Soluble Fraction Of Rat Liver Research Articles

Characteristics of regulation of the activity of glucokinase from rat liver

A simple method for isolation of glucokinase from a soluble fraction of rat liver was proposed, making it possible to obtain the enzyme preparation characterized by high specific activity, native cooperative characteristics of the enzyme, and actually complete absence of other molecular forms of hexokinase. Dependence of the enzyme activity on the glucose concentration shows a pronounced sigmoidal behavior: with in the whole range of substrate concentrations at 25 and 2°C, the Hill coefficients are equal to 1.76 and 1.35, correspondingly, while the S0.5 values of glucose are 6.90 and 20 mM, respectively. The role of the kinetic reaction mechanism in the regulation of glucokinase activity in liver is under discussion.

Characterization of a soluble mouse liver enzyme capable of hydrolyzing diisopropyl phosphorofluoridate

A novel mouse liver soluble fraction DFPase which has organophosphatase activities with sarin, soman and tabun, was purified and characterized. However, it lacks paraoxonase and arylesterase activities with paraoxon and phenyl acetate, respectively. This DFPase closely resembles and may be identical with the one purified by Little et al. in 1989 from the soluble fraction of rat liver, based on its substrate specificity, size (∼39 kDa) and its stimulation by several metal ions, namely magnesium, manganese and cobalt. Sequencing of our purified mouse liver DFPase showed it to be identical in its amino acid sequence with the recently identified senescence marker protein-30 (SMP-30) by Fujita et al. in 1996. Other senescence marker proteins possessing high structural homology with the mouse SMP-30 have also been found and sequenced from human and rat livers. There is no structural homology between the senescence marker protein family and the group of mammalian paraoxonases. Thus, it is clear that there are at least two distinct, unrelated families of mammalian liver enzymes that share DFPase activity.

Effect of hypothyroidism and thyroid hormone replacement on the level of protein kinase C and protein kinase A in rat liver

We investigated the influence of the thyroid hormone status on the levels of protein kinase C (PKC) and A (PKA) in the soluble fraction of rat liver. The immunodetectable PKC level in hypothyroid liver was elevated 7.7-fold, whereas the phorbol-ester binding capacity and the immunodetectable α-PKC level were increased 2.4- and 2.6-fold, respectively. Conversely, in hypothyroid livers the abundance of the regulatory type I and the catalytic subunits of PKA were lowered to 42% of the euthyroid level as determined by immonoblotting and by measuring the substrate specific phosphorylation rate of PKA. These changes in the PKC and PKA levels were reversible upon treatment with 0.5 μg T 4/100 g body weight for 2–21 days. The thyroid state dependent alterations in hepatic PKC and PKA levels may be responsible for the Known changes in the response of hepatocytes to other hormonal stimuli in hypothyroidism.

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.

Identity of alanine: glyoxylate aminotransferase with alanine: 2-oxoglutarate aminotrasferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a p I of 5.2 and an M t of approx. 110 000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The p I 6.0 enzye with an M t of approx. 220 000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the p I 8.0 enzyme with an M t of approx. 80 000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine:glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.

Biochemical and immunological similarity of soluble angiotensin II-binding proteins in different organs

Binding of angiotensin II has been detected in soluble extracts of rabbit liver, adrenal gland, aorta, brain, kidney and uterus. In each case, binding required p-chloromercuriphenylsulfonic acid and bound angiotensin II was released by treatment with dithiothreitol. These properties resemble those of the 75 kDa binding protein purified from liver. Immobilized guinea pig antiserum developed against the isolated hepatic protein removed binding activities from the different extracts in an immune-specific, quantitatively comparable manner. In addition, the activities were removed by a mouse monoclonal antibody which specifically recognized a protein of 75 kDa in the various preparations. An immunologically homologous angiotensin II-binding protein with similar characteristics was also identified in the soluble fraction of rat liver.

Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus.

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.

O-linked N-acetylglucosamine is attached to proteins of the nuclear pore. Evidence for cytoplasmic and nucleoplasmic glycoproteins.

Glycoproteins bearing a single N-acetylglucosamine (GlcNAc) residue attached by an O-glycosidic linkage to the polypeptide chain (Holt, G. D., and Hart, G. W. (1986) J. Biol. Chem. 261, 8049-8057) have been found to be enriched in the nuclear and soluble fractions of rat liver. Our goal was to determine the localization and membrane topography of proteins bearing O-linked GlcNAc using galactosyltransferase and wheat germ agglutinin (WGA) as membrane-impermeant probes. Latency of the enzyme mannose-6-phosphatase was used to quantitatively confirm the intactness of the nuclear envelope during incubations with galactosyltransferase or WGA. The O-linked GlcNAc residues of nuclei were fully accessible to modification by galactosyltransferase under conditions where the nuclear envelope mannose-6-phosphatase was 70% latent. Addition of detergent destroyed the permeability barrier but did not increase galactosylation of the O-linked GlcnAc. The major polypeptides bearing O-linked GlcNAc residues on nuclei were peripheral rather than integral membrane proteins with apparent molecular masses ranging from 210 to 54 kDa. The proteins were also detected on sealed nuclei using conjugates of WGA. WGA-rhodamine labeled intact nuclei when examined by immunofluorescence; WGA-peroxidase was used to identify the nuclear glycoproteins after transfer to nitrocellulose. WGA-ferritin selectively labels the cytoplasmic and nucleoplasmic faces of the nuclear pore complex when examined by electron microscopy. Taken together, these data strongly suggest that proteins bearing cytoplasmically oriented O-linked GlcNAc are components of the nuclear pore complex, thereby raising the possibility that cytoplasmic and nucleoplasmic glycoproteins are involved in the assembly or functioning of the nuclear pore.

Isozymes of S-adenosylmethionine synthetase from rat liver: isolation and characterization.

S-Adenosylmethionine synthetase exists in at least two distinct forms, alpha- and beta-forms, in adult liver. The beta-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified beta-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, alpha- and beta-forms, are identical. Thus, the alpha-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the beta-form. The molecular weights of the beta- and alpha-forms were determined to be 48,000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100,000, and 200,000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the beta-form consisted of two subunits of 48,000 daltons and the alpha-form of four subunits of 48,000 daltons. The sedimentation coefficient was calculated to be 5.5S for the beta-form and 8.0S for the alpha-form.

Effect of gold thioglucose on subcellular selenium distribution in rat liver and kidney

Reported are the subcellular distributions of selenium (Se), gold, glutathione peroxidase, and enzyme markers for nuclei, mitochondria, lysosomes, and soluble fraction of rat liver and kidney in normal and gold-injected rats. Gold thioglucose increased the Se content of kidney and liver and decreased Se-dependent gluthathione peroxidase activity. The Se in control liver and kidney was found predominantly in the soluble fraction: 73% in liver and 62% in kidney. The Se redistribution in these tissues closely followed the subcellular distribution of gold and Se concentrated mainly in fractions with high lysosomal enzyme-specific activity. The nuclear fraction of kidneys from gold-injected rats contained 46% of the gold and 30% of the Se and had an eightfold increase in lysosomal enzyme-marker activity over activity in the nuclear fraction from kidneys of control rats.

Determination of in vivo protein binding of homocysteine and its relation to free homocysteine in the liver and other tissues of the rat.

Low concentrations (0.5-6 nmol/g) of homocysteine (Hcy) have recently been demonstrated in acid extracts of various tissues of the mouse and rat (Ueland, P.M., Helland, S., Broch, O.-J., and Schanche, J.-S. (1984) J. Biol. Chem. 259, 2360-2364). This is referred to as free Hcy in tissues. This paper describes a method for the determination of protein-bound Hcy, which involves precipitation and washing of tissue protein with ammonium sulfate, release of Hcy from native proteins in the presence of dithioerythritol, and determination of free Hcy by a sensitive radioenzymic assay. Both free and bound Hcy decreased markedly in rat tissues within a few seconds following death of the animal. The amount of protein-bound Hcy was highest in liver, somewhat lower in kidney, brain, heart, lung, and spleen. The ratio between free and bound Hcy was between 1 and 2 in most tissues, except in cerebellum, containing a large excess of free Hcy (free/bound ratio of 18). Free Hcy was almost exclusively localized to the soluble fraction of rat liver, whereas protein-bound Hcy was about equally distributed between this fraction and the microsomes. Isolated rat hepatocytes contained free and protein-bound Hcy in proportions observed in whole liver, but a large amount of Hcy was exported into the extracellular medium. The half-lives, as determined from pulse-chase experiments with [35S] methionine, were 53 s for S-adenosylmethionine, 2 s for S-adenosylhomocysteine and 3 s for Hcy (free and bound regarded as a single pool). Furthermore, isotope equilibrium between these metabolites and between free and bound Hcy throughout the rapid chase period suggests the turnover rates of S-adenosylhomocysteine and Hcy to be production rate limited, and the dissociation rate of the Hcy-protein complex may greatly exceed the turnover rate of Hcy. Thus, the half-lives of Hcy are such that participation of both free and bound Hcy in metabolic regulation is feasible.

Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2.

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.

Ethanol oxidation in systems containing soluble and mitochondrial fractions of rat liver: Regulation by acetaldehyde

Systems containing soluble fraction of rat liver, with or without mitochondrial fraction, oxidised [l- 14C] ethanol to acetaldehyde, 14CO 2 and non-volatile 14C-products of which acetate was the principal, and possibly the only, component. Ethanol oxidation was stimulated by pyruvate which served as an electron sink thereby allowing rapid regeneration of NAD. When no mitochondria were present acetaldehyde accumulated, rapidly at first but eventually reaching a plateau. The rate of ethanol oxidation in these systems was much lower than the measured maximum activity of alcohol dehydrogenase (ADH) and it was concluded that ADH was inhibited by the accumulated acetaldehyde. Mitochondria, because of their relatively high aldehyde dehydrogenase (ALDH) activity, prevented the accumulation of acetaldehyde, or quickly removed acetaldehyde already accumulated. This action was accompanied by a sharp increase in the rate of ethanol oxidation, presumably due to the deinhibition of ADH. Cyanamide, an inhibitor of mitochondrial ALDH, blocked the stimulatory effect of mitochondria on ethanol oxidation. It was concluded that, in the reconstituted systems, acetaldehyde played a dominant role in controlling the rate of ethanol oxidation. The possible importance of acetaldehyde in governing ethanol oxidation in vivo is discussed.

Rapid oxidation of NADH via the reconstituted malate-aspartate shuttle in systems containing mitochondrial and soluble fractions of rat liver: Implications for ethanol metabolism

In an attempt to assess whether hydrogen shuttle capacity might serve as the rate-limiting factor in the hepatic oxidation of ethanol, the malate-aspartate shuttle was reconstituted in systems containing mitochondrial and soluble fractions of rat liver. Oxidation of NADH was stimulated slightly by the addition of either glutamate or malate but when both substrates were added the stimulation was far stronger. This effect was greatly enhanced by aspartate indicating that. when not added to the system, extramitochondrial aspartate was limiting. It was found that the rate of oxidation of NADH was directly related to the amount of mitochondrial protein present but extramitochondrial reactions became restrictive when the ‘soluble protein/mitochondrial protein’ ratio fell below 0.8. When calculated on a whole tissue basis the maximum rate of oxidation of NADH by the reconstituted shuttle was substantially higher than reported rates of ethanol oxidation in vino. The results are discussed in relation to the normal control of ethanol metabolism.

Studies on Metabolism of BHC Isomers and Related Organochlorine Compounds

Metabolic experiments of lindane and related compounds are reviewed. Lindane and other BHC isomers produced 2, 4, 6-trichlorophenol as the major oxidative metabolite by rat liver microsomes. A mechanism was shown to be direct oxygenation of the cyclohexane ring followed by dehydrochlorinations. Microsomes from rat liver and housefly abdomen effectively metabolized lindane to (36/45)- and (346/5)-pentachlorocyclohexene and (36/45)-hexachlorocyclohexene in addition to 2, 4, 6-trichlorophenol. A reaction mechanism was proposed which includes electrophilic attack by an activated oxygen to chlorine or hydrogen. Microsomes metabolized isomers of tetra-, penta-, and hexachlorocyclohexene to tetrachlorocyclohexenol isomers, 2, 4, 5-trichlorophenol and 2, 3, 4, 6-tetrachlorophenol, respectively. The (346/5)-isomer of pentachlorocyclohexene also gave an abundant amount of pentachlorocyclohexenol isomer. These oxidative reactions were shown to proceed mainly via ene-like hydroxylation accompanied by double bond migration. Reductive dechlorination of lindane and related polychlorocyclohexenes under anaerobic condition was found using microsomes and NADPH. 1, 2, 4-Trichlorobenzene was produced abundantly from (36/45)-hexachlorocyclohexene in this condition. Correlations of this biochemical reductive dechlorination with electrochemical reduction were illustrated. 4-Chloro-, 2, 4-dichloro-, 3, 4-dichloro-, 2, 3, 5-trichloro- and 2, 4, 5-trichlorophenylmercapturic acids were identified as the main mercapturic acid metabolites of lindane. Their formation from (346/5)-tetra-, (346/5)-penta-, (36/45)-penta-, and (36/45)-hexachlorocyclohexene was proposed based on in vitro experiments using rat liver soluble fraction and glutathione. A mechanism was proposed which includes substitution by glutathione of allylic chlorine with or without double bond migration. All these findings elucidated the in vivo metabolic pathways of lindane degradation in the rat and in the housefly.

Involvement of tissue sulfhydryls in the formation of a complex of methylmercury with selenium

Albumin-bound methylmercury was converted to a benzene-extractable form by the soluble fraction of rat liver, kidney or brain in the presence of selenite, but not in its absence. The factors in the soluble fraction causing this conversion were investigated by column chromatography. Sephadex G-25 chromatography showed that effective factors were present in non-protein and protein fractions. It was concluded from ion exchange and Sephadex G-200 chromatography that these factors in the non-protein and protein fractions were reduced glutathione (GSH) and protein sulfhydryl groups respectively. Because GSH and the soluble protein could be replaced by sulfhydryl compounds, such as cysteine and 2-mercaptoethanol, as well as by a purified protein with sulfhydryl groups, reduced ribonuclease (RNase), respectively, it was concluded that sulfhydryl groups of GSH and/or proteins in the soluble fraction were needed for selenite-induced conversion of methylmercury to a benzene-soluble form. Among the various selenium compounds tested, only H 2Se (the reduced metabolite of selenite) was found to react directly with methylmercury to form a benzene extractable mercury compound in the absence of the soluble fraction. These findings suggest that the conversion of methylmercury to a benzene-soluble form occurs by reaction of methylmercury with selenium (possibly H 2Se) reduced by GSH and/or protein sulfhydryl groups in the soluble fraction. Thin-layer chromatography showed that benzene-extractable mercury consists mainly of bis(methylmercuric) selenide (BMS). A minor component, trismethylmercuric selenonium, was also detected by mass spectrography.

Enhancement of hepatic microsome-mediated bacterial mutagenesis by the rat liver soluble protein fraction

Bacterial mutagenesis from aflatoxin B 1, 2-aminofluorene and benzo[ a]-pyrene, mediated by washed rat liver microsomes using the assay system of Ames, is elevated 2–4-fold by the liver soluble protein fraction. Enhancement results from an apparent stimulation of one (or possible more) biochemical steps leading to mutagenesis in this system. Enhancement is observed over a wide range of NADPH concentrations and is present in dialyzed preparations of liver soluble proteins. Enhancement activity is stable when the soluble protein fraction is stored for 18 h at pH 5–9 (4°C); when stored for 18 h at 25°C or 37°C (pH 7.8), and when heated for 10 min at 56°C. The activity is labile when heated for 10 min at 100°C.

Intracellular Ca 2+-dependent protease (CALPAIN) and its high-molecular-weight endogenous inhibitor (CALPASTATIN)

Ubiquitous distribution of Ca 2+-dependent neutral protease and its specific inhibitor in various tissues and cells is described. A proposal is made to call the protease calpain and the inhibitor calpastatin, both as generic names. Calpain, which implies a Ca 2+-dependent protease belonging to the thiol protease group, has been knoqn to be widely distributed under different names: e.g., phosphorylase kinase activating factor (KAF), mayofibrllar Z-line-removing protease, Ca 2+-activated neutral protease (CANP), recpetor transforming factor (RTF), etc. Some of the common features of these enzymes are discussed with special reference to the effect on them of a common, endogenous inhibitor, calpastatin. The existence of two types of calpain was discovered from rat liver and kidney and human erythrocytes. Calpain I requires only 40 μ m Ca 2+ for 50% activation, while calpain II responds only to a Ca 2+ concentration of m m order. Capain I is the minor component in rat liver, but it is the only type of calpain that can be found in human erythrocytes. Calpastatin was found to exist in the soluble fraction of rat liver, brain, kidney, spleen cells and thymocytes, human liver, erythrocytes and lymphocytes, and monkey and guinea pig erythrocytes. Calpastatin was partially purified by chromatography on DEAE-cellulose and Ultrogen AcA 34. It has a molecular weight of 280,000–300,000 and is extremely heat stable but readily digetible by trypsin. None of the proteases tested, except calpains I and II, were inhibited by calpastatin. Inhibition is not based on sequestering of Ca 2+ from the medium by the inhibitor, while the binding of calpain to calpastatin does need Ca 2+. Calpain frm one tissue can be inhibited by calpastatin not only from the same tissue but also from the other tissues of the same animal or even of a different species. The relative abundance of calpain to calpastatin found in one tissue differs greatly from that found in the other tissue. Teven in the same kind of tissue, the ratio seems to vary depending upon the sex, age and stag eof differentiation, possibly including neoplastic transformation.

Factors controlling the metabolism of phosphatidate by phosphohydrolase and phospholipase A-type activities: Effects of magnesium, calcium and amphiphilic cationic drugs

1. 1. The simultaneous deacylation and dephosphorylation of 1,2-diacyl- sn-[ 3H]glycerol 3-phosphate by the microsomal and soluble fraction of rat liver was studied. The substrate was either in the form of an emulsion or bound to microsomal membranes. 2. 2. Mg 2+ stimulated the deacylation and dephosphorylation of phosphatidate emulsions by both fractions, although the stimulation of both microsomal activities was less than that in the soluble fraction. The preparations of membrane-bound phosphatidate contained Mg 2+. Further addition of Mg 2+ inhibited dephosphorylation, whereas low concentrations of EDTA stimulated. Additional Mg 2+ had little effect on the deacylation of membrane-bound phosphatidate and EDTA inhibited it. 3. 3. Ca 2+ inhibited the phosphohydrolase reactions in both fractions, but had little effect on the deacylation of phosphatidate emulsions or membrane-bound phosphatidate. 4. 4. In the absence of Mg 2+, lower concentrations of amphiphilic cations (chlorpromazine and benfluorex) stimulated the deacylation and dephosphorylation of phosphatidate emulsions by the soluble fraction. They also stimulated deacylation by the microsomal fraction, but inhibited dephosphorylation. In the presence of 5 mM MgCl 2, these drugs inhibited the dephosphorylation and deacylation of phosphatidate emulsions, the deacylation reaction being slightly less sensitive. Chlorpromazine (0.4 and 0.8 mM) also inhibited the dephosphorylation of membrane-bound Mg 2+-phosphatidate by microsomal and microsomal plus soluble fractions. The deacylation was stimulated by 0.4 mM chlorpromazine and by 1 and 2 mM norfenfluramine. Chlorpromazine (0.8 mM) inhibited the deacylation by microsomal plus soluble fractions, but not by microsomal fractions alone. 5. 5. The possible importance of the deacylation of phosphatidate in the physiological and pharmacological control of glycerolipid synthesis is discussed.

Cytosolic location of an endo-N-acetyl-beta-D-glucosaminidase activity in rat liver and kidney.

Endo-N-acetyl-beta-D-glucosaminidase activity towards an oligomannosidic type glycoamino acid substrate was found in the soluble fraction of rat liver and kidney. No evidence for a lysosomal form of the activity was found.