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PubMed1975

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.

Adenine Nucleotides↗

PubMed1994

Monitoring of osteoblast activity with an immunoradiometric assay for determination of bone alkaline phosphatase mass concentration in patients receiving renal transplants.

We examined the diagnostic validity of an immunoradiometric assay for determination of mass concentration of bone alkaline phosphatase (EC 3.1.3.1) in 134 sera from 35 patients receiving renal transplants. Comparison between bone alkaline phosphatase concentration and total alkaline phosphatase activity yielded a strong correlation (r = +0.860; P < 0.001). Nine (17%) of 54 sera which were characterized by a total alkaline phosphatase activity between 100 units/l and the upper reference limit (178 units/l (males) and 160 units/l (females), respectively) showed an increased bone alkaline phosphatase concentration (> 21.3 micrograms/l (males) and > 15.0 micrograms/l (females), respectively). There was also a correlation between bone alkaline phosphatase values and parathyroid hormone levels both before (r = +0.640 (n = 23), P < 0.001) and after renal transplantation (r = +0.528 (n = 111), P < 0.0001). A follow-up of 15 patients after renal transplantation revealed that the median of bone alkaline phosphatase values increased from 5.5 micrograms/l before transplantation to 14.9 micrograms/l 3 months after transplantation (P < 0.0001). Nevertheless no correlation could be observed between parathyroid hormone concentrations and bone alkaline phosphatase values at any time following renal transplantation in these 15 patients (P > 0.1). Rise of bone alkaline phosphatase concentration following renal transplantation is most probably due to an activating effect of cyclosporin A upon osteoblasts.

Adult↗

PubMed1990

Comparison of tartrate resistant acid phosphatase in a giant cell bone tumor and a spleen infiltrated with hairy cells.

Acid phosphatase (E.C.3.1.3.2) in a giant cell bone tumor and a spleen infiltrated with hairy cells was extracted by citrate buffer and then by 0.3 mol/L NaCl. The cationic acid phosphatase in the crude extract was isolated by CM-cellulose chromatography, and further separated by high pressure liquid chromatography. The majority of the tartrate resistant acid phosphatase in the hairy cell spleen was unabsorbed on CM-cellulose and was insensitive to iron. A much larger portion of the acid phosphatase in the bone tumor, than in the spleen, was cationic and was eluted from the column by 0.8 mol/L NaCl. The cationic acid phosphatase was further separated into consecutive peaks of acid phosphatases with different sensitivity to iron. A major portion of acid phosphatase in the giant cell bone tumor was enhanced by iron, while the amounts of iron-enhanced and iron-insensitive acid phosphatase were about the same in the spleen. The differences of the phosphatases in these two types of pathologic specimens indicate the occurrence of two types of enzymes with different biological significance.

Acid Phosphatase↗

PubMed1985

Heat-stable regulatory factors are associated with polycation-modulable phosphatases.

Several protein phosphatases which we designated PCM-I, PCM-II and PCM-IId were identified in preparations from aortic smooth muscle. A unique feature of these enzymes is that phosphatase activities expressed against different substrates are subject to modulation by polycationic effectors such as polylysine and lysine-rich histone-H1. They can be distinguished from each other by virtue of significant quantitative differences regarding (a) relative substrate specificities exhibited against phosphorylated myosin light chains, phosphorylase, and inhibitor-1, (b) apparent molecular weights as determined by sucrose density centrifugation, and (c) differential susceptibility to polylysine-mediated modulation of phosphatase activities. Surprisingly, gel filtration of the very same PCM-phosphatase preparation yields either of two apparently different enzymes: namely PCM-II, or PCM-IId. The enzymes appear similar in that low concentrations of polylysine (0.03-0.13 microM) inhibit dephosphorylation of light chains by either enzyme and high concentrations inhibit dephosphorylation of phosphorylase a. However, PCM-II exhibits higher basal phosphatase activity against myosin light chains (480 U/mg) than against phosphorylase a (175 U/mg). In contrast, PCM-IId is more effective in dephosphorylating phosphorylase a (180 U/mg) than in dephosphorylating the light chains (88 U/mg). Moreover, phosphorylase phosphatase activity of PCM-II is biphasically stimulable by low concentrations of polylysine (0.01-0.5 microM), but no stimulation is seen with PCM-IId. In addition, earlier studies with PCM-I showed that, unlike either PCM-II or PCM-IId polylysine biphasically stimulated the dephosphorylation of both phosphorylase a and the myosin light chains. Nevertheless, in spite of these obvious differences between the enzymes other data suggests that the PCM-phosphatases may be related to each other. Incubation of PCM-II at 90 degrees C for 10 min apparently releases heat-stable regulatory proteins which reverse the modulatory effects of polylysine on light chain and phosphorylase phosphatase activities expressed by either PCM-II or PCM-I. Similarly, heat-stable proteins released from PCM-I also reverse polylysine-mediated modulation of both PCM-I and PCM-II. These findings are consistent with a working hypothesis suggesting that PCM-phosphatase may consist of a modulatory domain containing several regulatory proteins and a catalytic domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex.

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.

Adenosine Triphosphate↗

PubMed1978

Serum alkaline phosphatase elevation in female rats treated with ethinyl estradiol.

Ethinyl estradiol treatment to female rats resulted in increased levels of serum alkaline phosphatase, but was not associated with any other manifestation of toxicity such as increased serum transaminases or toxic lesions. Elevated serum alkaline phosphatase seen in rats treated with chloroform was associated with frank hepatotoxicity. Induction of hepatic drug metabolising enzymes in rats by phenobarbitone treatment did not result in raised serum alkaline phosphatase levels. Estradiol benzoate treatment to rats also did not increase serum alkaline phosphatase levels. Ethinyl estradiol also resulted in increased alkaline phosphatase content in the liver, intestine and bone. The raised intestinal alkaline phosphatase content of rats treated with phenobarbitone or estradiol benzoate was not associated with an increase in the serum levels. There was histochemical evidence of induction of canalicular alkaline phosphatase in the liver in Ethinyl Estradiol treatment. The study of the electrophoretic separation of serum alkaline phosphatase of ethinyl estradiol treated rats revealed the presence of a new fast moving fraction, similar to those seen in bile duct ligated rats. It is concluded that the serum alkaline phosphatase increase during ethinyl estradiol treatment at least in part is from the liver, due to new synthesis.

Alkaline Phosphatase↗

PubMed1994

Okadaic acid reverses the inhibitory effect of protein kinase C on alkaline phosphatase activity in osteoblast-like cells.

We previously reported that fetal calf serum-induced alkaline phosphatase activity is suppressed due to the activation of protein kinase C in osteoblast-like MC3T3-E1 cells (Miwa et al. (1991) Bone Miner. 14, 15-25; Kotoyori et al. (1994) Horm. Metab. Res. 26, 116-118). In the present study, we examined the effect of okadaic acid, a potent and specific inhibitor of protein phosphatase type 1 and 2A, on fetal calf serum-induced alkaline phosphatase activity in MC3T3-E1 cells. The pretreatment with okadaic acid enhanced the fetal calf serum-induced alkaline phosphatase activity in a dose-dependent manner in the range between 0.1 and 5 nM. 1-Norokadaone, a less potent analogue of okadaic acid, had little effect on the fetal calf serum-induced alkaline phosphatase activity. Okadaic acid partially reversed the suppression of fetal calf serum-induced alkaline phosphatase activity by 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C activator. The effect of okadaic acid was dose-dependent in the range between 0.1 and 5 nM. The patterns of the dose-dependency of both okadaic acid effects on fetal calf serum-induced alkaline phosphatase activity and on the suppression by 12-O-tetradecanoylphorbol-13-acetate were similar. These results strongly suggest that protein phosphatase type 1 and/or 2A act as a regulator of alkaline phosphatase activity at a point downstream from protein kinase C in osteoblast-like cells.

Alkaline Phosphatase↗

PubMed1986

Alterations of the microsomal glucose-6-phosphatase system evoked by ferrous iron- and haloalkane free-radical-mediated lipid peroxidation.

Alterations of catalytic activities of the microsomal glucose-6-phosphatase system were examined following either ferrous iron- or halothane (CF3CHBrCl) and carbon tetrachloride (CCl4) free-radical-mediated peroxidation of the microsomal membrane. Enzyme assays were performed in native and solubilized microsomes using either glucose 6-phosphate or mannose 6-phosphate as substrate. Lipid peroxidation was assessed by the amounts of malondialdehyde equivalents formed. Regardless of whether the experiments were performed in the presence of NADPH/Fe3+, NADPH/CF3CHBrCl, or NADPH/CCl4, with the onset of lipid peroxidation, mannose-6-phosphatase activity of the native microsomes increased immediately, while further alterations in catalytic activities were only detectable when lipid peroxidation had passed characteristic threshold values: above 2 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase activity of the native microsomes was lost, and at 10 nmol malondialdehyde/mg microsomal protein, glucose-6-phosphatase and mannose-6-phosphatase activity of the solubilized microsomes started to decline. It is concluded that the latter alterations are due to an irreversible damage of the phosphohydrolase active site of the glucose-6-phosphatase system, while the changes observed at earlier stages of microsomal lipid peroxidation may also reflect alterations of the transporter components of the glucose-6-phosphatase system. Virtually no changes in the catalytic activities of the glucose-6-phosphatase system occurred under anaerobic conditions, indicating that CF3CHCl and CCl3 radicals are without direct damaging effect on the glucose-6-phosphatase system. Further, maximum effects of carbon tetrachloride and halothane on lipid peroxidation and enzyme activities were observed at an oxygen partial pressure (PO2) of 2 mmHg, providing additional evidence for the crucial role of low PO2 in the hepatotoxicity of both haloalkanes.

Animals↗

PubMed1996

Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures.

Mineralization of cartilage and bone requires alkaline phosphatase activity. In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants. To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used. Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis. Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg. In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e. 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e. 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg). With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization. Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight. A hydrophilic form was obtained by treatment with a specific lipase. This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures. Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.

Alkaline Phosphatase↗

PubMed1990

A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine.

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.

Alkaline Phosphatase↗

PubMed2006

Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells.

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.

Animals↗

PubMed2005

Alkaline phosphatase is involved in the control of adipogenesis in the murine preadipocyte cell line, 3T3-L1.

OBJECTIVE: As alkaline phosphatase may play a role in cell differentiation, our aim was to study the possible role of this enzyme in the differentiation of preadipocytes (3T3-L1 cells) into adipocytes. RESEARCH METHODS AND PROCEDURES: 3T3-L1 cells were grown in medium containing insulin, dexamethasone and IBMX to induce adipogenesis. Adipogenesis was measured using the triglyceride-specific dye, oil red O at 0, 3, 7 and 11 days after initiation of adipogenesis in the presence or absence of the alkaline phosphatase inhibitors, levamisole, histidine and Phe-Gly-Gly. Intracellular localisation of the enzyme was detected using ELF-phosphatase, a fluorescent substrate and alkaline phosphatase gene expression was assessed using RT-PCR. RESULTS: Alkaline phosphatase activity was detected in untransformed cells (1.91+/-0.62 mU/mg protein) and activity increased 11.5+/-1.4-fold after 11 days treatment with transformation medium and 5.3+/-0.3-fold in transformation medium containing levamisole (p<0.05). Triglyceride content of cells increased 3.1+/-0.2-fold after 11 days treatment with transformation medium and 2.1+/-0.3-fold in the presence of levamisole (p<0.005). Histidine inhibited adipogenesis and alkaline phosphatase to a greater extent than did levamisole, but Phe-Gly-Gly had no effect on these variables. Alkaline phosphatase was localised around the lipid droplets of the cells. Gene expression of alkaline phosphatase increased during adipogenesis. DISCUSSION: This study demonstrates that tissue-nonspecific alkaline phosphatase is present in 3T3-L1 cells and that it may play a role in the control of adipogenesis.

3T3-L1 Cells↗

PubMed1987

Association of high-molecular-mass and electrophoretically atypical alkaline phosphatases.

Polyacrylamide gel electrophoresis of alkaline phosphatase may yield abnormally migrating fractions; these include high-molecular-mass alkaline phosphatase, which remains at the gel origin, and immunoglobulin-alkaline phosphatase complexes, which have a mobility approximately 1/3 that of liver isoenzyme. We performed a retrospective study of 19 patients whose sera exhibited atypical alkaline phosphatase fractions, defined as bands whose mobility was slower than bone, liver, or intestinal alkaline phosphatase; 17 had a mobility approximately 1/3 that of liver isoenzyme and 16 also exhibited gel origin enzyme activity or high-molecular-mass bands. The strong association of the atypical and high-molecular-mass alkaline phosphatases suggests that they may be structurally related, both consisting of either immunoglobulin-enzyme complexes or membrane-alkaline phosphatase complexes. This hypothesis is supported by (1) one serum available for investigation containing alkaline phosphatase-immunoglobulin complexes in both abnormally migrating fractions, but on detergent treatment showing no evidence of membrane-bound enzyme; (2) detergent treatment of serum from patients with only high-molecular-mass alkaline phosphatase creating bands with a mobility of approximately 1/3 that of the liver isoenzyme.

Adolescent↗

PubMed1998

Allotype immunoglobulin enhances alkaline phosphatase activity: implications for the inflammatory response.

To understand the interactions among components of the immune/inflammation response, we studied the effects of immunoglobulins on the phosphatase activity of alkaline phosphatase in vitro. Bovine intestinal alkaline phosphatase was incubated with substrate in the presence of allotypic and xenotypic immunoglobulin. We found that bovine but not rabbit immunoglobulin enhanced the phosphatase activity of bovine intestinal alkaline phosphatase. Similarly, human but not bovine immunoglobulin G enhanced human placental alkaline phosphatase activity. By enhancing alkaline phosphatase activity, immunoglobulins bound to alkaline phosphatase may assist physiologic transport functions and enhance resolution of the inflammatory response. Further, in clinical conditions with high immunoglobulin concentrations, the serum alkaline phosphatase recorded may have spuriously high values.

Alkaline Phosphatase↗

PubMed1987

Prospective comparison between serum monoclonal prostate specific antigen and acid phosphatase measurements in metastatic prostatic cancer.

Prostate specific antigen, prostatic acid phosphatase antigen and acid phosphatase activity were measured on 175 serum samples serially collected from 80 patients with metastatic stage D adenocarcinoma of the prostate. Prostate specific antigen and prostatic acid phosphatase antigen concentrations were measured with a monoclonal radioimmunometric assay, and acid phosphatase activity was measured enzymatically. The over-all frequency of abnormal levels of prostate specific antigen (76 per cent) was significantly greater than abnormal prostatic acid phosphatase antigen (60 per cent) and acid phosphatase activity (49 per cent) results (p less than 0.001). These differences were greater among the subset of patients in clinical remission. Levels greater than 10 times normal were observed in 68 per cent of prostate specific antigen, 43 per cent of prostatic acid phosphatase antigen and 31 per cent of acid phosphatase activity measurements (p less than 0.001). Three or more serial prostate specific antigen measurements in 17 patients demonstrated excellent correlation with independently assessed clinical disease activity. These results suggest that prostate specific antigen is a more sensitive and potentially more useful tumor marker than acid phosphatase measurements in patients with metastatic prostatic carcinoma.

Acid Phosphatase↗

PubMed1985

Immunoreactive prostatic acid phosphatase in prostatic cancer: diagnosis and followup of patients.

We compared the measurements of serum acid phosphatase activity to those obtained by radioimmunoassay of prostatic acid phosphatase in the sera of 126 untreated prostatic cancer patients. The catalytic activity of prostatic acid phosphatase was elevated in 32 per cent of the patients and the serum concentration of prostatic acid phosphatase was elevated in 66 per cent. Of these 126 patients 16 had stage T0-2M0N0-x disease, and enzyme activity and prostatic acid phosphatase concentration were increased in 0 and 38 per cent, respectively, in this group. Of the 110 patients with proved extracapsular cancer the corresponding figures were 36 and 70 per cent, respectively. We followed 109 of these 126 patients for 1 or more years after orchiectomy. A salient finding was that return of elevated serum prostatic acid phosphatase concentration to the health-associated reference interval within 7 days following castration indicated no progression of the disease at 1 year irrespective of the initial staging. The same was not detected by the measurement of catalytic activity of serum acid phosphatase. Our findings substantiate data showing that the measurement of circulating prostatic acid phosphatase is achieved better by immunological techniques than by measurements of catalytic activity of the enzyme. A novel aspect is the usefulness of immunological prostatic acid phosphatase measurements in evaluation of the prognosis of patients with metastatic prostatic carcinoma following ablative endocrine treatment.

Acid Phosphatase↗

PubMed1997

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43.

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phosphatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.

Acid Phosphatase↗

PubMed2000

Enzymological properties of the LPP1-encoded lipid phosphatase from Saccharomyces cerevisiae.

The product of the LPP1 gene in Saccharomyces cerevisiae is a membrane-associated enzyme that catalyzes the Mg(2+)-independent dephosphorylation of phosphatidate (PA), diacylglycerol pyrophosphate (DGPP), and lysophosphatidate (LPA). The LPP1-encoded lipid phosphatase was overexpressed 681-fold in Sf-9 insect cells and used to examine the enzymological properties of the enzyme using PA, DGPP, and LPA as substrates. The optimum pH values for PA phosphatase, DGPP phosphatase, and LPA phosphatase activities were 7. 5, 7.0, and 7.0, respectively. Divalent cations (Mn(2+), Co(2+), and Ca(2+)), NaF, heavy metals, propranolol, phenylglyoxal, and N-ethylmaleimide inhibited the PA phosphatase, DGPP phosphatase, and LPA phosphatase activities of the enzyme. The inhibitory effects of N-ethylmaleimide and phenylglyoxal on the LPP1-encoded enzyme were novel properties when compared with other Mg(2+)-independent lipid phosphate phosphatases from S. cerevisiae and mammalian cells. The LPP1-encoded enzyme exhibited saturation kinetics with respect to the surface concentrations of PA (K(m)=0.05 mol%), DGPP (K(m)=0.07 mol%), and LPA (K(m)=0.08 mol%). Based on specificity constants (V(max)/K(m)LPA (1.3 units/mg/mol%). DGPP (K(i)=0.12 mol%) was a competitive inhibitor with respect to PA, and PA (K(i)=0.12 mol%) was a competitive inhibitor with respect to DGPP. This suggested that the binding sites for these substrates were the same. The enzymological properties of the LPP1-encoded enzyme differed significantly from those of the S. cerevisiae DPP1-encoded lipid phosphatase, a related enzyme that also utilizes PA, DGPP, and LPA as substrates.

Animals↗