Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

The biosynthesis of a choline nucleotide by a cell-free extract from Streptococcus pneumoniae.

Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.     

Kinetic and biochemical properties of CTP:choline-phosphate cytidylyltransferase from the rat brain

In order to investigate the mechanisms involved in some brain disorders at the membrane level, we studied the kinetics and biochemical properties of brain CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15), the rate-limiting enzyme of the two-step biosynthesis of phosphatidylcholine. This enzyme catalyzes the biosynthesis of CDPcholine from choline phosphate and CTP. We found that its subcellular localization (mainly in microsomal and cytosolic fractions) was different from that of phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17), the enzyme of the alternative pathway for phosphatidylcholine synthesis. CTP:choline-phosphate cytidylyltransferase showed a Km of 10 mM for CTP and 0.3 mM for choline phosphate and exhibited a random mechanism. CDPcholine, the reaction product, was a competitive inhibitor of choline phosphate and CTP utilization and had a Ki of 0.090 mM. Both particulate and soluble enzymes required Mg2+ and exhibited an optimal pH at about 7. Cytosolic activity was enhanced by addition of unsaturated fatty acids or phospholipids extracted from brain membranes. Such an enhancement was increased with the centrifugation time used for preparing the soluble enzyme.     

The effects of peripubertal testosterone on ethylmorphine demethylase activity in adult rats testectomized neonatally

The physiologic activity of ethylmorphine demethylase (EMDM) is 2.5 times greater in adult male rats than in females. Defeminization, effected neonatally by testicular androgens, causes 65% of the increase while masculinization causes the rest. Castration of the intact adult does not alter the defeminized activity of the enzyme but abolishes its masculinized activity. Testosterone restores the latter. Adults that are not defeminized, because of neonatal castration, have enzymes unresponsive to testosterone. Thus, defeminization seems necessary for the expression of masculine activity. This was studied by castrating males at birth (day 1) and implanting capsules of testosterone propionate on day 35. On day 71, the activity had increased 4.4-fold to physiologic levels, compared with rats receiving no steroid (1.9 nmol.min-1.mg-1). Removal of the implant on day 71 decreased the activity on day 84 to that of the no-steroid rats. Thus, defeminization is neither caused by androgen in peripubertal rats nor needed to effect full physiologic activity of the enzyme. The activation of EMDM by peripubertal testosterone may reflect its regulation of growth hormone; first, testosterone (days 35-71) failed to increase enzyme activity in males hypophysectomized on day 35; second, growth hormone infused (5 micrograms.h-1) over days 71-76 blocked the activation, actually decreasing activity 3.7-fold; third, the activity induced by testosterone was further increased (26%) by growth hormone release-inhibiting hormone (5 micrograms.h-1, days 71-76).     

Dexamethasone increases the activity but not the amount of choline-phosphate cytidylyltransferase in fetal rat lung

The activity of choline-phosphate cytidylyltransferase is increased by glucocorticoids in late gestation fetal lung in association with increased phosphatidylcholine biosynthesis. Previous indirect data had suggested that the stimulatory effect of the hormone was due to activation of existing enzyme rather than synthesis of new cytidylyltransferase protein. Using a rabbit antibody raised against purified rat liver choline-phosphate cytidylyltransferase, we have now quantitated the amount of the enzyme in fetal rat lung explants cultured with and without dexamethasone. Our results show that the hormone increased the activity of the enzyme but not the amount of cytidylyltransferase protein. Thus the stimulatory effect of dexamethasone on cytidylyltransferase is due to activation of existing enzyme rather than induction of enzyme synthesis.     

Studies of diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities in Tetrahymena microsomes

Microsomes isolated from Tetrahymena pyriformis synthesized phosphatidylcholine and phosphatidylethanolamine by CDPcholine: 1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1), utilizing ethanol-dispersed dioleoglycerol. Cholinephosphotransferase and ethanolaminephosphotransferase activities have similar dependences on MgCl2 and MnCl2, but the latter was more effective than the former for both enzyme activities. The V values for 1,2-dioleoylglycerol obtained at optimal conditions were 1.8 nmol/min per mg microsomal protein for cholinephosphotransferase and 0.6 nmol/min per mg microsomal protein for ethanolaminephosphotransferase. Both enzymes could not utilize 1,3-dioleoylglycerol or 1-oleoylglycerol as substrates. Cholinephosphotransferase had an apparent Km for CDPcholine of 11.7 microM with 1,2-dioleoylglycerol and was inhibited by CDPethanolamine competitively. On the other hand, ethanolaminephosphotransferase has an apparent Km for CDPethanolamine of 8 microM and CDPcholine was a noncompetitive inhibitor of ethanolaminephosphotransferase activity. Furthermore, despite the marked alteration of phospholipid composition occurring during the temperature acclimation of Tetrahymena cells, both enzyme activities showed similar dependences on growth and incubation temperatures. This may imply that the final step of de novo synthesis of two major phospholipids does not participate in the thermally induced modification of the profile of phospholipid polar head group in membranes.     

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.     

Isolation and purification of glutathione S-transferases from Brachionus plicatilis and B. calyciflorus (Rotifera)

1. The enzyme glutathione S-transferase (GST), a critical element in xenobiotic metabolism, was isolated from the marine rotifer Brachionus plicatilis and its freshwater congener B. calyciflorus. 2. In B. plicatilis, GST comprised 4.2% of cytosolic protein and was present as three separate isozymes with mol. wts 30,000, 31,400 and 33,700. Specific activity of crude homogenates was 56 nmol min-1 mg-1 protein, while that of affinity chromatography purified GST was 1850. 3. In B. calyciflorus, GST was present as two isozymes with mol. wts of 26,300 and 28,500, representing 1.0% of cytosolic protein. Crude GST specific activity was 1750 nmol min-1 mg-1 protein and purified was 72,400. 4. Rotifer GSTs are unusual because they are monomers whereas all other animals thus far investigated posses dimeric GSTs.     

Human insulin receptor beta-subunit transmembrane/cytoplasmic domain expressed in a baculovirus expression system: purification, characterization, and polylysine effects on the protein tyrosine kinase activity.

We have expressed, purified, and characterized the insulin receptor protein tyrosine kinase (PTK) retaining the transmembrane and downstream domains. The proteins expressed in insect cells using a baculovirus expression system were identified as membrane-bound by immunofluorescence staining and biochemical characterization. One-step purification by immunoaffinity chromatography from Triton X-100 cell extracts resulted in a approximately 360-fold increase in the specific kinase activity with a yield of approximately 50%. An appMr = approximately 60,000 protein was the major component identified by both silver staining of the purified enzyme and immunostaining of the crude extracts after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Using nondenaturing conditions, the molecular weight was estimated to be approximately 250,000 and approximately 500,000 by glycerol gradient centrifugation and gel permeation chromatography, respectively, suggesting that oligomers of the beta-subunit domains such as tetramers and octamers are formed. The basal PTK activity of this enzyme was much higher than those of previously reported soluble-form insulin receptor PTKs expressed in insect cells or the native receptor. Km and Vmax for two substrates, src-related peptide and poly(Glu, Tyr) (4:1), were 2.4 mM and 2.5 mumol min-1 mg-1 and 0.26 mM and 1.2 mumol min-1 mg-1, respectively. Specific activities measured under two previously reported conditions using histone H2B as a substrate were 100 or 135 nmol min-1 mg-1, in contrast to those of soluble PTKs which were reported to be 20 or 70 nmol min-1 mg-1, respectively. The purified enzyme was autophosphorylated at Tyr residues. Autophosphorylation activated the enzyme approximately 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trifluoperazine and chlorpromazine inhibit phosphatidylcholine biosynthesis and CTP:phosphocholine cytidylyltransferase in HeLa cells

The influence of chlorpromazine and trifluoperazine on phosphatidylcholine biosynthesis in HeLa cells was investigated. HeLa cells were prelabeled with [Me-3H]choline for 1 h. The cells were subsequently incubated with various concentrations of drugs. Both compounds were potent inhibitors of phosphatidylcholine biosynthesis, with 50% inhibition by 5 micron of either drug. Analysis of the radioactivity in the soluble precursors indicated a block in the conversion of phosphocholine to CDPcholine catalyzed by CTP:phosphocholine cytidylyltransferase (CTP:cholinephosphate cytidylyltransferase, EC 2.7.7.15). Inhibition by these drugs was slowly reversed after incubation for more than 2 h, or was immediately abolished when 0.4 mM oleate was included in the cell medium or when the drug-containing medium was removed. The subcellular location of the cytidylyltransferase was unaffected by either drug, nor did the drugs alter the rate of release of cytidylyltransferase from HeLa cells by digitonin treatment. The drugs had a direct inhibitory effect on cytidylyltransferase activity in HeLa cell postmitochondrial supernatants. Half-maximal inhibition was achieved with 30 microM trifluoperazine and 50 microM chlorpromazine. These drugs did not change the apparent Km of the cytidylyltransferase for CTP or phosphocholine. Inhibition of cytidylyltransferase by these compounds was reversible with exogenous phospholipid or oleate in the enzyme assay. The data indicate that both drugs inhibit phosphatidylcholine synthesis by an effect on the cytidylyltransferase. The mechanism of action remains unknown at this time.     

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.     

Physiologic 1,2-diacylglycerol levels induce protein kinase C-independent translocation of a regulatory enzyme

Phorbol esters and 1,2-diacylglycerols have been used interchangeably to study protein kinase C action. This laboratory first suggested that 1,2-diacylglycerols may also act independent of protein kinase C using protein kinase C-"down-modulated" cells (Kolesnick, R. N., and Paley, A. E. (1987) J. Biol. Chem. 262, 9204-9210). Unfortunately, down-modulation was never complete. The present studies establish an in vitro system of enzyme translocation to resolve this issue. Choline phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme for phosphatidylcholine biosynthesis, was utilized. Cytidylyltransferase translocation from cytosol to membranes mediates phorbol ester-induced phosphatidylcholine synthesis in GH3 pituitary cells. In the present studies, 1,2-diacylglycerols similarly induced phosphatidylcholine synthesis and cytidylyltransferase translocation. 1,2-Diacylglycerol-induced phosphatidylcholine synthesis, however, was not concentration-dependent but proportional to the moles of 1,2-diacylglycerol added per cell, i.e. subject to surface dilution. For instance, at constant cell number (1.67 x 10(6)/sample) and 1,2-dioctanoylglycerol concentration (diC8; 20 micrograms/ml), 32Pi incorporation into phosphatidylcholine varied from 150 to 350% above control as the incubation volume increased from 0.3 to 1.2 ml. Hence, the effective diC8 concentrations 0.5-30 micrograms/ml are preferably referred to as doses and reported as 0.25-15 nmol/10(6) cells. These doses increased cellular 1,2-diacylglycerol levels within a few fold of basal (374 pmol/10(6) cells). In vitro, diC8 also induced translocation of purified cytidylyltransferase devoid of protein kinase C to microsomes. Translocation was again subject to surface dilution. Translocation occurred with the same ratio of diC8 to microsomal membrane as phosphatidylcholine synthesis in intact cells (1-10 nmol of diC8/10(6) cell membranes). Despite stimulating cytidylyltransferase translocation in intact cells, phorbol esters failed to stimulate translocation in vitro. Hence, 1,2-diacylglycerols are not always interchangeable with phorbol esters and at physiologic levels may stimulate enzyme translocation by an alternative mechanism to protein kinase C.     

The coordination of sterol and phospholipid synthesis in cultured myogenic cells: Effect of cholesterol synthesis inhibition on the synthesis of phosphatidylcholine

The coordination of biosynthesis of cholesterol and phosphatidylcholine has been investigated in a myoblast cell line, L₆, grown in lipid-depleted medium. The addition of 25-hydroxycholesterol or compactin to this medium inhibits cholesterol synthesis by over 95%. The rate of [³H]choline incorporation into phosphatidylcholine begins to decline after 6 h and eventually falls to 45% of control. Measurements of choline flux through the CDPcholine pathway and of the pool sizes of choline-containing intermediates indicate that the formation of CDPcholine is the rate-limiting step in phosphatidylcholine synthesis in L₆. The rate of CDPcholine synthesis was measured in vivo by pulse-chase experiments. Culturing cells with 25-hydroxycholesterol or compactin results in an inhibition of this step, which parallels the inhibition of incorporation of [³H]choline into phosphatidylcholine. The specific activities of the enzymes of phosphatidylcholine synthesis were assayed under optimal substrate conditions. Growth in the presence of sterol-synthesis inhibitors for 24 h has a significant, but variable, effect on the activity of microsomal and cytosolic cholinephosphate cytidylyltransferase. Inhibition is seen in approximately one-half of the preparations and ranges up to 60%. The degree of inhibition of the enzyme in vitro correlates with an elevation of cytosolic triacylglycerol and phospholipid levels, and is not eliminated by the inclusion of excess stimulatory phospholipids in the assay. The pool sizes of the substrates, cholinephosphate and CTP, are unaffected by cholesterol synthesis inhibition. In contrast to the effects on cholinephosphate cytidylyltransferase, the microsomal enzymes glycerol-3-phosphate acyltransferase and choline phosphotransferase are stimulated 2-fold or more. Choline kinase specific activity was inhibited 2-fold after 24 h of treatment with 25-hydroxycholesterol; however, no effect on this step was observed in vivo. These results indicate that the coordination of cholesterol and phosphatidylcholine synthesis involves regulation at the cytidylyltransferase-catalyzed step.     

Comparison of ornithine transcarbamylase from rat liver and intestine. Evidence for differential regulation of enzyme levels

Ornithine transcarbamylase (OTCase) was purified from the small intestine of rat and the properties of the gut enzyme were compared with those of the enzyme from liver. The enzymes from both sources bound to the transition-state analog inhibitor, delta-N-(phosphonoacetyl)-L-ornithine, immobilized on Sepharose and eluted with carbamyl phosphate as a homogeneous preparation. The specific activities of the pure enzymes were 966 mumol min-1 mg-1 and 928 mumol min-1 mg-1 from liver and gut respectively, and the molecular mass, based on electrophoretic mobility, was 38 000 Da. The isoelectric point of the enzymes from both sources was 7.3. The enzymes from both sources cross-react to the same extent with antibodies against the liver enzyme on Western transfers and the size of the mRNA was identical on Northern transfers probed with a cDNA for the liver enzyme. Although OTCase is apparently the same gene product in both liver and gut, the enzyme levels respond differently to alterations in the protein content of the diet. OTCase in liver increased from 0.76 mumol min-1 microgram-1 DNA on 15% casein to 1.3 mumol min-1 microgram-1 DNA on 60% casein (P less than 0.01) whereas in small intestine the level decreased from 8.8 nmol min-1 microgram DNA on 15% casein to 5.7 nmol min-1 microgram-1 DNA on 60% casein (P less than 0.05). When expressed on a fresh-weight basis, the enzyme activity in liver shows the characteristic increase with increasing protein, whereas the activity in gut does not. The connection between these differences in gene expression and the different physiological roles of OTCase in liver and gut is discussed.     

Diacylglycerol signals the translocation of CTP:choline-phosphate cytidylyltransferase in HeLa cells treated with 12-O-tetradecanoylphorbol-13-acetate.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated phosphatidylcholine biosynthesis in HeLa cells was investigated. TPA caused a 3-fold increase in particulate CTP:phosphocholine cytidylyltransferase activity in HeLa cells which correlated with decreased cytidylyltransferase activity in the cytosol. The increase in membrane-associated cytidylyltransferase was confirmed by immunoblotting. Immunoprecipitation studies suggested that TPA had no effect on the phosphorylation state of cytidylyltransferase. Enhanced binding of cytidylyltransferase to diacylglycerol-enriched membranes has previously been shown. Diacylglycerol levels in TPA-treated HeLa cells increased approximately 2-fold (2.29 to 4.02 nmol/mg of protein) after 1 h of TPA treatment. A time course experiment showed a temporal relationship in which production of diacylglycerol appeared to signal translocation of cytidylyltransferase to membranes followed by a stimulation of phosphatidylcholine biosynthesis. Diacylglycerol was further evaluated as a translocator of cytidylyltransferase by depleting HeLa cells of protein kinase C and incubating with dioctanoylglcerol. This treatment increased both membrane-associated cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis approximately 2-fold. A time course experiment with dioctanoylglycerol showed a strong positive correlation (r2 = 0.89) between the amount of particulate cytidylyltransferase activity and the rate of phosphatidylcholine biosynthesis. Therefore, TPA stimulates phosphatidylcholine biosynthesis by causing a translocation of cytidylyltransferase from the cytosol to membranes, which appears to be mediated by increased diacylglycerol.     

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.     

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.     

CTP:phosphocholine cytidylyltransferase is a substrate for cAMP-dependent protein kinase in vitro

The role of phosphorylation/dephosphorylation in the regulation of CTP:phosphocholine cytidylyltransferase activity was investigated. Incubation of post mitochondrial supernatant with cAMP-dependent protein kinase (50 units) led to an increased (28%) recovery of the cytidylyltransferase in the cytosolic fraction, while incubation with an intestinal alkaline phosphatase (20 units) led to an increased (61%) recovery in the microsomal fraction. When pure cytidylyltransferase was incubated with washed microsomes in the presence of cAMP-dependent protein kinase (133 units), the enzyme associated with the supernatant fraction increased (3.12 +/- 0.02 to 3.77 +/- 0.03 nmol/min/ml) while that of the microsomal fraction decreased (1.36 +/- 0.01 to 0.56 +/- 0.05 nmol/min/ml) by 2.5-fold. The increase in the cytidylyltransferase activity in the supernatant corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (40 units) decreased the cytidylyltransferase activity in the supernatant (3.61 +/- 0.08 to 2.88 +/- 0.07 nmol/min/ml) while the activity in the microsomal fraction increased (0.56 +/- 0.08 to 1.16 +/- 0.06 nmol/min/ml) by 2-fold. The decrease in the cytidylyltransferase activity in the supernatant corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of cytidylyltransferase with phosphatidylcholine vesicles in the presence of cAMP-dependent protein kinase (110 units) decreased the cytidylyltransferase activity by 30%. The decrease in cytidylyltransferase activity corresponded to an increase in 32P incorporation into the cytidylyltransferase. Treatment with alkaline phosphatase (20 units) resulted in a 41% increase in the cytidylyltransferase activity. The increase in cytidylyltransferase activity corresponded to a decrease in 32P incorporation into the cytidylyltransferase. Incubation of the cytidylyltransferase with [gamma-32P] ATP and cAMP-dependent protein kinase led to incorporation of 32P into the serine residues of cytidylyltransferase. If the cytidylyltransferase were preincubated with alkaline phosphatase prior to incubation with cAMP-dependent protein kinase, 2-fold more 32P (0.2 mol P/mol cytidylyltransferase) was incorporated into the cytidylyltransferase. Collectively, this data is in agreement with a role for reversible phosphorylation in the regulation of cytidylyltransferase.     

Ca2+-dependent ATPases in the basolateral membrane of rat kidney cortex

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.