP-xylose. It remains to be established whether t’he UDP-xylose inhibition is an allosteric effect involving subunit interactions; conformational changes are not necessarily t’he basis for sigmoid rate curves (37). REFERENCES W. Z. (1969) Science 166, 137. 1. H~SSID, 2. LAMPORT, D. T. A. (1970) Ann. Rev. Plant Physiol. 21, 235. J. L., MAXWELL, E. S., AXELROD, 3. STROMINGER, J., AND KALCKAR, H. M. (1957) J. Biol. Chem. 224, 79. H., ANREL, E., AND FEINGOLD, D. S. 4. ANKEL, (1966) Biochemistry 6, 1864. F. A., FRERMAN, F. E., AND HEATH, 5. TROY, E. C. (1971) J. Biol. Chem. 246, 118. 6. NEUFELD, E. F., AND HALL, C. W. (1965) Biothem. Biophys. Res. Commun. 19, 456. A., AND FEINGOLD, D. S. (1968) Bio7. BDOLAH, chim. Biophys. Acta 169, 176. 8. ABDUL-BAKI, A. A., AND RAY, P. M. (1971) Plant Physiol. 47, 537. T. (1970) Annu. Rev. 9. PREISS, J., AND KOSUGE, Plant Physiol. 21, 433.

61

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