Activation by calmodulin of inositol-1,4,5-trisphosphate 3-kinase in guinea pig peritoneal macrophages

The activity of inositol-1,4,5-trisphosphate 3-kinase in the cytosol fraction of guinea pig macrophages was assayed with special reference to the dependence on the free Ca2+ concentration. The enzyme activity, as assessed by the production of inositol 1,3,4,5-tetrakisphosphate was reversibly activated by free Ca2+ concentrations ranging from 10(-7) to 10(-6)M. The calmodulin antagonists, W-7 and chlorpromazine, inhibited the Ca2+-activated enzyme activity in a dose-dependent fashion, thereby indicating that calmodulin may be involved in the activation by Ca2+. The content of calmodulin in the cytosol fraction (about 2.8 micrograms/mg of cytosol protein) was markedly reduced to less than 0.03 microgram/mg of proteins by subfractionation by ammonium sulfate, followed by an anion-exchange chromatography. The subfraction obtained by the chromatography showed no Ca2+ dependence in the enzyme activity, while an exogenous addition of calmodulin with 10(-6)M Ca2+ increased the enzyme activity. The enzyme activity was retained on a calmodulin-affinity column in the presence of Ca2+, and was eluted from the column by lowering the free Ca2+ concentration by adding ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. These results clearly indicate that calmodulin activates the inositol-1,4,5-trisphosphate 3-kinase activity.     

酿酒酵母胞内无机焦磷酸酶的分离纯化及性质

An inorganic pyrophosphatase (EC3.6.1.1) from Saccharomyces cerevisiae was purified to PAGE homogeneity by sonication disruption. (NH4)2SO4 fractionation and DEAE-cellulose colunm chromatography. The optimum pH and temperature of the enzyme were 7.4～7. 8 and 60℃, respectively. The Km was 19.3 mmol / L. The enzyme required Mg2+ as a cofactor for hydrolysis of pyrophosphate and was inhibited by Ca2+, Hg2+, Pb2+, Mn2+.     

Ca2+/calmodulin independent inositol 1,4,5-trisphosphate 3-kinase activity in guinea pig peritoneal macrophages

1. The Ca2+/calmodulin (CaM) independent activity of inositol 1,4,5-trisphosphate (InsP3) 3-kinase in macrophages could be separated from the dependent activity by serial column chromatography, gel filtration, Orange A and DEAE-5PW. 2. An InsP3 analog which has an aminobenzoyl group on the 2nd carbon of the inositol ring inhibited the conversion of [3H]InsP3 to [3H]InsP4 (inositol 1,3,4,5-tetrakisphosphate) in a dose-dependent manner. The concentration required for half-maximal inhibition (IC50) with the Ca2+/CaM independent enzyme activity was also dependent on the free Ca2+ concentration, as with the dependent activity. 3. These results suggest that a conformational change in the enzyme occurs in response to a change in free Ca2+ concentration, and thus the potency to recognize the InsP3 analog would change, even when the Ca2+/CaM independent enzyme activity was used.     

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Purification and characterization of inositol 1,4,5-trisphosphate 3-kinase from pig aortic smooth muscle.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase, which phosphorylates InsP3 to form inositol 1,3,4,5-tetrakisphosphate, was purified to apparent homogeneity by (NH4)2SO4 fractionation and sequential chromatographic steps on DEAE-sepharose, calmodulin-Affi-Gel and DEAE-5PW h.p.l.c. The purified enzyme had a specific activity of 24.4 nmol of inositol tetrakisphosphate formed/min per mg of protein, which represented a purification of approx. 195-fold with a 0.29% recovery, compared with the cytosol fraction of the muscle. SDS/polyacrylamide-gel electrophoresis showed a single protein-staining band of Mr 93,000. Moreover, the major protein peak, of Mr 84,000, was detected by TSK gel G3000SW gel-permeation chromatography of the purified sample. As this value was approximately consistent with the Mr determined by SDS/polyacrylamide-gel-electrophoretic analysis, the InsP3 3-kinase might be a monomeric enzyme. The purified enzyme had a Km for InsP3 of 0.4 microM, with an optimum pH range of 5.8-7.7. The enzyme was maximally activated by calmodulin, with a stoichiometry of 1:1.     

Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate.

Phosphatidylinositol 4-phosphate (PtdIns4P) kinase was purified from cytosolic and particulate material of rat brain. The purification procedure of the enzyme from cytosol consisted of (NH4)2SO4 precipitation. DEAE-cellulose column chromatography and preparative isoelectric focusing. Other methods after DEAE-cellulose column chromatography failed to achieve further purification of the PtdIns4P kinase, probably caused by the tendency of the enzyme to aggregate with contaminating proteins. The final purification was 67-fold, and the recovery was 0.6%. After isoelectric focusing the fraction containing the highest PtdIns4P kinase activity showed only one protein as visualized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and silver staining. The apparent Mr of this protein was 45 kDa and the isoelectric point about 5.8. The activity of PtdIns4P kinase was dependent on the concentration of divalent cations in the incubation medium. PtdIns4P kinase activity was found to be optimal at 10-30 mM-Mg2+. In an attempt to compare the cytosolic with the membrane-derived kinase activity, a Triton/KCl extract from synaptic membranes was subjected to the same purification procedure as the cytosolic enzyme. A difference in isoelectric focusing was observed, possibly due to a higher tendency to form aggregates. However, we tend to conclude that also in the membranes the PtdIns4P kinase activity is present as a 45 kDa protein, identical with that found in the cytosol.     

Calmodulin-mediated adenylate cyclase from mammalian sperm

Calmodulin (CaM), the calcium binding protein that modulates the activity of a number of key regulatory enzymes, is present at high levels in sperm. To determine whether CaM regulates adenylate cyclase in mammalian sperm, the actions of EGTA and selected CaM antagonists on a solubilized adenylate cyclase from mature equine sperm were examined. The activity of equine sperm adenylate cyclase was inhibited by EGTA in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 2 mM. Equine sperm adenylate cyclase was also inhibited in a concentration-dependent manner by the CaM antagonists chlorpromazine and calmidazolium (IC50 = 400 and 50 microM, respectively). The inhibition of enzyme activity by these agents correlated with their known potency and specificity as anti-CaM agents. The activity of the enzyme in the presence of 200 microM calmidazolium was restored by the addition of authentic CaM (EC50 = 15 microM); full activity was restored by the addition of 50 microM CaM. La3+, an ion that dissociates CaM from tightly bound CaM-enzyme systems, inhibited equine sperm adenylate cyclase (IC50 = 1 mM). Incubation of equine sperm adenylate cyclase with La3+ dissociated endogenous CaM from the enzyme so that most of the enzyme bound to a CaM-Sepharose column equilibrated with Ca2+. Specific elution of CaM-binding proteins from the CaM-Sepharose column with EGTA yielded a CaM-depleted adenylate cyclase fraction that was stimulated 2-fold by the addition of exogenous CaM.     

Studies on the properties of a soluble phosphatidylinositol-phosphodiesterase of rabbit iris smooth muscle

Some properties of the soluble phosphatidylinositol phosphodiesterase (monophosphatidylinositol inositolphosphohydrolase, EC 3.1.4.10) of rabbit iris smooth muscle are described. Studies on its subcellular distribution showed that in this tissue the phosphodiesterase is not exclusively cytosolic. Thus, under our experimental conditions about 58% of the enzyme activity was found in the soluble fraction and the remainder was particulate. When the latter was treated with deoxycholate about 59% of the enzyme activity, compared to 86% of that of ATPase, was still bound to the particulate fraction. The kinetic properties of the enzyme (30--50% (NH4)2SO4 fraction) were examined. Maximum breakdown was 7.7 mumol/h per mg protein and occurred at pH 5.6. The products of [14C]arachidonic acid-labelled phosphatidylinositol were 1,2-diacylglycerol and a mixture of 86% myoinositol 1-phosphate and 14% myoinositol 1,2-(cyclic)phosphate. The enzyme has an absolute requirement for Ca2+. Addition of Ba2+, La3+, Mg2+, Mn2+, EGTA or EDTA at 0.05--5 mM concentrations; Sr2+ at higher concentrations (greater than 0.25 mM) markedly inhibited the phosphodiesterase activity and this inhibition was completely reversed by Ca2+. The enzyme is specific for the phosphoinositides.     

Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase in rat and bovine brain tissues

Inositol 1,4,5-trisphosphate (Ins P3) 3-kinase catalyzes the ATP-dependent phosphorylation of Ins P3 to Inositol 1,3,4,5-tetrakisphosphate (Ins P4). Ca2+/calmodulin (CaM)-sensitivity of Ins P3 3-kinase was measured in the crude soluble fraction from rat brain and different anatomic regions of bovine brain. Kinase activity was inhibited in the presence of EGTA (free Ca2+ below 1 nM) as compared to Ca2+ (10 microM free Ca2+) or Ca2+ (10 microM free Ca2+) and CaM (1 microM). Ca2+-sensitivity was also seen for the cAMP phosphodiesterase measured under the same assay conditions, but was not for the Ins P3 5-phosphatase. DEAE-cellulose chromatography of the soluble fraction of rat brain or bovine cerebellum resolved a Ca2+/CaM-sensitive Ins P3 3-kinase (maximal stimulation at 1 microM Ins P3 substrate level was 2.0-3.0 fold).     

The inositol (1,4,5)-trisphosphate 3-kinase of Xenopus oocyte is activated by CaMKII and involved in the regulation of InsP3-mediated Ca2+ release

The effect of Ca2+ on inositol (1,4,5)-trisphosphate 3-kinase (3-kinase) activity was measured on Xenopus oocyte cytosolic extracts. The Ca2+-evoked elevation in 3-kinase activity appeared to be mediated by calmodulin (CaM) and the calmodulin-dependent protein kinase II (CaMKII). The results observed in vitro were totally retrieved in intact oocytes and tend to demonstrate the involvement of a CaMKII-mediated phosphorylation in the regulation of 3-kinase activity. Finally, electrophysiological recordings of InsP3-elicited chloride current transients in the presence of CaM/CaMKII inhibitors allowed to postulate an involvement of 3-kinase activity in the regulation of InsP3-mediated Ca2+ release.     

Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.     

Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride.

Arysulphatase A was purified from rabbit testis. The purification was accomplished by a four-step procedure involving (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, SP(sulphopropyl)-Sephadex and affinity chromatography on concanavalin A-Sepharose. The specific activity of purified preparation was 135 mumol/min per mg of protein, which represented an increase of 900-fold above that of the crude homogenate. The purified enzyme (20-50 micrograms) was found to move electrophoretically as a single band on polyacrylamide gel at pH 7.2 and 8.4. The homogeneous enzyme was shown to be a glycoprotein with 0.8% (w/w) of N-acetylneuraminic acid and 20% neutral sugar. The treatment of purified enzyme with bacterial neuraminidase had no effect on enzyme activity or kinetic properties, but it changed the elution prolife of rabbit testis arylsulphatase A through DEAE-Sephadex. The purified enzyme was strongly inhibited by Cu2+, Fe3+ and Ag+. It hydrolysed several sulphate esters including cerebroside 3-sulphate, ascorbic acid 2-sulphate and steroid sulphates. Pure arysulphatase was effective in dispersing the cumulus cells of rabbit ova.     

Studies on the biosynthesis of hepatic pyruvate kinase and its correlation with enhanced hepatic lipogenesis in meal-trained rats.

Metabolic and enzymic changes were measured in meal-trained rats fed on high-carbohydrate diet. Rates of hepatic fatty acid synthesis are probably greater than rates of gluconeogenesis throughout the 24 h day provided that animals are fed. The daily enhancement of fatty acid synthesis on meal feeding coincided with the maximum activation of hepatic pyruvate kinase. Maximum activation of this enzyme was reflected in increased total catalytic activity (Vmax.), increased activity at 0.5 MM-phosphoenolpyruvate (V0.5), decreased Vmax./V0.5 ratio and a decrease in co-operativity of phosphoenolpyruvate binding as measured by the Hill coefficient (h). The latter changes are consistent with a decrease in enzyme phosphorylation during activation of the enzyme. To estimate changes in enzyme protein, quantitative enzyme precipitation with rabbit antisera was used. Giving a high-carbohydrate diet to meal-trained animals induced enzyme synthesis within a few hours. Adaptations in diet that enhanced fatty acid synthesis (chow to high carbohydrate; starved to high carbohydrate) led to an increased steady-state concentration of pyruvate kinase protein. An approximate estimate of the half-life of hepatic pyruvate kinase was 56 h. Whenever pyruvate kinase specific activity was measured in liver tissue extracts it was always considerably less (20--100 mumol/min per mg of protein, depending on dietary status) than the specific activity of pure pyruvate kinase (200 mumol/min per mg of protein). Antigenically active, catalytically inactive protein was removed during enzyme purification from cytosol at the stage of (NH4)2SO4 fractionation. The fraction precipitated by 30--45%-satd. (NH4)2SO4 was enzymically active, antigenically reacting protein was identified in the remaining (NH4)2SO4 fractions (0--30%- and 45--85%-satd.) and this contained no enzyme activity. These may correspond to inactive proteolytic fragments of pyruvate kinase. The rate-determining step in adjusting enzyme concentration seems to be proteolysis.     

Cloning and expression in Escherichia coli of a rat brain cDNA encoding a Ca2+/calmodulin-sensitive inositol 1,4,5-trisphosphate 3-kinase.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase activity was stimulated by Ca2+ in the presence of calmodulin (CaM) and the protein was associated with two silver-stained bands which migrated with an apparent Mr of approx. 50,000 on SDS/polyacrylamide gels. Upon limited proteolysis with trypsin, the native InsP3 3-kinase was converted into polypeptides of Mr 44,000 and 36,000. Both tryptic fragments displayed InsP3 3-kinase activity that was Ca2+/CaM-sensitive. A cDNA clone, C5, that encodes the C-terminal part of the InsP3 3-kinase, was isolated by immunoscreening of a rat brain cDNA library. The 5' end of this clone was used in turn to probe the same library, yielding a clone (CP16) containing the entire coding sequence of InsP3 3-kinase. The encoding protein of 459 amino acids (calculated Mr 50,868) has several putative phosphorylation sites for cyclic AMP-dependent protein kinase, protein kinase C and CaM-dependent protein kinase II. When clone C5 was expressed in Escherichia coli, the truncated fusion protein showed Ca2+/CaM-sensitive InsP3 3-kinase activity. Our data demonstrate that the N-terminal part of the protein is not essential for either enzymic or CaM-regulatory properties.     

Escherichia coli S-adenosylhomocysteine/5''-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action.

Ca2+-activated protein phosphatase activity was demonstrated in mouse pancreatic acinar cytosol with alpha-casein and skeletal-muscle phosphorylase kinase as substrates. This phosphatase activity preferentially dephosphorylated the alpha subunit of phosphorylase kinase. After DEAE-cellulose chromatography, the Ca2+-activated phosphatase activity became dependent on exogenous calmodulin for maximal activity. Half-maximal activation was achieved at 0.5 +/- 0.1 microM-Ca2+. Trifluoperazine completely inhibited Ca2+-activated phosphatase activity, with half-maximal inhibition occurring at 8.5 +/- 0.6 microM. Mn2+, but not Mg2+, at 1 mM concentration could substitute for Ca2+ in eliciting full enzyme activation. The apparent Mr of the phosphatase as determined by Sephadex G-150 chromatography was 93000 +/- 1000. Submitting active fractions obtained after Sephadex chromatography to calmodulin affinity chromatography resulted in the resolution of a major protein of Mr 55500 +/- 300. In conclusion, Ca2+-activated protein phosphatase activity has been identified in exocrine pancreas and has several features in common with Ca2+-activated calmodulin-dependent protein phosphatases previously isolated from brain and skeletal muscle. It is possible that this Ca2+-activated phosphatase may utilize as substrates certain acinar-cell phosphoproteins previously shown to undergo dephosphorylation in response to Ca2+-mediated secretagogues.     

Production and properties of alpha-glucosidase from Lactobacillus acidophilus.

Lactobacillus acidophilus IFO 3532 was found to produce only intracellular alpha-glucosidase (alpha-D-glucoside glucohydrolase; EC 3.2.1.20). Maximum enzyme production was obtained in a medium containing 2% maltose as inducer at 37 degrees C and at an initial pH of 6.5. The enzyme was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. Enzyme production was strongly inhibited by 4 microM CuSO4, 40 microM CoCl2, and beef extract; MnSO4 and the presence of proteose peptone and yeast extract in the medium greatly enhanced enzyme production. A 16.6-fold purification of alpha-glucosidase was achieved by (NH4)2SO4 fractionation and DEAE-cellulose column chromatography. The enzyme showed high specificity for maltose. The Km for alpha-p-nitrophenyl-beta-D-glucopyranoside was 11.5 mM, and the Vmax for alpha-p-nitrophenyl-beta-D-glucopyranoside hydrolysis was 12.99 mumol/min per mg of protein. The optimal pH and temperature for enzyme activity were 5.0 and 37 degrees C, respectively. The enzyme activity was inhibited by Hg2+, Cu2+, Ni2+, Zn2+, Ca2+, Co2+, urea, rose bengal, and 2-iodoacetamide, whereas Mn2+, Mg2+, L-cysteine, L-histidine, Tris, and EDTA stimulated enzyme activity. Transglucosylase activity was present in the partially purified enzyme, and isomaltose was the only glucosyltransferase product. Amylase activity in the purified preparation was relatively weak, and no isomaltase activity was detected.     

Phosphatidylinositol 4,5-bisphosphate hydrolysis in human sperm stimulated with follicular fluid or progesterone is dependent upon Ca2+ influx.

Hydrolysis of the phospholipid phosphatidylinositol 4,5-bisphosphate is thought to be intimately involved in agonist-induced changes in intracellular Ca2+ levels. Recently we have shown that human preovulatory follicular fluid, which induces exocytosis in human sperm, can stimulate a rapid, transient increase in sperm cytosolic [Ca2+] [Thomas & Meizel (1988) Gamete Res. 20, 397-411]. We report here that both a Sephadex G-75 column fraction, derived from follicular fluid, and progesterone (a component of both the G-75 fraction and whole follicular fluid) stimulate rapid hydrolysis of PtdIns(4,5)P2 and PtdIns4P in human sperm. We also report that progesterone stimulates a rapid influx of Ca2+ in human sperm. Human spermatozoa were labelled for 24 h with myo-[3H]inositol and then treated with either the G-75 fraction or progesterone. A 30-65% loss of label was detected in PtdIns(4,5)P2 and PtdIns4P within 15 s of stimulus addition; no changes were observed in PtdIns during 2 min of treatment. The loss of label from both lipids was accompanied by an increase in water-soluble inositol phosphates. Production of both InsP3 and InsP2 was seen within 10 s; however, InsP3 was rapidly removed and had reached control levels by 1 min. Similarly, formation of InsP2 reached a peak by 30 s and then began a decline accompanied by a corresponding increase in InsP. No increases in InsP4 were seen in sperm treated in this fashion. Stimulated hydrolysis of the phosphoinositides and release of inositol phosphates were both blocked by the Ca2+ antagonist La3+. Likewise, the progesterone-induced increase in intracellular Ca2+ was inhibited by La3+, and phosphoinositide hydrolysis stimulated by this hormone was dependent upon the presence of extracellular Ca2+.     

Kinetic and physicochemical characteristics of an endogenous inhibitor to progesterone--receptor binding in rat placental cytosol.

This study describes the kinetic behaviour and physicochemical aspects of an endogenous inhibitor of progesterone--receptor binding in trophoblast cytosol from day-12 embryos. The progesterone cytosol receptor was partially purified and isolated from the inhibitor as the 0--50%-satd. (NH4)2SO4 fraction. The inhibitory substance was shown to reside in the 50--70%-satd. (NH4)2SO4 fraction. Equilibration of the inhibitor preparation with the receptor fraction increased the Kapp.D of the ligand--receptor binding reaction in a concentration-dependent manner (26 +/- 3-fold increase in Kapp.D per mg of protein of the (NH4)2SO4 fraction, n = 16). However, the inhibitor did not alter the concentration of binding sites. Studies of other physicochemical aspects of the inhibitor showed it to be non-diffusible, excluded from Sephadex G-25, stable at 35 degrees C for 30 min, but irreversibly denatured at 70 degrees C for 30 min. The Stokes' radius was estimated by gel chromatography to be 2.8 +/- 0.11 nm (n = 5). Inhibitory activity was destroyed by HgCl2, suggesting that disulphide bridges play an essential role in the biological activity of this molecule. The inhibitor is a macromolecule which does not bind progesterone and differs from albumin. The kinetic mechanism by which the inhibitor enhanced Kapp.D was investigated by measuring association and dissociation rate constants and the energy of activation (Ea) for each reaction. The association rate (k+1) for progesterone and receptor was (1.3 +/- 0.2) x 10(4) M-1 . s-1 but declined to (0.4 +/- 0.1) x 10(4) M-1 . s-1 (n = 5) when exposed to the inhibitor (P less than 0.01). The dissociation rate (k-1) was (3.2 +/- 0.6) x 10(-5) s-1 for progesterone--receptor complex and was unchanged by the inhibitor. The Ea for the association of complex was 33.6 +/- 4.2 kJ/mol and was increased to 63.0 +/- 8.4 kJ/mol by the inhibitor (P less than 0.05). The Ea of dissociation was unaltered. Thus, an inhibitor is present in trophoblast cytosol which specifically enhances Kapp.D without altering availability of binding sites. The mode of action of inhibitor is to increase the energy of activation for association of complex without influencing the dissociation reaction.     

Guanosine triphosphate cyclohydrolase activity in rat tissues.

The GTP cyclohydrolase activity of rat tissues has been studied by means of the measurement of formic acid release and neopterin synthesis from GTP. After gel filtration of a 45%-satd.-(NH4)2SO4 fraction of liver homogenates, three enzyme fractions were separated and named A1, A2 and A3 according to the order of their elution. Fractions A1 and A3 displayed an 8-formyl-GTP deformylase activity; no proof of cyclized product has yet been established. This activity was heat-labile and required Mg2+ for maximal activity. Fraction A2 displayed a 'neopterin-synthetase' activity, with dihydroneopterin triphosphate and formic acid formed in stochiometric amounts. Fraction A1 isolated from heat-treated homogenates also produced dihydroneopterin triphosphate. Neopterin synthetase activity in fractions A1 and A2 was heat-resistant and inhibited by Mg2+. In liver the A2 fraction represented 70-75% of the neopterin synthetase capacity and was inhibited by reduced pterines (sepiapterin, dihydrobiopterin and tetrahydrobiopterin) and to a lesser extent by reduced forms of folic acid. In kidney and brain, fraction A1 and A3 GTP 8-formylhydrolase activities were found in significant amounts, in contrast with the neopterin synthetase activity, which was low and appeared to be confined to the A1 fraction.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.