Mg2-dependent phosphatidate phosphohydrolase of rat lung: development of an assay employing a defined chemical substrate which reflects the phosphohydrolase activity measured using membrane-bound substrate

An assay of pulmonary phosphatidate phosphohydrolase activity has been developed that employs a chemically defined liposome substrate of equimolar phosphatidate and phosphatidylcholine. Enzyme assays employing this substrate resolved two distinct activities based upon their requirements for Mg2+. Assays were performed in the presence and absence of 2 mM MgCl2 and the Mg2+-dependent phosphatidate phosphohydrolase activity calculated by difference. The Mg2+-independent phosphatase activity resembled that found using aqueous dispersions of phosphatidate (PAaq). Approximately 90% of the Mg2+-dependent phosphatidate phosphohydrolase activity was recovered in the cytosol and the remainder was associated with the microsomal fraction. The Mg2+-dependent phosphatidate phosphohydrolase activity has kinetic parameters of Km = 55 microM, Vmax = 1.6 nmol/min/mg protein for the microsomal fraction, and Km = 215 microM, Vmax = 6.8 nmol/min/mg protein for the cytosolic fraction. These parameters resembled those found using the microsomal membrane-bound (PAmb) substrate. In addition, the pH optima and sensitivity to detergents and thermal inactivation are equal to those for the PAmb-dependent phosphatidate phosphohydrolase activity. In the course of these studies the microsomal and cytosolic activities were qualitatively equal, indicative of a single enzyme in two subcellular locations. In conclusion, the assay of Mg2+-dependent phosphatidate phosphohydrolase activity measured using equimolar phosphatidate and phosphatidylcholine liposomes is equivalent to that activity previously described using microsomal membrane-bound substrate. However, the chemically-defined system provides a more simplified starting point for further studies on this important enzyme.     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.     

Pulmonary phosphatidic acid phosphohydrolase. Developmental patterns in rabbit lung

1. The developmental patterns of the phosphatidic acid phosphohydrolase activities in developing rabbit lung were determined using both aqueously dispersed phosphatidic acid (PAaq) and membrane-bound phosphatidic acid (PAmb) as the substrates. 2. The specific activities and the total activities of the PAmb-dependent phosphohydrolase activities in the microsomes and to a lesser extent in the homogenates increased between 26 and 30 days gestation (term 31), but decreased in the adult. The PAaq-dependent activities demonstrated a smaller increase during late gestation and a decrease in the adult. 3. There was little change in either the Paaq- or the Pamb-dependent activities in the cytosol between 25 and 30 days gestation. The total activities per g lung were increased in the adult. 4. Fractionation of adult cytosol on Bio-Gel A5m revealed PAaq-dependent activities in the void volume (Vo) (50% total), a peak with an apparent molecular mass (Mr) = 150 kdaltons (25% total) and a peak with Mr = 110 kdaltons (25% total). The PAaq-dependent peak with Mr = 150 kdaltons was not detected in the fetal cytosols. 5. Gel filtration revealed PAmb-dependent activity in the Vo (15% total), a major peak with an apparent Mr = 390 kdaltons (44% total) and minor peaks with Mr = 240 kdaltons (16% total) and Mr = 110 kdaltons (24% total). Little change was observed during development. 6. Thermal denaturation studies on he PAmb-dependent activities in the cytosols produced biphasic curves with a rapidly inactivated component and a relatively heat-stable component. The thermal denaturation profiles for the PAmb-dependent activities remained relatively unaltered throughout fetal development. The thermal denaturation profiles of the PAaq-dependent activities in the fetal cytosols were also biphasic. In contrast, the inactivation profiles of the PAaq-dependent activities in adult cytosol were monophasic.     

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.     

Glycerolipid synthesis in rat adipose tissue. II. Properties and distribution of phosphatidate phosphatase

The properties and subcellular distribution of phosphatidate phosphatase (EC 3.1.3.4) from adipose tissue have been investigated. The enzyme was assayed using both aqueous phosphatidate and membrane-bound phosphatidate as substrates. When measured with aqueous substrate, activity was detected in the mitochondria, the microsomes, and the soluble fraction. Mg(2+) at low concentration stimulated the phosphatidate phosphatase from soluble and microsomal fractions but had no effect on the mitochondrial phosphatidate phosphatase. At higher concentration Mg(2+) was inhibitory. In the presence of Mg(2+), the phosphatidate phosphatase from soluble and microsomal fractions was active against membrane-bound phosphatidate. No activity was demonstrated with membrane-bound substrate in the absence of Mg(2+). Mitochondria did not contain activity toward the membrane-bound substrate. The rate of utilization of aqueous phosphatidate was always higher than that of membrane-bound substrate. These results indicate that there are at least two different phosphatidate phosphatases in adipose tissue.     

Microsomal phosphatidate phosphatase in maturing safflower seeds

An assay system comprising sodium phosphatidate, phosphatidylcholine, and bovine serum albumin has been developed for the reproducible determination of phosphatidate phosphatase activity in maturing seeds of safflower (Carthamus tinctorius L.). The activity was detected in both membrane and soluble fractions, and the microsomal phosphatidate phosphatase was characterized. The optimum pH for Pi release was 6.7, and the activity depended on the concentration of Mg²⁺. Phosphatidylcholine and bovine serum albumin stimulated the phosphatase reaction. This phosphatase was highly specific for phosphatidate; lysophosphatidate, and water-soluble phosphate esters did not serve as substrate. The specific activity was approximately 20 nanomoles per minute per milligram of protein, which was close to that of glycerol-phosphate acyltransferase and higher than that of diacylglycerol acyltransferase. Furthermore, the activity per seed was enough to account for the rate of triacylglycerol accumulation in vivo. The step of diacylglycerol formation by phosphatidate phosphatase does not appear to be rate-limiting for triacylglycerol synthesis during seed maturation.     

Regulation of phosphatidate phosphatase activity by inositol in Saccharomyces cerevisiae.

Regulation of phosphatidate phosphatase (EC 3.1.34) activity was examined in Saccharomyces cerevisiae cells supplemented with phospholipid precursors. Addition of inositol to the growth medium of wild-type cells resulted in a twofold increase in phosphatidate phosphatase activity. The increase in phosphatidate phosphatase activity was not due to soluble effector molecules, and inositol did not have a direct effect on enzyme activity. The phosphatidate phosphatase activity associated with the mitochondrial, microsomal, and cytosolic fractions of the cell was regulated by inositol in the same manner. Cells supplemented with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared with unsupplemented cells. Serine, ethanolamine, and choline did not significantly affect the phosphatidate phosphatase activity of cells grown in the absence or presence of inositol. Enzyme activity was not regulated in inositol biosynthesis regulatory mutants, suggesting that regulation by inositol is coupled to regulation of inositol biosynthesis. Phosphatidate phosphatase activity was pleiotropically expressed in structural gene mutants defective in phospholipid biosynthesis. These results suggested that phosphatidate phosphatase was regulated by inositol at a genetic level.     

Intracellular translocation of phosphatidate phosphatase in maturing safflower seeds: a possible mechanism of feedforward control of triacylglycerol synthesis by fatty acids

Phosphatidate phosphatase activity was found both in the cytosol and in the microsomal membrane of maturing safflower seeds. The combined and relative activities of these two forms varied with seed maturation. During the period of rapid triacylglycerol accumulation in the cell, most of the phosphatidate phosphatase activity was membrane-bound; at the initial and last stages of seed development when triacylglycerol synthesis was at an insignificant level, the majority of the activity was soluble. The potassium salts of palmitic, stearic and oleic acids, which are the fatty acid products of proplastids, caused the translocation of the cytosolic phosphatidate phosphatase to the microsomal membrane, while laurate and linoleate, which are not products of proplastids, showed no effect. Oleoyl-CoA did not convert the soluble form of the enzyme into the membrane-bound form. The translocation induced by oleate was reversible. The cytosolic phosphatidate phosphatase of safflower seeds was not transferred to the microsomal membranes prepared from soybean, a plant species of Leguminosae, and from rapeseed, a species of Cruciferae, but was transferred to that from sunflower, which belongs to the same family as safflower, Compositae. These observations suggest that in maturing oil seeds the rate of fatty acid synthesis in proplastids may regulate the species-specific translocation of phosphatidate phosphatase between the cytosol and the endoplasmic reticulum membrane where triacylglycerol synthesis occurs and that in turn the translocation of this ambiquitous enzyme could control the rate of triacylglycerol synthesis in the cell.     

Secretion of a lipolytic protein aggregate by barley aleurone and its dissociation by starchy endosperm

Acidic and basic lysophospholipase activities (LPL) have been separated by ion-exchange chromatography of barley extracts. The basic activity predominates in the starchy endosperm of germinating barley and in the medium of hormone-stimulated half-seeds; the acidic activity is the predominant form in the medium of hormone-stimulated aleurone layers. Addition of either starchy endosperm or EDTA to the acidic activity produces the basic activity. The two activities display the same pH optimum and have similar Km values. Inactivation profiles of LPLs with immunoglobulin G (IgG) prepared against the purified basic LPL are the same. The acidic LPL obtained from the incubation medium from stimulated aleurone layers appears in the void volume on gel filtration with Bio-Gel P100. Acid phosphatase and alpha-amylase in the same incubation medium appear at their expected elution volumes on this column. Gel filtration in the presence of EDTA results in the acidic activity eluting in a volume characteristic of the basic LPL (Mr, 40,000). On Bio-Gel P300 the acidic activity peak is centered at Mr, 160,000. SDS-gel electrophoresis of fractions across this peak shows a simple distribution of proteins eluting with Mr greater than or equal to 160,000. The potential role of an aggregate in the secretion of lipolytic proteins is discussed.     

The influence of exogenous and of membrane-bound phosphatidate concentration on the activity of CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase.

Rat liver microsomes were treated with phospholipase D to obtain microsomal membranes with varying amounts of membrane-bound phosphatidate. This treatment did not impair the activity of two microsomal-bound enzymes acting with phosphatidate as substrate, i.e. CTP: phosphatidate cytidylyltransferase and phosphatidate phosphohydrolase. The dependency of the activity of these enzymes on the concentration of membrane-bound phosphatidate was determined. Both enzymes showed a linear increase in activity with membrane-bound phosphatidate concentrations up to at least 100 nmol phosphatidate/mg microsomal protein. These results indicate that both enzymes have a large reserve capacity and suggest that the enzymes are operating intracellularly, i.e. at phosphatidate concentrations of 5-10 nmol/mg endoplasmic reticulum protein, far below their maximal capacity. The ratio of phosphatidate conversion into CDP-diglyceride and 1,2-diglyceride seems to be constant for a large range of membrane-bound phosphatidate concentrations. The membrane-bound enzymes cannot utilize phosphatidate substrate present in heat-denatured membranes, but are active on phosphatidate incorporated into membranes of phospholipid vesicles.     

Partial purification, properties, and subcellulsr distribution of rat liver phosphatidate phosphatase.

Phosphatidate phosphatase (EC 3.1.3.4Y was purified 15- to 20-fold from the soluble fraction of rat liver. The purification procedure involved calcium phosphate gel adsorption and elution, ammonium sulfact precipitation, and molecular-sieve chromatography. For the enzyme assay, and aqueous dispersion of phosphatidate, rather than "membrane-bound" phosphatidate, was used as substrate. The partially purified enzyme depends almost entirely on the presence of Mg2+ for its activity. Morover, the activity of the enzyme is stimulated by phosphatidylcholine. The enzyme exhibits a high substrate specificity for phosphatidate. The apparent Km for phosphatidate is approximately 0.05 mM. The optimum pH is between 7.4 and 7.6. The enzyme is inhibited by fluoride and by p-chloromercuribenzoate. The subcellular distribution of phosphatidate phosphatase in rat liver was studied by assaying the activity of the enzyme in the presence of Mg2+ and phosphatidylcholine. In contrast ot the results of previous studies, most of the enzyme activity was found in the soluble fraction.     

Pulmonary surfactant synthesis. A highly active microsomal phosphatidate phosphohydrolase in the lung.

Lung cell-free homogenate, which contains about twice the units of phosphatidate phosphohydrolase per mg of protein compared to liver, was fractionated by differential centrifugation and the fractions were assayed for phosphatidate phosphohydrolase and marker enzymes of endoplasmic reticulum, mitochondria, and lysosomes. Over 60% of the lung phosphatidate phosphohydrolase was associated with the endoplasmic reticulum, compared to 50% of the total liver enzyme. Thus a major portion of the more active lung enzyme is potentially involved in lipid biosynthesis by the endoplasmic reticulum. Less than 0.2% of the total lung enzyme was found in a lamellar body fraction, consistent with previous findings. The lung microsomal phosphohydrolase was specific for lipid substrates, showing equal activity towards phosphatidic acid or lysophosphatidic acid and relatively low activities towards glycerophosphates. It had a neutral pH optimum, similar to the liver enzyme, but differed somewhat in its relative activity at extremes of pH. Stability at 65 degrees C was greater for the lung enzyme. Fluroide inhibited lung (or liver) microsomal phosphatidate phosphohydrolase, while tartrate, MgCl2, or EDTA had no effect. The presence of a high activity of phosphatidate phosphohydrolase in lung endoplasmic reticulum is consistent with the rapid synthesis of pulmonary surfactant phosphatidylcholine.     

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.     

Stimulation and inhibition of the activity of rat liver cytosolic phosphatidate phosphohydrolase by various phospholipids.

The influence of phospholipids on the activity of the soluble phosphatidate phosphohydrolase from rat liver was studied. Phosphatidylethanolamine stimulated the enzyme activity whereas phosphatidylglycerol, phosphatidylserine, and phosphatidylinositol were inhibitory. At a phospholipid concentration of 0.7 mg/ml, phosphatidylglycerol inhibited phosphatidate phosphohydrolase activity by 75%, while the enzyme activity was stimulated twofold in the presence of phosphatidylethanolamine. Both lysophosphatidylglycerol and lysophosphatidylethanolamine inhibited phosphatidate phosphohydrolase activity as did octylglucoside, sodium cholate, and Tween 20. The finding that phospholipids influence hepatic phosphatidate phosphohydrolase activity indicates that changes in the lipid environment may modulate the enzyme activity.     

Catalytic subunit of the polycation-stimulated protein phosphatase. Effect of proteolysis on polycation stimulation

The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher Mr value than peak 1 (35,500 vs. 35,000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to Mr and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent Mr of 35,500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca2+-dependent proteinase decreased the apparent Mr by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the 'nicked' catalytic subunit was similar to that obtained with the holoenzyme.     

Kinetic analysis of yeast phosphatidate phosphatase toward Triton X-100/phosphatidate mixed micelles

A detailed kinetic analysis of purified yeast membrane-associated phosphatidate phosphatase was performed using Triton X-100/phosphatidate mixed micelles. Enzyme activity was dependent on the bulk and surface concentrations of phosphatidate. These results were consistent with the "surface dilution" kinetic scheme (Deems, R. A., Eaton, B. R., and Dennis, E. A. (1975) J. Biol. Chem. 250, 9013-9020) where phosphatidate phosphatase binds to the mixed micelle surface before binding to its substrate and catalysis occurs. Phosphatidate phosphatase was shown to physically associate with Triton X-100 micelles in the absence of phosphatidate, however, the enzyme was more tightly associated with micelles when its substrate was present. The enzyme had 5- to 6-fold greater affinity (reflected in the dissociation constant nKsA/chi) for Triton X-100 micelles containing dioleoyl-phosphatidate and dipalmitoyl-phosphatidate when compared to micelles containing dicaproyl-phosphatidate. The Vmax for dioleoyl-phosphatidate was 3.8-fold higher than the Vmax for dipalmitoyl-phosphatidate, whereas the interfacial Michaelis constant chi KmB for dipalmitoyl-phosphatidate was 3-fold lower than the chi KmB for dioleoyl-phosphatidate. The specificity constants (Vmax/chi KmB) of both substrates were similar which indicated that dioleoyl-phosphatidate and dipalmitoyl-phosphatidate were equally good substrates. Based on catalytic constants (Vmax and chi KmB), dicaproyl-phosphatidate was the best substrate with an 11- and 14-fold greater specificity constant when compared to dioleoyl-phosphatidate and dipalmitoyl-phosphatidate, respectively.     

Comparative effects of dietary fish oil and carbohydrate on plasma lipids and hepatic activities of phosphatidate phosphohydrolase, diacylglycerol acyltransferase and neutral lipase activities in the rat

In rats fed a fish oil-enriched diet, plasma triacylglycerols were lowered 51%. At the same time there was a mean 45% reduction in Mg2+-dependent phosphatidate phosphohydrolase activity in liver microsomes and a mean 20% decrease in microsomal triacylglycerol (neutral) and diacylglycerol hydrolase activities, but not of diacylglycerol acyltransferase. These observations support the hypothesis that decreases in the activities of phosphatidate phosphohydrolase and of both lipases are involved in the expression of the inhibitory effects of fish oil feeding on hepatic lipoprotein triacylglycerol secretion. Conversely, the feeding of a sucrose-enriched diet resulted in a mean 39% rise in plasma triacylglycerols, a 19% increase in triacylglycerol hydrolase and a mean 45% increase in Mg2+-dependent microsomal phosphohydrolase activity. The effects of the two nutritional interventions on phosphatidate phosphohydrolase activity confirm a key function for this enzyme in triacylglycerol formation.     

Problems encountered in measuring the activity of phosphatidate phosphohydrolase.

The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.     

The effects of Triton X-100 and chlorpromazine on the Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities of rat lung.

Lung contains both Mg2+-dependent and Mg2+-independent phosphatidate phosphohydrolase activities. Addition of Triton X-100 (0.5%) or chlorpromazine (1 mM) leads to a marked increase in the total phosphatidate phosphohydrolase activity in rat lung microsomes (microsomal fractions), but a decrease in the Mg2+-dependent activity. These observations suggest that the Mg2+-independent activity is stimulated, whereas the Mg2+-dependent activity is inhibited. However, the possibility exists that Triton X-100 could stimulate the Mg2+-dependent enzymic activity in an Mg2+-independent manner. In addition, the positively charged amphiphilic drug could be replacing the enzyme's requirement for Mg2+. These two possibilities were examined by using subcellular fractions in which the Mg2+-dependent phosphatidate phosphohydrolase had been abolished by heat treatment at 55 degrees C for 15 min. Heat treatment does not affect the microsomal Mg2+-independent phosphohydrolase to any great extent. Since the 6-8-fold stimulations due to Triton X-100 and chlorpromazine are retained after heat treatment of this fraction, the Mg2+-independent activity must be involved. Addition of Triton X-100 and chlorpromazine to cytosol virtually abolishes the Mg2+-dependent phosphatidate phosphohydrolase activity and decreases the Mg2+-independent activity by half. Heat treatment also abolishes the Mg2+-dependent activity and decreases the Mg2+-independent activity by over half. The Mg2+-independent phosphatidate phosphohydrolase activity remaining after heat treatment was not affected by Triton X-100 or chlorpromazine. These studies demonstrate that Triton X-100 and chlorpromazine specifically stimulate the heat-stable Mg2+-independent phosphatidate phosphohydrolase activity in rat lung microsomes. In contrast, the heat-labile Mg2+-independent phosphatidate phosphohydrolase activities in cytosol are inhibited by these reagents. Triton X-100 and chlorpromazine inhibit the Mg2+-dependent phosphatidate phosphohydrolase activities in both rat lung microsomes and cytosol. These results are consistent with the view that a single Mg2+-dependent phosphatidate phosphohydrolase present in both microsomes and cytosol is specifically involved in glycerolipid metabolism.     

Porcine follicular fluid contains several low molecular weight inhibitors of follicle-stimulating hormone binding to receptor

Porcine follicular fluid (PFF) inhibited the binding of 125I-human follicle-stimulating hormone (hFSH) to receptor in vitro in a dose-dependent fashion. PFF (2.5 l) was fractionated on the basis of apparent molecular weight (Mr) by ultrafiltration using hollow fibers and membranes of precalibrated pore size. Desalted, low Mr (500-5000) subfractions containing FSH-binding inhibitor (FSH-BI) activity were further purified by Sephadex G10 gel filtration and anion-exchange high-performance liquid chromatography (HPLC). This resulted in the partial purification of several low Mr FSH-BIs. Three major peaks of FSH-BI were resolved on the Sephadex G10 column eluted with water; G10-1 [elution volume (Ve)/exclusion volume (Vo) = 1.1] had only FSH-BI activity, while G10-2 (Ve/Vo = 1.4) and G10-3 (Ve/Vo = 1.5) had both FSH-BI and luteinizing hormone (LH)-BI activities. A fourth strongly retarded peak (G10-4; Ve/Vo = 2.7) was also obtained. This latter fraction had only FSH-BI activity and represented less than 1% of the FSH-BI activity applied to the column. No separation of these fractions was obtained when the column was eluted with 10 mM ammonium acetate instead of water, suggesting resolution was due to ion-exchange or hydrophobic interactions with the Sephadex. Anion-exchange (Polyanion SI) HPLC of G10-1, G10-2 or G10-3 samples resolved several fractions with FSH-BI activity. A fraction unretained at either pH 5.0 or 7.0 (HPLC-1) was present in all samples. A fraction strongly retained by the column (HPLC-2) and a fraction eluted between 0.13 to 0.24 M acetate (HPLC-3) were present in G10-1 and G10-2 but not in G10-3. HPLC-4, eluted between 0.32 to 0.36 M acetate at pH 5.0, was detected only in G10-3 samples. The most potent low Mr FSH-BI obtained (HPLC-2) inhibited FSH binding by 50% at a dose of 10 micrograms and was enriched approximately 2500-fold relative to whole follicular fluid. These results indicate that PFF contains several low (500-5000) Mr inhibitors of FSH binding to receptor in vitro which differ on the basis of charge, hormone specificity and possibly molecular size and hydrophobicity.