Properties of cytidinediphosphodiacyl-sn-glycerol: Myoinositol transferase of bovine mammary tissue

In!

J. Bmhrm.

1977. Vol. 8. pp. 449 to 456. Pergrrmon PWSP. Printed in Grrar Brilotn

PROPERTIES OF CYTIDINEDIPHOSPHODIACYLsn-GLYCEROL:MYOINOSITOL TRANSFERASE OF BOVINE MAMMARY TISSUE J. A. Department of Food

WOOTTON AND J. E. KINSELLA

Science, Stocking Cornell University, (Received

Hall, College of Agriculture Ithaca. NY 14853. U.S.A. 20 Octohrr

and Life Sciences,

1976)

Abstract-l. CDP-diacyl-sn-glycerol:inositoltransferase (E.C. 2.7.8.11) which catalyzes the final reaction in dr nom synthesis of phosphatidylinositol (PI) is located in the microsomal fraction of bovine mammary tissue and showed specific activities ranging from 2 to 9 nmoles PI formed/mg protein per min. 2. Optimum pH was between 8.5 and 9.0 with CDP-diacyl-sn-glycerol (CDPDG) and 7.7 when CTP and phosphatidic acid were the source of substrate CDPDG. 3. CDP-didecanoyl-sn-glycerol was the preferred substrate with CDP-dioleyland CDP-dipalmitylsn-glycerol showing lower rates of PI synthesis. 4. Apparent K, values of 60 PM and 0.71 mM were obtained for CDP-didecanoyl-sn-glycerol and myoinositol. respectively. 5. Manganese, at 3 mM, was required for activity though magnesium at very high concentrations. 48 mM, could replace Mn’+. 6. Mercaptoethanol (5 mM) enhanced activity. 7. EGTA [ethyleneglycol-bis (B amino ethyl ether) NW-tetraacetic acid)] in the absence of added cations.permitted PI synthesis. 8. Calcium, at 2 mM, inhibited PI synthesis. 9. CDPDG:phosphohydrolase in mammary microsomes reduced PI synthesis.

INTRODUCHON

In other secretory tissues rapid turnover of PI has been associated with protein secretion and regulation of membrane permeability and calcium levels, though a cause and effect relationship has not been demonstrated (Lapetina & Michell, 1973; Michell, 1975; Hawthorne & White, 1975). In mammalian tissues PI is made from cytidinediphosphodiacyl-srl-glycerol (CDPDG) and myoinositol (I) in a reaction catalyzed by CDPDG:inositol phosphatidyltransferase (E.C. 2.7.8.11) (Paulus & Kennedy 1960: Ben,jamins & Agranoff, 1969; Bishop & Strickland, 1970). In view of the rapid turnover of PI and its possible role in secretory activity we have examined some properties of the enzyme(s) involved in PI synthesis in bovine mammary tissue.

Phosphatidylinositol

(PI) occurs universally in membranes of eucaryotic cells but its function(s) has not been determined (Hawthorne, 1973; Hawthorne & White, 1975). PI metabolism has been associated with several diverse physiological functions such as ion transport. phagocytosis, nerve impulse conduction, lipid mobilization and secretion of intracellular materials in response to neuroendocrinological stimulation (Clements & Rhoten. 1976; Hayashi et al., 1974; Hawthorne, 1975; Lapetina & Michell. 1973; Michell, 1975). The secretory tissue of bovine mammary gland contains approx 1.OmM PI and PI comprises 7 and 6% of mammary and milk phospholipids, respectively (Kinsella & Infante. 1977). The relative concentration of PI is 18, 32, 64 and 98 pg per mg protein in endoplasmic reticulum, Golgi apparatus, plasma membrane and fat globule membrane, respectively (Keenan & Huang, 1972), which indicate that as these membranes evolve they become enriched in PI. Approximately So/ of mammary PI is secreted into milk per 24 hr. However, despite its low rate of secretion PI is rapidly labeled in lactating mammary tissue by a variety of radioactively labeled precursors, i.e. glycerol, fatty acids, inorganic phosphorus (Kinsella. 1968; Patton et al., 1973; Patton, 1975) indicating its dc ~XNVJsynthesis. Data from specific activity time curves reveal a rapid breakdown of labeled PI (t,,2 < 1 hr) indicative of a more rapid turnover of PI than accounted for solely by the rate of secretion.

MATERIALS AND METHODS Materials Sucrose, myoinositol, buffer materials, phosphatidic acid. ethyleneglycoi-bis (p amino ethyl ethe;) NN’-tetraacetic acid (EGTA). ethvlene diaminotetraacetic acid (EDTA) were purchased from Sigma Chemical Co. (St. Louis, MO: U.S.A.). Cytidinediphosphodiacyl-sn-glycerol (CDPDG) species were obtained from Serdary Research (Ontario, Canada); phosphatidylinositol. silica gel G and chromatographic supplies were brought from Applied Science Laboratories (State College. PA. U.S.A.). Radioactive myoinositol [2--‘H(N)], specific activity 3 Ci/ m-mole, Triton X-100 and radiochemical supplies were obtained from New England Nuclear (Boston. MA, U.S.A.). 449

450

J. A. WOOI-CON AND J. E. KINSELLA

All common chemicals were of reagent grade or of the highest purity of commercially available materials. Glass re-distilled and deionized water was used in all solutions and buffers.

Microsoml preparatiorl. Mammary tissue was obtained from lactating Holstein cows immediately after slaughter. In some cases mammary tissue was frozen at -20°C prior to use. Secretory tissue was carefully excised, connective tissue was removed and the tissue was minced by scissors. This material was susnended in sucrose (0.25 M) in a 1:4 (v/v) ratio and homogenized at top speed in a Waring Blendor for 30 sec. This homogenate was further re-homogenized in a small mill (Polyscience Corp., IL. U.S.A.) for 30 sec. All manipulations were carried out at 4°C. A portion of the crude homogenate was filtered through two layers of cheesecloth and centrifuged in a refrigerated Sorvail (RC-2B) centrifuge using a GSA. rotor (radius 14.6 cm) at 15,OOOy for 20 min at 4°C. The supernatant was decanted through two layers of cheesecloth to separate the fat layer and centrifuged in a Beckman model L2-65 preparative ultracentrifuge using a type-21 fixed angle rotor (radius 9 cm) at 44,000 9 for 75 min. The supernatant was decanted and the microsomal pellets, after washing with sucrose (0.25 M), were resuspended in sucrose 0.25 M using a TenBroeck tissue homogenizer. The microsomal material was used fresh. stored for short periods (l-5 days) at O’C prior to use, or it was quickly frozen in a round bottom flask using a dry ice-acetone bath and lyophilized in a Virtis freeze-drier (at 0.03 mm Hg pressure at 24°C for 24 hr). The resulting powder was ground to a fine consistency and stored in plastic-capped vials at -25°C. In some cases freshly prepared microsomes were dialyzed for 24 hr at 4°C against potassium phosphate buffer (70 mM, pH 8). Remaining portions of the crude homogenate in sucrose were dialyzed against 40 vol of potassium phosphate buffer (70 mM, pH 8.0) for 24 hr at 4°C. Microsomes were recovered from this dialyzed homogenate by centrifugation and used fresh. or stored frozen or freeze-dried as described above. These were referred to as dialyzed microsomes. Protein in these preparations was quantified by the method of Lowry et al. (1951). Assay conditions. Standard assays which were done in capped culture-tubes (15 x 120 mm) in a total volume of 0.5 ml of Tris-HCl buffer (pH 8.3. 175 mM) contained CDPDG (300 PM) 3H-myoinositol (1.4 mM _ 500,000 counts/min), manganese chloride (3.0 mM) or magnesium chloride (2448 mM) and microsomal enzyme (15@200 ng protein). Optional ingredients were EGTA (500 PM). mercaptoethanol (5 mM), bovine serum albumin 2.5 mg and potassium chloride (10 mM). Initially the CDPDG was carefully added in chloroform which was then evaporated leaving a film of CDPDG on the bottom (1 cm) of the tube. This method was superior to adding CDPDG in emulsified form after adding all the appropriate ingredients, including enzyme. Freshly made MnCI, was added last, about 10 set after enzyme addition. This was necessary to avoid precipitation of CDPDG and to minimize oxidation of the manganese prior to initiating the assay. After all ingredients had been added the mixture was sonicated at 4°C in a water bath sonicator (Model 8845-3 Cole-Palmer Instrument Co., Chicago, IL, U.S.A.), and incubated at 37°C for 30 min in a reciprocating rotary incubator (Evapo-mix. Buchler. Fort Lee. NJ, U.S.A.). Zero time and appropriate blank controls were run with each experiment. The reaction was stopped by the addition of 50~1 of TCA f I M) or 1.5 ml of 0.1 N methanolic HCl. Then unlabeled ‘phosphatidylinositol (0.3 mg) was added in chloroform (3 ml). The contents were thoroughly mixed on a Vortex for 10 set and 1 ml of potassium chloride (1.5 M)

containing myoinositol (0.2 M) was added. After further mixing, the tubes plus contents were centrifuged. The clear lower chloroform layer was recovered using a pastcur pipette. Two additional extractions were made on the upper phase using chloroform. The pooled chloroform extracts were then thrice washed with Folch (Folch t’t trl.. 1957) ‘upper’ phase solvents (0.75 ml each of methanol and aqueous KCl), containing 0.2 M myoinositol. to remove contaminating labeled (3H) inositol. The lower chloroform layer containing soluble radioactive products was recovered following centrifugation and reduced to a colume of 1 ml by evaporation. This extraction procedure was very efficient (> 95”/,) in recovering radioactive phosphatidylinositol and in minimizing carryover of labeled inositol. Radioactivity in aliquots of the chloroform extract was measured in a Packard (Model 3385) liquid scintillation counter using standard methods. The product(s) of the reaction were identified by thinlayer chromatography by cochromatography with authentic standards. Activated silica gel G and a solvent system of chloroform:methanol. water and ammonium hydroxide (65:35:4:0.15v/v) gave discrete spots for PI and inositol with R, values of 0.5 and 0.15. respectively, as detected by iodine staining and radioactivity. The radioactive product cochromatographed with authentic phosphatidylinositol using the two-dimensional thin-layer chromatographic system of Parsons and Patton (1967). Products were also resolved by paper chromatography using Whatman No. 33 MM paper and a solvent system of butanol:ethanol: water (50: 33: 16 v/v). In this system inositol and PI had R, values of 0.2 and 0.7. respectively. Radioactivity was determined by liquid scintillation spectrometry (Kinsella & Wootton, 1977). In addition to measuring the incorporation of mdioactive inositol, CDPDG:inositol transferase activity was also measured spectrophotometrically by determining amount of CMP released from CDPDG in standard assays (Benjamins & Agranoff. 1969). A zero time control and a control assay devoid of inositol were included in each experiment. The standard assays were stopped by the addition of l.Oml 7% TCA at 2°C. The precipitated protein and CDPDG were pelleted by centrifugation and the supernatant was removed. The pellet was rewashed with 1.0 ml TCA at 2°C. centrifuged and the supernatants were pooled. The concentration of CMP in the supernatant was determined spectrophotometrically using the difference between absorbancies at 280 and 3 10 nm, respectively. A molar absorbancy of 13 x lOh mole-‘cm’ was used to calculate the amount of CMP present (Benjamins & Agranoff. 1969). RESULTS The CDPDG phosphatidylinositol transferase activity was located predominantly in the microsomal fraction of bovine mammary tissue. PI formation was linear with protein levels up to 200 pg per assay (Fig. 1). Enzyme activity in fresh microsomes was higher (2&30x) than that observed in frozen microsomes and significantly higher than that of freeze-dried microsomes. With lOCk250 pg microsomal protein the rate of PI formation with microsomes from dialyzed homogenate was linear for over 30 min in standard assays. At protein concentrations above 300 pg/ml the rate of PI formation was not linear, conceivably because of substrate (CDPDG)/protein binding and because of degradation of CDPDG by an endogenous pyrophosphatase. The specific activity of microsomes from different cows ranged from 2 to 9 nmoles PI formed per min/mg microsomal protein. Dialysis of the homo-

Phosphatidylinositol

u i5 E c

C

p!

I

100

I 200

I

300

Protein.

pg

Fig. I. The rate of phosphatidylinositol synthesis by increasing quantities of microsomal protein from bovine mammary tissue. (A) Freshly prepared microsomes; (B) denotes microsomes prepared from dialyzed homogenate of frozen mammary tissue; and (C) are freeze-dried microsomes prepared from (B). Assays contained ‘H-inositol

I .3mM : manganese chloride 3 mM ; CDP-didecanoyl diacyiglycrrol I X0 PM: mercaptoethanol 5 mM; EGTA 0.4 mM in 0.5 ml Tris-HCl 0.1 M. pH 9.0. and were incubated at 37°C for 30 min.

A

Ees

l

l

Hepes

0 Glycine

0 Tris

451

synthesis

genate against phosphate buffer, prior to centrifugation and recovery of microsomes, resulted in a modest increase in specific acti#ty @.a.) of the resultant microsomes. However, this improvement varied with the nature of cation used in assays, as reported below. Dialysis of the microsomes after centrifugation gave a negligible increase in s.a. Microsomes prepared from dialyzed homogenate retained maximum activity for 12-14 days when stored in concentrated solution (l&l5 mg protein/ml) at 1°C. Enzyme activity in freeze-dried microsomes remained stable during storage at -20°C for over 6 months. Because of the unavailability of fresh bovine mammary tissue on a routine basis, most experiments were done with either frozen dialyzed. or freeze-dried microsomes. The optimum pH for reaction with exogenous CDPDG was between 8.5 and 9.0, whereas when CDPDG was generated in the reaction mixture from phosphatidic acid and CTP, the apparent pH optimum was 7.7 (Fig. 2). The addition of CDPDG was essential for the formation of PI. However, the inclusion of its appropriate precursors, i.e. CTP (1.0 mM) and phosphatidic acid (1.5 mM) in the presence of magnesium (10 mM) also supported PI synthesis indicating that the microsomes could synthesize CDPDG. The rate, however, was lower than that obtained with CDPDG (Figs. 2 and 3). Phosphatidylinositol was the only radioactive lipid product as confirmed by both thin-layer and paper chromatography (Fig. 3). Several compounds affected the activity of the

Etcine

A Glycylglycine

E 9c

CDPDG

R f ;i E

c

_

6C

2 .+

z

PA +CTP

b

L

3c 7

Inositol

Fig. 2. The effect of pH on rate of phosphatidylinositol formation from CDPDG (A) and phosphatidic acid plus cytidinetriphosphate (B). Assays contained myoinositol 1.3 mM: enzyme (100 bg freeze-dried microsomes) MnCl, 3.0 mM, in 500~1 of the appropriate buffer (0.5 M). In addition assays A contained CDPDG 0.18 mM (prepared from egg lecithin). while assays B contained phosphatidic acid 1.3 mM. CTP 1 mM and MgC12 2.5 mM. Buffers used were NJ-bis-(2 hydroxyethyl-2-amino ethanesulfonic acid (BES); N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES); NJ-bis-(2-hydroxyethyl glycine (Bicine); Tris+hydroxymethylaminomethane (Tris); glytine; and glycylglycine.

Phosphotidylinositol

Fig. 3. Chromatogram showing the radioactive product (phosphatidylinositol) and precursor 3H-myoinositol following the incubation. under standard conditions, of bovine mammary microsomes with 3H-myoinositol and CDPdiolein. 300 PM (CDPDG) or C’TP (1.0 mM) and phosphatidic acid, 1.3 mM (PA). Chromatography was on Whatman 33 MM paper using a solvent system composed of butanol:ethanol:water (50:33.5: 16.5 v/v). Distribution of radioactivity in zones of the chromatograms was determined by liquid scintillation spectrometry using Aquasol. Phosphatidylinositol formed from phosphatidic acid (PA) and CTP is shown in black.

J. A. WOOTTON

452 Table

1. The effect of various and microsomes

AND J. E. KINSELLA

compounds on CDPDG:phosphatidylinositol transferase of freshly prepared from a dialyzed homogenate prepared from frozen bovine mammary tissue

microsomes

Rate of phosphatidylinositol synthesis Fresh Frozen/dialyzed (nmoles/mg

Addition EGTA (0.4 mM) Mn’+ (3 mM) Mn*+ (3 mM) + mercaptoethanol Mnzf (3 mM) + mercaptoethanol All assays contained of microsomal protein

(5 mM) (5 mM) + EGTA

(0.4 mM)

4

8

48

24

12 Cation

concentration.

mM

Fig. 4. The effect of manganese and magnesium on the CDPDG:inositol transferase activity in preparations of bovine mammary microsomes. The response of inositol transferase in dialyzed, non-dialyzed and freeze-dried microsomes to manganese is shown by curves A, B. C and to magnesium by D, E, F, respectively. Standard assays contained 3H-inositol 1.3 mM, CDPDG 300 PM. mercaptoethanol 4 mM and 0.5 ml Tris-HCI (0.1 M) pH 8.5.

per 30 min) 80 85 140 185

125 180 210 240

myoinositol (1.3 mM), CDP-didecanoin (0.3 mM), Tris-HCI in 0.5 ml and were incubated at 37°C for 30 min.

CDPDG:inositol transferase (Table 1). Cations were required for inositol transferase. Increasing concentrations of manganese up to 3 mM progressively increased inositol transferase activity but above this inhibition occurred (Fig. 4). This behavior was consistently observed with all enzyme preparations. Magnesium at much higher concentrations could replace manganese and optimum concentration occurred at 48mM, at which concentration maximum activity exceeded that obtained at optimum manganese levels. Magnesium, above 48 mM, inhibited inositol transferase slightly. Dialysis of the mammary homogenate against phosphate buffer prior to isolation of microsomes altered the response of inositol transferase to cations. Thus it enhanced and reduced the maximum rates obtained with Mn*+ and Mg2+, respectively (Fig. 4). Freeze drying of the dialyzed microsomes reduced the response patterns to Mg2’ stimulation. EGTA, in the absence of added cations. was capable of supporting PI synthesis and a concentration of 1.5 mM was optimum (Fig. 5). These data suggested the presence of an endogenous inhibitor even in dialyzed microsomes. The inclusion of EGTA in assay tubes reduced the effect of Mg at low concentrations, presumably by chelation, and enhanced the

protein

buffer

(0.1 M. pH 9.0) and

100 pg

effect of Mg *+ in the concentration range 3-12 mM (Fig. 6) though maximum s.a. was not changed significantly. EGTA almost completely restored the inositol transferase activity in freeze-dried microsomes to that of the original freshly dialyzed microsomes. The addition of EGTA to assay tubes containing Mn*+ (3 mM) had a negligible effect on the inositol transferase. EDTA failed to activate the inositol transferase and it counteracted the effect of EGTA when both were present in reaction mixtures. The stimulatory effect of EGTA was consistent with the presence of inhibitory cation(s). Calcium, a possible contaminant in mammary microsomes, significantly inhibited inositol transferase (Fig. 7) particularly in the assay tubes containing Mg*+, where Ca’+ at 2 mM completely inhibited the transferase irrespective of order of addition. Microsomes prepared from dialyzed mammary homogenate were slightly less inpreparations in hibited by Ca2+ than non-dialyzed assays containing Mn2+. Addition of Ca2+ after the Mn’+ had been added to the assays failed to inhibit the enzyme suggesting that it failed to displace the bound Mn2 + The apparent differential stimulation of inositol transferase by Mn2+ and Mg*+ could possibly be attributed to the existence of two discrete enzymes.

I_. 1n -

0.25 0.5

EGTA.

3.5

6.0

mM

Fig. 5. The effect of ethyleneglycol-bis (/3 amino ethylether) NW-tetraacetic acid (EGTA) on phosphatidylinositol synthesis in the absence of added cations. Freshly prepared microsomes from dialyzed bovine mammary homogenate were used in standard assays containing CDP-didecanoyl sn-glycerol (300 PM) and 3H-inositol (1.3 mM).

Phosphatidylinositol

I 3

I 6

1

I

12

24

Magnesrum,

synthesis

453

,

-is

mM 40

Fig. 6. The effect of magnesium with and without EGTA on the CDPDG:inositol transferase activity of freshly prepar-cd (A and B and freer+dried microsomes (C and D) prepared from dialyzed mammary homogenate. Standard assays with CDP-didecanoyl-sn-diacylglycerol (300 PM) were used. EGTA (0.5 mM) was included in assays A and C. ( x ~~~ x ) indicates the activity in microsomes from non-dialyzed fresh samples of mammary tissue. However, thermal inactivation studies tended to discount this (Fig. 8). Both activities decreased in a par-

allel manner. The data revealed that this enzyme is relatively stable to heat. Of the various molecular species of CDPDG used, CDP-didecanoin was the most effective substrate (Table 2). The observed differences in rates for the various CDPDG species may reflect the relative solubility or dispersibility of these amphipathic molecules. Above concentrations of 300 PM the various CDPDG species were inhibitory. Detergents (Tween-20, Tween-80 and Triton X- 100) over a range of concentrations had little effect on enzyme activity while sodium deoxycholate was inhibitory. Apparent

Preheotlng

60

80

temperature,

Fig. 8. Thermal instability of CDPDG:inositol transferase from bovine mammary microsomes. Microsomes prepared from dialyzed mammary homogenate were held at the indicated temperatures for 20 min and the transferase activity was then determined under standard conditions in the presence of magnesium (48 mM) or manganese (3 mM). K, values for CDP-didecanoin of 66 and 60 PM were obtained for freeze-dried and frozen dialyzed microsomes, respectively (Fig. 9). There was no incorporation of 3H-myoinositol in assays lacking the glycerolipid acceptor (CDPDG) indicating the lack of inositol exchange with endogenous PI. The apparent K, measured under initial velocity conditions for inositol was 0.70 and 0.71 mM for freeze-dried and fresh microsomes. respectively. Because cytidine monophosphate (CMP) is a product of inositol transferase we quantified CMP release by the spectrophotometric method of Benjamins and Agranoff (1969). In the complete assay lacking only inositol there was a significant release of CMP (Table 3) indicating the presence of CDPDG phosphohydrolase in microsomal preparations. The presence of CMP in these preparations was confirmed by the positive identification of CMP following paper chromatographic resolution of the products. Further evidence for the presence of CDPDG phosphohydrolase Table 2. The relative rate of phosphatidylinositol from various cytidine diphosphodiacyl-sn-glycerol by bovine mammary microsomes

Substrate CDP-didecanoin” CDP-diolein CDP-diacylglycerol” CDP-dipalmitin Calcium,

“C

Relative A 100 51 45 20

synthesis species

rates B 100 60 48 18

mM

Fig. 7. The inhibitory effect of calcium on the activity of CDPDG:inositol transferase of dialyzed freeze-dried (A, B. D) and freeze-dried (C) bovine mammary microsomes. Standard assays were used. In assays A calcium was added after equilibration of enzyme with manganese (3 mM), in B and C the calcium was added before the manganese and in D addition of calcium before or after magnesium (12 mM) gave similar inhibition.

’ Rate obtained with CDP-didecanoin was 180 (A) and 130 (B) nmoles PI formed/mg protein per 30 min for fresh dialyzed and freeze-dried microsomal preparations respectively. h Prepared from diacyl-sn-glycerol moiety of egg lecithin. Assay tubes contained inositol 1.3 mM. CDPDG 300 PM. MnCI, 3 mM, mercaptoethanol 2 mM, EGTA 0.4 and IOOpg microsomal protein in 0.5 ml of Tris-HCI buffer 0.1 mM, pH 9.0.

J. A. WOOTFON

KM

=60pM

V,,,=

IO

AND

J. E.

KINSELLA

.

nmoles

/mq

.

per mln

/

. / . /

4’

/

.*

.‘*

CDP

I

I

I

20

40

60

dldecanoyl

w-glycerol,

mW

Fig. 9. Lineweaver-Burk plot showing activity of CDPDG: inositol transferase of bovine mammary microsomes with CDP-didecanoyl-sn-glycerol. Assay system contained inositol 1.4 mM, mercaptoethanol 5 mM, EGTA 0.4mM, MnCI, 6 mM and 100 pg dialyzed microsomal enzyme in

0.5 ml Tris-HCI 0.1 M. pH 8.5.

was obtained in a study involving pre-incubation of microsomes with CDPDG prior to assay under normal conditions. There was a progressive decline in PI formation with duration of incubation (Fig. 10). i.e. by over 40% following a 30 min pre-incubation. Whether this decline was caused by inhibitory levels of CMP or by the limited availability of free substrate, warrants further study. Depletion of substrate appeared unlikely because an excess of CDPDG was added to each assay tube and only 4-6 nmoles was hydrolyzed. The data obtained by measuring CMP release (Table 3) indicated that PI formation was greater than that measured by net incorporation of labeled inositol into PI. The discrepancies in these results suggested that some degradation of synthesized PI was occurring in the assays. The enzyme phosphatidylinositol inositolphosphohydrolase, which is probably responsible for hydrolyzing PI to 1,2 diacylglycerol and inositolphosphate, is active in lactating mammary tissue (Kinsella & Wootton, 1977).

Table

3. Formation upon

of cytidine incubation

Pre-incubation,

CDP-didecanoin W)

+ inositol

min

Fig. 10. The effect of pre-incubation of microsomes with substrate CDPDG on subsequent synthesis of phosphatidylinositol. Assay tubes contained Tris buffer (pH 8.5) 0.1 M, bovine serum albumin 2.5 mg, manganese chloride CDP-didecanoyl-sn-glycerol 300 PM ; EGTA 6 mM, 0.4 mM ; mercaptoethanol 5 mM and dialyzed microsomal enzyme (70 pg protein) in a total volume of 0.5 ml. After pre-incubation periods of 0, lo,20 and 30 min, respectively (37°C) myo-inositol (H’) 1.4 mM was added and the contents were incubated at 37°C for 30 min with continuous agitation. The incorporation of inositol into phosphatidylinositol was determined as described in Methods.

DlSCUSSION

Paulus and Kennedy (1960) first established that PI synthesis involved a transferase reaction between inositol and CDPDG and the present data are consistent with this. Our results indicate that inositol transferase of bovine mammary tissue has properties similar to those of other tissues, i.e. brain (Benjamins & Agranoff, 1969; Thompson rt al., 1963; Bishop & Strickland, 1968). platelets (Lucas et ul., 1970), reticulocyte membrane (Percy et al., 1973), pancreas (Prottey & Hawthorne, 1967) and heart (Hill, 1974). As previously observed by Benjamins and Agranoff

monophosphate and concurrent of bovine mammary microsomes CMP

Microsomal enzyme

30

20

IO

phosphatidylinositol synthesis from CDP-didecanoin in the presence and absence of inositol

released Phosphatidylinositol A

- inositol

formed* B

nmoles Freeze-dried Dialyzed

300 300 600

15.5 31.5 30.5

4.5 4.0 15.0

11.0 27.5 15.5

7.0

17.0 14.0

’ Assay tubes contained substrates indicated with MnCl, 3 mM; EGTA 0.4 mM, mercaptoethanol 5 mM, BSA 2.5 mg, Tris-HCI buffer 0.1 M, pH 8.5, and 100 pg microsomal protein in a total volume of 0.5 ml. Assays were terminated after 30 min at 37°C with TCA (see Methods). The CMP was measured by difference in spectrophotometric readings at 280 and 310 nm and using a molar absorbancy of 13 x 166 mole-’ cm2. “Phosphatidylinositol formation was estimated from the inositol stimulated release of CMP (A) and by the direct measurement of the incorporation of [H3] inositol into labeled product (B).

Phosphatidylinositol

(1969) and Bishop and Strickland (1970) for brain, and Van Golde et al. (1974) for liver, the mammary enzyme was localized in the crude microsomal fraction and negligible activity was detected with the high speed supernatant or mitochondrial fractions. The 4-fold increase in specific activity obtained by Benjamins and Agranoff (1969) by dialyzing the tissue (rat brain) homogenate before isolation of the microsomes was not achieved with bovine mammary tissue. Dialysis did improve activity when manganese was the cation species included in the assay but it reduced the activity when magnesium was the cation used. Inactivation of some of the enzyme may have occurred in some instances because Rao and Strickland (1974) observed that a partially purified inositol transferase from rat brain was inactivated following dialysis. Several researchers reported that detergent or bovine serum albumin was necessary to activate membrane bound inositol transferase (Thompson et al., 1963; Benjamins & Agranoff, 1969). However, detergent was not required to activate the inositol transferase in microsomes of bovine mammary tissue. Bovine serum albumin did stimulate the activity in freeze-dried microsomes. It had been observed that freezing and thawing of the enzyme from rdt brain reduced activity (Salway et cd., 1968). This was prevented by the addition of dithioerythritol and substrate CDPDG. Apparently free sulfhydryl (SH) groups are necessary for optimum enzyme activity (Rao & Strickland, 1974). Direct incorporation of inositol by exchange with the inositol moiety of endogenous PI species was negligible in bovine mammary microsomes as indicated by lack of incorporation of 3H-inositol in the absence of added CDPDG. This exchange has been observed by Paulus and Kennedy (1960) and several others, but Holub (1974) reported that the rate was low compared to rate of de MIUOformation. The apparent k, of CDPDG:inositol transferase for inositol. i.e. 0.7 mM. would indicate that the enzyme is saturated in vitlo because the concentration of inositol, which is facilely synthesized in lactating mammary tissue (Burton & Wells, 1974). is around 4-5 mM in rat mammary tissue (Dawson & Freinkel. 1961). The inositol concentration in bovine milk is around 0.6 mM (Jenness, 1974) while in rat milk it reaches a maximum of I .6 mM (Burton & Wells. 1974). CDP-didecanoyl-sn-glycerol was twice as active as CDP-dioleyl- and CDP-diacyl-sn-glycerol species. derived from egg lecithin, and 5 times as effective as CDP-dipalmityl-sn-glycerol as inositol acceptors. Similar preferences were also observed by Benjamins and Agranoff (1969). Bishop and Strickland (1970) showed that the CDP-dioleyl-srl-glycerol was about five-fold more effective as a substrate than CDP-dipalmityl-sn-glycerol. The apparent specificities observed in vitro may reflect the relative poor state of dispersibility of these substrates in the assay systems. In the present studies the inclusion of detergents (1 mg/ml), to facilitate dispersibility of substrate actually impaired enzyme activity. Above 4OOjlm CDPDG species became inhibitory. presumably via their detergency properties. Inhibition by substrate was also noted by Prottey and Hawthorne (1967). Benjamins and Agranoff (1969) and Bishop and Strickland (1970).

synthesis

455

Despite the low apparent K, observed for the mammary enzyme the availability of CDPDG may limit the synthesis of PI. CDPDG is a transient intermediate whose concentration in tissue is low, i.e. 617 nmolc/g liver (Thompson & McDonald, 1975). The mammary tissue contains the enzyme CTP:phosphatidic acid cytidyltransferase (E.C. 2.7.7.41) which synthesizes CDPDG from phosphatidic acid (PA) and CTP. Thus, the activity of PA phosphohydrolase, which controls the concentration of mammary PA, may indirectly influence PI synthesis in mammary tissue by controlling availability of CDPDG. Evidence for this has been obtained from studies with liver tissue wherein PI synthesis was enhanced when PA phosphohydrolase was inhibited by drugs (Brindley & Bowley. 1975). Mn’+ gave a sharp maximum with all microsomal preparations in comparison with freeze-dried microsomes, whereas Mg”+ produced a plateau at its optimum concentration 40-50 mM. Incidentally, the high concentrations of Mg’+ required in vitro are at least 4-fold the average intracellular magnesium levels. The observed activation by this cation may be related to its effect on substrate enzyme interactions; on the state of aggregation of the enzyme or substrate, or on the physical state of the product PI. Rao and Strickland (1974) reported that in the presence of detergent, Mg”+ precipitated an active form of the enzyme from microsomal membranes. Speculation concerning the function of the cations is redundant until appropriate kinetic studies are made. The inositol transferase of mammary tissue revealed greater sensitivity to calcium inhibition than did that of pig brain brain or blood platelets (Benjamins & Agranoff, 1969; Lucas et a/., 1970). The Mg”+ stimulated activity associated with microsomal mammary enzyme was more sensitive to calcium inhibition than when Mn” was used. Hill (1974) also reported that the inositol transferase of heart was much more susceptible to calcium inhibition when Mg’+ was the stimulatory cation. The stimulatory effect of EGTA could be attributed to its chelation of inhibitory bound calcium ions in the various enzyme preparations studied. Its effects compared to EDTA may have been due to its possible ‘loosening’ effect on the structure of the membrane wherein the enzyme was located, thereby facilitating enzyme substrate accessibility.

SUMMARY

The enzyme CDP-diacyl-sn-glycerol :inositoltransferase which is respnsible for the de nouo synthesis of phosphatidylinositol is located in the microsomal fraction of bovine mammary tissue. The optimum pH was 8.5 and apparent K, values of 60 PM and 0.71 mM were obtained for CDP didecanoin and myoinositol, respectively. Optimum cation concentration was 3 mM for manganese and 48 mM for magnesium. EGTA supported PI synthesis in the absence of manganese or magnesium. Mercaptoethanol stimulated enzyme activity indicating the involvement of sulfhydryl groups in enzymatic activity. Calcium, 2mM, inhibited the activity of CDP-diacyl-sn-glycerol inositol transferase.

J. A.

456

W~~TTON AND

Aclinowlrdgements-This work was supported in part by grants 37174 and PCM75-19123 from National Science Foundation.

REFERENCES BENJAMINS J. A. & AGRANOFFB. W. (1969) Distribution and properties of CDP diglyceride:inositol transferase from brain. J. Neurochern. 16. 513. BISHOPH. H. & STRICKLAND K. P. (1970) On the specificity of cytidine diphosphate diglycerides in monophosphoinositide biosynthesis in rat brain preparations. Can. J. Biochem. 48. 269.

BRINDLEY D. N. & BOWLEYM. (1975) Drugs affecting the synthesis of glycerides and phospholipids in rat liver. Biochem. J. 148, 461. BURTONL. E. & WELLSW. W. (1974) Development pattern of the enzymes converting glucose 6-phosphate to myoinositol in the rat. Develop. Biol. 37. 35. CLEMENTS R. S. & RHOTENW. B. (1976) Phosphoinositide metabolism and insulin secretion from isolated rat pancreatic cells. J. clin. Invest. 57. 684. DAWSONR. M. C. & FREINKELN. (1961) The distribution of free mesoinositol in mammalian tissues including observations on lactating rat. Biochem. J. 78. 606. FILCH J.. LEES M. & SLOANE-STANLEY G. S. (1957) A simple method for the isolation and purification of total lipids from animal tissue. J. hiol. Chem. 226, 497. HAWTHORNE J. N. (1973) In Form and Ftmctions of Phospholipids (Edited by ANSELLG., HAWTHORNE J. N. & DAW~ONR. M. C.). Elsevier Press, New York. HAWTHORNEJ. N. & WHITE D. A. (1975) Myoinositol lipids. Warns Horm. 33. 529. HA;ASHI E., MAEDAT. & TOMITAT. (1974) Effect of inositol deficiency on lipid metabolism in rats. Biochim. biophys. Acta 360. 134-146.

J. E.

KINSELLA

pholipids in mammary tissue. In Lactatiorl (Edited by LARSONB. L.). Vol. 4. Academic Press, New York. KINSELLAJ. E. & WCMX-TON J. A. (1977) Phosphatidylinositol inositolphosphohydrolase activity in mammary tissue. Inr. J. Biochem. 8. 457459. LAPETINAE. G. & MICHELLR. H. (1973) Phosphatidylinositol metabolism in cells receiving extracellular stimulation. FEBS Lett. 31. 1. LOWRY 0. H., ROSEBROUGH N. J., FARR A. L. & RAXDALL R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193. 265. LUCASC. T., CALLF. L. & WILLIAMSW. J. (1970) Biosynthesis of phosphatidylinositol in human platelets. J. clin. Incest. 49. 1949. MICHELLR. H. (1975) Inositol phospholipids and all surface receptor function. Biochim. biophys. Acta 415. 81. PARWNSJ. G. & PATTONS. (1967) Two-dimensional thinlayer chromatography of polar lipids from milk and mammary tissue. J. Lipid Res. 8. 896. PATTON S.; MCCARTHY-R. D., PLANTZ P. E. & LEE R. F. (1973), Phosoholinids incornoration into nlasma membrane of lactaiing mammary cell with special reference to sphingomyelin. Nature, Land. 241. 241. PA~ON S. (1975) Detection of labeled phosphatidic acid in lactating mammary gland of rat. J. Dairy Sci. 58. 560. PAULUSH. & KENNEDYS. P. (1960) Enzymatic synthesis of inositol monophosphatide. J. biol. Chem. 235. 1303. PERCYA. K.. SCHMELLE.. EARLESB. J. & LENNARZW. J. (1973) Phospholipid biosynthesis in the membrane of immature and mature red blood cells. Biochenlistry 12. 2456. PROTTEYC. 8~ HAWTHORNE J. N. (1967) The biosynthesis of nhosnhatidic acid and ohosnhatidvlinositol in mammaiian bancreas. Biochem.‘J. 1bS. 379. RAO H. R. & STRICKLAND K. P. (1974) On the solubility. stability and partial purification of CDP-diacyl-sn-glycerol inositol transferase from rat brain. Biochim. bio-

HII.I_W. H. (1974) CDP-diglyceride inositol transferase of -al heart. Ph.D. Thesis, Tulane University. phys. Acta 348, 306. H~L~IBB. J. (1974) The Mn activated incorporation of in- SALWAYJ. G., HARWOODJ. L., KAI M., WHITE G. L. & ositol into molecular species of phosphatidylinositol in HAWTHORNE J. N. (1968) Enzymes of phosphoinositide metabolism during rat brain development. J. Neurochem. rat liver microsomes. Biochim. biophys. Acta 369, 111. JENNESS R. The composition of milk. In Lactation: A Com15. 221. prehensive Treat&e (Edited by LARSON B. L. & SMITH THOMPSON W. & MCDONALDG. (1975) CDP-diacyl-sn-glyV. R.). Academic Press. New York. cerol concentrations in rat liver. J. biol. Chem. 250. 6779. KEENANT. W. & HUANG C. M. (1972) Membranes of THOMPSON W.. STRICKLAND K. P. & ROSITER R. J. (1963) mammary gland. Lipid and protein composition of Biosynthesis of phosphatidylinositol in rat brain. BioGolgi apparatus and rough endoplasmic reticulum from them. J. 87. 136. bovine mammary gland.-J. Dair; Sci. 55. 1586. VAN GOLDE L. M. G., RABEN J., BA~ENRUKGJ. J.. KINSELLAJ. E. (1968) Incornoration of glvcerol into lipids FLEISCHER B.. ZAMBRANO F. & FLEISCHERS. (19741 Bioby dispersed ‘bovine mammary cells- kochim. hiol;hvs. synthesis of lipids in Golgi complex and other sudcelluActa 164. 540. lar fractions from rat liver. Biochim. biophys. Acta 360. 179. KINSELLAJ. E. & INFANTEJ. P. (1977) Synthesis of phos-