Enzymic characteristics of ecto-phosphoprotein phosphatase in goat epididymal intact spermatozoa

Intact washed spermatozoa from goat cauda epididymis possess an ecto-phosphoprotein phosphatase that causes dephosphorylation of phosphoserine and phosphothreonine residues of exogenous 32P-labelled histones. The cell-bound ecto-enzyme has high affinity for proteins (histones, casein, phosvitin, and protamine) rather than phosphate esters, such as p-nitrophenyl phosphate, beta-glycerophosphate, AMP, and ATP. The activity of the enzyme is inhibited by 4 mM Mg2+, Ca2+, Mn2+, or Co2+. Pi (10 mM), NaF (10 mM), and Zn2+ (1 mM) inhibit the enzyme by approximately 50, 35, and 100%, respectively. Polyamines such as spermine and spermidine at 10 mM each caused significant inhibition (60 and 30%, respectively) of the cell-bound phosphoprotein phosphatase activity, whereas cAMP, orthovanadate, and calmodulin (with or without Ca2+) had no appreciable effect. Under the standard assay conditions, spermatozoa remain intact as evidenced by assay of cytosolic enzyme markers. Both the washed and "native" intact spermatozoa showed nearly the same specific activity of the ectoenzyme. The product of the reaction (Pi) was found in the extracellular medium. Sonication doubled the enzymic activity of the intact cells. The specific activity of the enzyme was nearly fourfold higher in the intact forwardly motile cells than the "composite" spermatozoa. These data provide further support for the localization of a phosphoprotein phosphatase on the external surface of spermatozoa and that the ectoenzyme may have a role in the regulation of flagellar motility.     

Evidence for the occurrence of an ecto-(adenosine triphosphatase) in rat epididymal spermatozoa.

Intact spermatozoa from rat cauda epididymis possess a Mg2+-dependent ATPase activity that hydrolyses externally added [gamma-32P]ATP. The ATPase reaction was linear with time for approx. 6 min and there was no detectable uptake of ATP by these cells. The ATPase activity of the whole spermatozoa was not due to leakage of the intracellular enzymic activity, contamination of the broken cells or any possible cell damage during incubation and isolation of spermatozoa. The activity of the enzyme was strongly inhibited (approx. 85%) by p-chloromercuribenzenesulphonic acid (50 microM) or the diazonium salt of sulphanilic acid (50 microM), which are believed not to enter the cells, whereas ouabain (0.5 mM), NaF (10 mM), NaN3 (2.5 mM) and oligomycin (5 microM) had no appreciable effect on the activity of the spermatozoal APTase. There was little loss of ATPase activity from the cells when washed with 0.5 mM-EDTA and an iso-osmotic or hyperosmotic medium. These data are consistent with the view that the observed ATPase activity is located on the external surface of spermatozoa. The sperm ecto-ATPase activity is resistant to the action of proteinases (50 micrograms/ml), namely trypsin, chymotrypsin and Pronase. Studies with various unlabelled phosphate esters indicate that the sperm ecto-ATPase is not a non-specific phosphatase and it has high degree of substrate specificity for ATP.     

Occurrence of a cyclic AMP-dependent protein kinase on the outer surface of rat epididymal spermatozoa.

Endogenous protein kinase activity was detected on the outer surface of rat cauda epididymal spermatozoa. The kinase activity of the intact sperm cells catalyses the transfer of the terminal phosphate of exogenous [γ³²-P] ATP to the alkali labile phosphoester bonds of exogenous calf thymus histones. There was little uptake of [γ³²-P] ATP and phosphorylation of endogenous proteins by intact spermatozoa. The amount of histones phosphorylated by the peripherial kinase is directly proportional to the sperm numbers and the reaction is linear for approx. 5 min. Cyclic AMP (2.5 μM) activates the kinase (approx. 120%) and also causes the release of the enzyme from spermatozoa into the medium. Approx. 80% of the peripherial kinase activity is released after 30 seconds of incubation of spermatozoa.     

Characterization of nuclear phosphoprotein phosphatases from goat testis

Two nuclear phosphoprotein phosphatases (PPases I and II) that cause dephosphorylation of [32P]histone, have been partially purified from goat testis. The enzymic activity is associated with nucleoplasm and chromatin. PPase I is markedly stimulated (approx. 200-600%) by Mg2+ or Mn2+ (1 mM) whereas Ca2+ (1 mM) causes slight stimulation (approx. 35%) of the enzyme. On the contrary, PPase II is only slightly activated (20-40%) by these metal ions (5 mM). Both the phosphoprotein phosphatase isoenzymes are maximally active at pH 6-7. PPases I and II are strongly inhibited (approx. 60-100%) by ZnCl2 (1 mM), P1 (5 mM) and thiol reagents. NaF (5 mM) inhibits (approx. 40%) specifically the activity of PPase I rather than PPase II. PPases are strongly inhibited by relatively high concentration of NaCl (0.4 M), isoenzyme II being more sensitive (approx. 80%) than isoenzyme I (approx. 50%). In addition to histones, both the isoenzymes can as well cause dephosphorylation of protamine, casein, and testicular nuclear proteins. Enzymic characteristics of the testicular nuclear PPases are clearly different from those of the cytosolic enzyme previously characterized.     

Phosphorylation of external cell-surface proteins by an endogenous ecto-protein kinase of goat epididymal intact spermatozoa

Intact spermatozoa from goat cauda epididymides possess an ecto-(cyclic AMP-independent protein kinase) activity that causes transfer of the terminal phosphate of exogenously added [gamma-32P]ATP to the serine and threonine residues of several endogenous plasma-membrane phosphoproteins located on the external cell surface. Cyclic AMP, cyclic GMP, calmodulin and muscle cyclic AMP-dependent protein kinases I and II had no appreciable effect on the rate of phosphorylation of ecto-proteins by the intact cells. The ecto-enzyme is not derived from the catalytic subunit of a cyclic AMP-dependent kinase. Sperm ecto-kinase activity is not due to contamination of broken cells or any possible cell damage during incubation and isolation of spermatozoa. The phosphorylation reaction was linear for approx. 1 min and there was no detectable uptake of ATP by these cells. The activity of the ecto-kinase was strongly inhibited by proteinases and by the membrane-nonpenetrating surface probes. The products of the reaction were associated with the intact cells and the 32P of the labelled cells was largely lost when treated with Triton X-100 or proteinases: trypsin and pronase. These data are consistent with the view that the observed protein kinase and the phosphoproteins are located on the external surface of spermatozoa. Vigorously forward-motile whole spermatozoa showed a relatively high capacity to phosphorylate ecto-proteins that undergo rapid turnover. The results suggest the occurrence of a novel coupled-enzyme system (ecto-protein kinase and phosphoprotein phosphatase) on the sperm external surface that may modulate sperm physiology by determining the phosphorylated states of the ecto-proteins.     

Purification and characterization of an acid phosphatase that displays phosphotyrosyl-protein phosphatase activity from bovine cortical bone matrix

An acid phosphatase activity that displayed phosphotyrosyl-protein phosphatase has been purified from bovine cortical bone matrix to apparent homogeneity. The overall yield of the enzyme activity was greater than 25%, and overall purification was approximately 2000-fold with a specific activity of 8.15 mumol of p-nitrophenyl phosphate hydrolyzed per min/mg of protein at pH 5.5 and 37 degrees C. The purified enzyme was judged to be purified based on its appearance as a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (silver staining technique). The enzyme could be classified as a band 5-type tartrate-resistant acid phosphatase isoenzyme. The apparent molecular weight of this enzyme activity was determined to be 34,600 by gel filtration and 32,500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of reducing agent, indicating that the active enzyme is a single polypeptide chain. Kinetic evaluations revealed that the acid phosphatase activity appeared to catalyze its reaction by a pseudo Uni Bi hydrolytic two-step transfer reaction mechanism and was competitively inhibited by transition state analogs of Pi. The enzyme activity was also sensitive to reducing agents and several divalent metal ions. Substrate specificity evaluation showed that this purified bovine skeletal acid phosphatase was capable of hydrolyzing nucleotide tri- and diphosphates, phosphotyrosine, and phosphotyrosyl histones, but not nucleotide monophosphates, phosphoserine, phosphothreonine, phosphoseryl histones, or low molecular weight phosphoryl esters. Further examination of the phosphotyrosyl-protein phosphatase activity indicated that the optimal pH at a fixed substrate concentration (50 nM phosphohistones) for this activity was 7.0. Kinetic analysis of the phosphotyrosyl-protein phosphatase activity indicated that the purified enzyme had an apparent Vmax of approximately 60 nmol of [32P]phosphate hydrolyzed from [32P]phosphotyrosyl histones per min/mg of protein at pH 7.0 and an apparent Km for phosphotyrosyl proteins of approximately 450 nM phosphate group. In summary, the results of these studies represent the first purification of a skeletal acid phosphatase to apparent homogeneity. Our observation that this purified bovine bone matrix acid phosphatase was able to dephosphorylate phosphotyrosyl proteins at neutral pH is consistent with our suggestion that this enzyme may function as a phosphotyrosyl-protein phosphatase in vivo.     

An ecto-cyclic AMP-independent protein kinase in goat spermatozoa and its change of activity during forward motility

Goat cauda-epididymal intact spermatozoa have been shown to possess an ecto-cyclic AMP-independent protein kinase activity on the external surface that causes phosphorylation of the serine and threonine residues of exogenous phosvitin. The enzyme is neither a tyrosine kinase nor a catalytic subunit of the cyclic AMP-dependent protein kinase. It is not activated by Ca2+, calmodulin and phosphatidylserine. The intact-cell enzyme is capable of phosphorylating a variety of proteins including sperm plasma membrane-bound phosphoprotein(s). The enzymic activity of the intact spermatozoa was not due to contamination of broken or "leaky" cells. The kinase activity of the whole cells was strongly inhibited by the non-penetrating surface probes: p-chloromercuriphenylsulphonic acid (10 microM) and proteases (125 micrograms/ml). The specific activity of the ecto-kinase increased nearly 100% during vigorous forward progression of spermatozoa.     

Plasma membrane-associated phosphatase activities hydrolyzing [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate

We describe a procedure of preparing [32P]phosphotyrosyl histones with minimal contamination by 32P-labeled lipids; the latter was usually found to be mixed with the phosphoproteins when the cell membrane-enriched fraction of A-431 cells was used as a source of tyrosine kinase. The phosphatase activities previously found to be associated with the plasma membranes of a human astrocytoma were resolved using purified [32P]phosphotyrosyl histones and [32P]phosphatidylinositol phosphate. In comparison with the phosphotyrosyl protein phosphatase, the phosphatidylinositol phosphate phosphatase activity is more active over a broad range of pH values, and its activity is inhibited by fluoride, zinc chloride, and lower concentrations of vanadate.     

Phosphoprotein phosphatase inhibits flagellar movement of Triton models of sea urchin spermatozoa

Phosphoprotein phosphatase prepared from bovine cardiac muscle was used to study the roles of axonemal phosphoproteins in the flagellar motility of sea urchin spermatozoa. When isolated axonemes were incubated with cyclic AMP-dependent protein kinase, gamma-[32P]ATP and cyclic AMP, more than 15 polypeptides were phosphorylated. Most were dephosphorylated by treatment with phosphoprotein phosphatase. When Triton models of sea urchin spermatozoa were treated with phosphoprotein phosphatase followed by an addition of ATP, the flagellar motility of the models was drastically reduced in comparison with that of the untreated models. The motility of the phosphatase-treated Triton models was partially restored by an addition of cyclic AMP and cyclic AMP-dependent protein kinase. These data give strong support to the idea that the motility of eukaryotic flagella is controlled by a protein phosphorylation-dephosphorylation system.     

Phosphoprotein phosphatase in bovine tracheal smooth muscle. Multiple fractions and multiple substrates.

Phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) from bovine tracheal smooth muscle extracts was isolated and its activity determined using two [32P]phosphorylated proteins as substrates, i.e. phosphorylated histone (H-P) and a phosphorylated muscle specific substrate protein (MS-P) for the tracheal smooth muscle protein kinase. The enzyme was purified by the use of DEAE-cellulose followed by a two stage chromatography on a histone-Sepharose affinity column. Elution from the affinity column resolved the phosphoprotein phosphatase into four activity fractions. While fractions expressed phosphatase activity against both tested substrates the relative amounts of either activity varied. The ratio of activity towards H-P to activity towards MS-P changed from 11.5 to 0.12. The characterization of four phosphoprotein phosphatase fractions was based on the differences found in the following parameters: substrate specificity; sensitivity to NaF; influences of nucleotides (ATP, 5'-AMP, cyclic AMP, cyclic GMP) and the requirement of Mn2+ for maximal activity. Mg2+, Ba2+ or Ca2+ could not substitute for Mn2+.     

Modulation of calcineurin phosphotyrosyl protein phosphatase activity by calmodulin and protease treatment

Calcineurin (CN) dephosphorylated [32P] phosphotyrosyl glutamine synthetase, a model phosphoprotein substrate containing approximately 1 mol of phosphotyrosine per mol subunit. Phosphatase activity with and without calmodulin (CaM) was greatly stimulated by Mn2+; with Ca2+, even in the presence of CaM, activity was very low. CaM-stimulated phosphatase activity exhibited deactivation with time; initial rates declined markedly after 2-3 min. The Michaelis constant for substrate (3 microM) was identical whether 2 or 12 min assays (with CaM) were used suggesting that the decreased rate of hydrolysis did not result from a decrease in affinity for the phosphoprotein substrate. Limited proteolysis of CN by chymotrypsin increased phosphatase activity 2-3 times that of CaM-supported activity; however, addition of CaM to assays with protease-activated CN reduced activity to that observed for non-proteolyzed enzyme. These data suggest that, in addition to stimulation, CaM can inhibit certain activated conformations of the phosphatase.     

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.     

Interconversion of Tetrahymena pyriformis ornithine decarboxylase from inactive to active form by phosphorylation

A protein kinase and an acidic phosphoprotein phosphatase were purified from Tetrahymena pyriformis which phosphorylate and dephosphorylate the purified ornithine decarboxylase (ODC) of this microorganism. The protein kinase and the phosphoprotein phosphatase are copurified with ODC and can be separated in three distinct peaks only by a hydrophobic column of phenyl-Sepharose. The purified kinase is not dependent on cAMP, requires Mg2+ for its catalytic activity and has a molecule mass of 45 kDa. Incubation of [32P]ODC with the purified phosphoprotein phosphatase results in a complete loss of 32P and its catalytic activity. Phosphorylation of the inactive phosphatase-treated ODC by endogenous kinase or rat liver casein kinase-2 results in 100 or 40% reactivation of the initial untreated ODC activity, respectively.     

Purification and properties of a phosphoprotein phosphatase from rat liver.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) has been partially purified from rat liver homogenates by (NH4)2SO4 and ethanol precipitations followed by DEAE-cellulose and Sepharose 6B chromatography. The phosphoprotein phosphatase is capable of cleaving [32P]phosphate from radiolabelled phosphopyruvate kinase (type L) (EC 2.7.1.40), phosphohistones, and phosphoprotamine. However, it did not detectably dephosphorylate ATP, ADP, DL-phosphorylserine or beta-glycerophosphate. Dephosphorylation of [32P]phosphopyruvate kinase was stimulated by divalent cations and inhibited by ATP, ADP, Fru-1,6-P2, and orthophosphate. Divalene cations could reverse inhibition induced by ADP or ATP. At least one function of the phosphoprotein phosphatase may be to remove phosphate groups from the phosphorylated form of pyruvate kinase in the liver.     

Phosphoprotein-phosphatase activity associated with human placental alkaline phosphatase.

Human placental alkaline phosphatase, a marker protein for some nontrophoblastic neoplasms, was found to have phosphoprotein phosphatase activity. This was demonstrated by the dephosphorylation of ³²P-labeled histones, protamine, glycogen synthetase, casein, and phosvitin at various pH values. Unlike the general phosphoprotein phosphatase, the placental alkaline phosphatase does not have phosphorylase $\text{a}$ phosphatase activity.     

Occurrence of a coupled-enzyme system on the intact-sperm outer surface that phosphorylates and dephosphorylates ecto-proteins

Goat cauda-epididymal intact sperm ecto [32P] proteins phosphorylated in presence of exogenous [gamma-32P]ATP by an endogenous ecto-cyclic AMP-independent protein kinase (CIK), have been found to lose 32P when the labelled cells are incubated at 37 degrees C in a modified Ringer's solution. Analysis of the 32P-labelled products of the turnover of the ecto-phosphoproteins show that 32Pi rather than 32P-labelled peptides, is released from the cell-surface phosphoproteins indicating that the turnover of the ecto-phosphoproteins is mediated by an endogenous sperm outer-surface phosphoprotein phosphatase (ecto-PPase). The ecto-PPase is not a non-specific phosphatase since unlabelled p-nitrophenyl phosphate, beta-glycerophosphate or ATP at a relatively high concentration (1 mM each) has no appreciable effect on the dephosphorylation of the cell-surface proteins. The intact-sperm ecto-proteins phosphorylated and then dephosphorylated by the endogenous ecto-CIK and PPase respectively, undergo rephosphorylation by the cell-surface CIK. The data are consistent with the view that sperm external surface possesses a novel coupled-ecto-CIK and PPase enzyme system that regulates the phosphorylated states of the intact-sperm ecto-proteins by a cyclic mechanism of protein phosphorylation and dephosphorylation.     

[Phosphotyrosine]protein phosphatase in rat brain. A major [phosphotyrosine]protein phosphatase is a 23 kDa protein distinct from acid phosphatase.

A [phosphotyrosine]protein phosphatase (PTPPase) was purified almost to homogeneity from rat brain, with [32P]p130gag-fps, an oncogene product of Fujinami sarcoma virus, as substrate. The characteristics of the purified preparation of PTPPase were as follows: the enzyme was a monomer with a molecular mass of 23 kDa; its optimum pH was 5.0-5.5; its activity was not dependent on bivalent cations; its activity was strongly inhibited by sodium vanadate, but was not inhibited by ZnCl2, L(+)-tartrate or NaF; it catalysed the dephosphorylation of [32P]p130gag-fps, [[32P]Tyr]casein, p-nitrophenyl phosphate and L-phosphotyrosine, but did not hydrolyse [[32P]Ser]tubulin, L-phosphoserine, DL-phosphothreonine, 5'-AMP, 2'-AMP or beta-glycerophosphate significantly. During the purification, most of the PTPPase activity was recovered in distinct fractions from those of conventional low-molecular-mass acid phosphatase (APase), which was reported to be a major PTPPase [Chernoff & Li (1985) Arch. Biochem. Biophys. 240, 135-145], from DE-52 DEAE-cellulose column chromatography, and those two enzymes could be completely separated by Sephadex G-75 column chromatography. APase also showed PTPPase activity with [32P]p130gag-fps, but the specific activity was lower than that of PTPPase with molecular mass of 23 kDa, and it was not sensitive to sodium vanadate. These findings suggested that PTPPase (23 kDa) was the major and specific PTPPase in the cell.     

Dephosphorylation and activation of exogenous glycogen synthase by adipose-tissue phosphatase

Exogenous purified rabbit skeletal-muscle glycogen synthase was used as a substrate for adipose-tissue phosphoprotein phosphatase from fed and starved rats in order to (1) compare the relationship between phosphate released from, and the kinetic changes imparted to, the substrate and (2) ascertain if decreases in adipose-tissue phosphatase activity account for the apparent decreased activation of endogenous glycogen synthase from starved as compared with fed rats. Muscle glycogen synthase was phosphorylated with [gamma-(32)P]ATP and cyclic AMP-dependent protein kinase alone, or in combination with a cyclic AMP-independent protein kinase, to 1.7 or 3mol of phosphate per subunit. Adipose-tissue phosphatase activity determined with phosphorylated skeletal-muscle glycogen synthase as substrate was decreased by 35-60% as a consequence of starvation. This decrease in phosphatase activity had little effect on the capacity of adipose-tissue extracts to activate exogenous glycogen synthase (i.e. to increase the glucose 6-phosphate-independent enzyme activity), although there were marked differences in the activation profiles for the two exogenous substrates. Glycogen synthase phosphorylated to 1.7mol of phosphate per subunit was activated rapidly by adipose-tissue extracts from either fed or starved rats, and activation paralleled enzyme dephosphorylation. Glycogen synthase phosphorylated to 3mol of phosphate per subunit was activated more slowly and after a lag period, since release of the first mol of phosphate did not increase the glucose 6-phosphate-independent activity of the enzyme. These patterns of enzyme activation were similar to those observed for the endogenous adipose-tissue glycogen synthase(s): the glucose 6-phosphate-independent activity of the endogenous enzyme from fed rats increased rapidly during incubation, whereas that of starved rats, like that of the more highly phosphorylated muscle enzyme, increased only very slowly after a lag period. The observations made here suggest that (1) changes in glucose 6-phosphate-independent glycogen synthase activity are at best only a qualitative measure of phosphoprotein phosphatase activity and (2) the decrease in glycogen synthase phosphatase activity during starvation is not sufficient to explain the differential glycogen synthase activation in adipose tissue from fed and starved rats. However, alterations in the phosphorylation state of glycogen synthase combined with decreased activity of phosphoprotein phosphatase, both as a consequence of starvation, could explain the apparent markedly decreased enzyme activation.     

Phosphoprotein phosphatase associated with rat liver plasma membrane. Properties of phosphorylase phosphatase and phosphohistone phosphatase

Plasma membrane isolated from rat liver contained activities of phosphoprotein phosphatase dephosphorylating [32P]phosphorylase a or [32P]phosphohistone. The properties of the membrane-bound phosphatase were examined using these exogenous substrates. The optimal reaction rate was at pH near neutrality. At concentrations as low as 0.1-1.0 mM, Mg2+ or Mn2+ slightly stimulated the activity for phosphorylase a or phosphohistone, respectively; at higher concentrations, they were inhibitory with both substrates. Co2+ was inhibitory with both substrates, while Ca2+ had no significant effect. The phosphatase activities were inhibited by ATP, ADP, or AMP; the extents of inhibition were in opposite order with the two substrates. Phosphorylase phosphatase activity was strongly inhibited by KF or Pi. Phosphorylase phosphatase activity could be completely solubilized by incubating the membrane with 0.5 M NaCl or trypsin, and this was associated with several-fold activation. While Vmax values were increased, Km values for phosphorylase a were not much affected by these treatments. Unlike the soluble phosphatase, freezing in the presence of mercaptoethanol or by precipitation with ethanol failed to activate or to solubilize the membrane-bound phosphatase. The molecular weights of the NaCl-and the trypsin-solubilized phosphatase were estimated on gel filtration to be about 42,000 and 32,000, respectively. The present results indicate that the phosphoprotein phosphatase associated with liver plasma membrane shares several properties in common with phosphatases from other sources reported, and that, like those in the soluble fraction, it may be bound to some inhibitory proteins.     

小鼠腹水型肝癌H22a细胞浆内磷蛋白磷酸酶活力调节的研究

小鼠腹水型肝癌细胞胞浆内磷蛋白磷酸酶对磷酸化的组蛋白、酪蛋白、鱼精蛋白具有脱磷酸化活力,而对小分子底物P-Ser、P-Thr、P-Tyr、PNPP等无活力。二价金属离子Mn~(2+)、Co~(2+)、Mg~(2+)对酶有明显激活作用,而Zn~(2+)、F~-、Pi对酶有明显抑制作用。代谢中间物G-6-P、G-1-P、F-6-P、F-1.6-2P、ATP、ADP、GTP对酶有抑制作用,而磷酸化氨基酸和环核苷酸对酶活影响很小。还试验了碱性蛋白质和酸性蛋白质对酶活力的影响,肝素和组蛋白均对酶活力有抑制作用,当两者混和后,其抑制作用会相互抵消。