Role of the carbohydrate part of yeast acid phosphatase

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.     

Oviduct dolichyl phosphate phosphatase: estrogen effects and a possible biosynthetic role

Estrogen-induced chick oviduct differentiation is accompanied by an increased capacity for protein glycosylation. A portion of this increase has been attributed to increased levels of dolichyl phosphate (Dol-P). Hormone withdrawal leads to an apparent decrease in Dol-P. Dol-P metabolism in the oviduct has been studied, and one of the enzymes having a direct effect on Dol-P, Dol-P phosphatase is herein described. Dol-P phosphatase has a pH optimum of 6.0, does not require a metal ion, and is inhibited by Mn2+ at concentrations greater than 5 mM. Inhibitor studies indicate that Dol-P hydrolysis is inhibited by polyprenyl phosphates having both saturated and unsaturated alpha-isoprene residues, but not by the corresponding alcohols. The enzyme is also inhibited by phosphatidic acid unless 2 mM Mn2+ is included in the incubations. Under these conditions Dol-P hydrolysis is only slightly inhibited (less than 10%), but phosphatidate inhibition is totally eliminated. Oviduct membranes also possess phosphatidate phosphatase, but this enzyme is distinct from Dol-P phosphatase based on thermolability, metal ion sensitivity, and sulfhydryl reagent sensitivity. Studies of enzyme activity in response to estrogen treatment reveal that both Dol-P phosphatase and phosphatidate phosphatase have maximal specific activity early in the differentiation process (peaking after 3 days of treatment), and low specific activity in fully differentiated oviducts, including laying hen oviduct. Hormone withdrawal elicits a small increase in specific activity of both phosphatases. The hormone effects suggest that Dol-P phosphatase may be a biosynthetic enzyme.     

Autocatalytic inactivation of plant cytochrome P-450 enzymes: selective inactivation of the lauric acid in-chain hydroxylase from Helianthus tuberosus L. by unsaturated substrate analogs

Lauric acid in-chain hydroxylation is inhibited in microsomes from Jerusalem artichoke tubers (Helianthus tuberosus L.) incubated with 9-decenoic, 11-dodecenoic, or 11-dodecynoic acids. 9-Decenoic acid is at best a weak competitive inhibitor of the in-chain hydroxylase, but inactivates the enzyme in a time-dependent, pseudo-first-order process with a rate constant of approximately 1.1 X 10(-3) s-1. In contrast, 11-dodecenoic acid causes a slower, time-dependent loss of the hydroxylase activity, but is a potent competitive inhibitor of the enzyme (Ki = 2 microM). Neither agent decreases the microsomal concentration of cytochrome b5, NADH-cytochrome b5 reductase, or NADPH cytochrome P-450 reductase. Cinnamic acid 4-hydroxylation, catalyzed by a cytochrome P-450 enzyme, is not affected by concentrations of 9-decenoic acid that suppress lauric acid hydroxylation. 11-Dodecenoic acid is much less specific and, at higher concentrations, markedly reduces the microsomal cytochrome P-450 content, and the hydroxylation of both lauric and cinnamic acids.     

Bone remodelling induced by physical stress is prostaglandin E2 mediated

In vitro cultured bone cells were found to be responsive to hormones and physical forces. A simple device has been developed which enables the direct application of physical forces to tissue culture dishes to which cells are firmly attached. The physical forces created a deformation of the dish. It was found that prostaglandin E₂ synthesis underwent a rapid increase, reaching a maximum after 20 min and then declined. Concurrent with the increase in prostaglandin E₂ was an increase in cyclic AMP production, having a maximum around 15 min. The increase in cyclic AMP was blocked by indomethacin, the prostaglandin E₂ synthesis inhibitor, indicating the dependence of cyclic AMP production on the de novo synthesis of prostaglandin E₂. Prostaglandin E₂ added to cells mimicked the effect of physical forces on the production of cyclic AMP. The increase in cyclic AMP resulted from an early rise in adenyl cyclase activity (within 5 min) and a later (10 min) increase in phosphodiesterase activity. The same physical forces also stimulatedthe incorporation of thymidine into DNA after 24 h. On addition of prostaglandin E₂ the increase in DNA synthesis was also mimicked. Pretreatment of the cells with indomethacin abolished the effect of physical forces on DNA synthesis.The results suggest a stimulus receptor mechanism for physical forces which, like hormonal effectors, are mediated by prostaglandins and stimulate cyclic AMP and DNA synthesis.We believe that physical forces stimulate bone remodelling through such a stimulus receptor system, mediated by prostaglandins.     

pH-dependent leaving group effects on hydrolysis reactions of phosphate and phophonate esters catalyzed by wheat germ acid phosphatase.

The substrate specificity of carefully purified wheat germ acid phosphatase was examined and the Michaelis constants for substrates having widely varying leaving groups were determined at pH values 4.6, 8.0, and 9.2. The pH-dependent leaving group effects were consistent with the formation of a covalent phosphoryl histidine intermediate in the reaction process catalyzed by this enzyme. In addition, the enzyme was found to hydrolyze nitrophenyl esters of methyl-, chloromethyl-, and phenylphosphonic acids at rates comparable to those observed for phosphomonoester hydrolysis. The data are most simply interpreted on the basis of a nucleophilic displacement by an active-site histidine residue to form an intermediate N′-phosphonyl histidine species, followed by decomposition of this intermediate by nucleophilic attack by water, analogous to the decomposition process of the N′-phosphoryl enzyme species.     

Dephosphorylation of the hepatic insulin receptor: absence of intrinsic phosphatase activity in purified receptors

We have compared here the reversibility of phosphorylation of insulin receptors either partially purified by lectin chromatography, or highly purified by specific immunoprecipitation with anti-receptor antibodies. We found that the beta subunit of partially purified insulin receptors was rapidly dephosphorylated (t 1/2 = 15 min). In contrast, the level of phosphorylation of immunoprecipitated receptors remained unchanged for up to 4 hours at 37 degrees C. However, cytosolic phosphatases, which are inhibited by vanadate, were able to induce a complete dephosphorylation of immunoprecipitated receptors. These results show that 1. phosphorylation of insulin receptors is reversible; and 2. no phosphatase activity is contained in the insulin receptor structure itself.     

Mitochondrial ATPase inactivation by interaction with its substrate

Purified F₁-ATPase is slowly inactivated by interaction, in a preincubation medium, with its substrate MgATP. Interaction with Mg²⁺ before addition of ATP to the preincubation medium is essential to induce the inactivation. This inactivation is different from other Mg²⁺-induced inhibitions previously described. Free ATP concentration is implicated in the inactivation process and a linear relationship can be established between this concentration and the number of turnovers which are necessary for total inactivation. ITP, 2′-dATP, and GTP can also induce inactivation. Although ITP and GTP are hydrolyzed at a lower rate than ATP and 2′-dATP, they induce inactivation after a smaller number of turnovers than the latter. This process closely follows a kinetics of the type described for suicide enzymes. A reaction scheme is suggested and discussed.     

A homogeneous isoenzyme of human liver acid phosphatase.

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K_i is 4.3 × 10^?7m at pH 4.8 in 50 mm sodium acetate/100 mm sodium chloride. K_i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K_m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K_i for l-(+)-tartrate. The pH dependence of both K_m and V indicate the effect of substrate ionization (pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.     

A fast block to polyspermy in frogs mediated by changes in the membrane potential

The role of the egg membrane potential in the prevention of polyspermy in Rana pipiens was studied with intracellular microelectrodes and ion-substituted media. At fertilization, the egg membrane potential shifts from a resting value of ?28 to +8 mV in a single step of less than 1 sec. A second, slower shift reaches a maximum amplitude of +17 mV; the membrane potential is positive for a total of 21 min. When the membrane potential of unfertilized eggs exposed to sperm was held at +1 to +22 mV for 30 min by injecting current through a second intracellular electrode, the initiation of the first cleavage furrow was delayed about 20 min, suggesting that the eggs were not fertilized while the membrane potential was positive. Injection of a similar amount of current after fertilization did not delay cleavage. Furthermore, fertilization in ion-substituted media suggests a correlation between the maximum amplitude of the positive-going shift and the incidence of polyspermy. Up to 25% of eggs were polyspermic when inseminated in the presence of NaI, and the maximum amplitude was reduced to ?20 mV when eggs were fertilized in 40 mM NaI. In contrast, fertilization in 40 mM NaCl reduced the maximum amplitude only to +6 mV, and produced no polyspermy. In solutions of NaBr, intermediate effects on the membrane potential and polyspermy were seen. Comparable results were obtained with the toad, Bufo americanus. We conclude that the membrane potential shift prevents polyspermy.     

Irreversible inactivation of human erythrocyte pyruvate kinase by 2,3-butanedione

Human erythrocyte pyruvate kinase was found to be irreversibly inactivated by butanedione in the dark. The second-order rate constants for inactivation at pH 8.0 and 25 degrees C were 2.14 and 2.74 M-1 min-1 in the absence and presence of 50 mM borate, respectively. The pH profile of the inactivation indicated the involvement of a residue with an apparent pK alpha of 8.1-8.3. ADP and phosphoenolpyruvate acted as partial inhibitors of the inactivation process. Certain details of the inactivation, spectral studies, and fluorometric determinations gave evidence for arginine as the only target residue. A total of 23 +/- 3 residues per subunit were modified within the period required for inactivation. In the same period the presence of 4 mM ADP reduced the extent of inactivation by 70% and the number of modified residues to 18 +/- 4. The number of the arginine residues protected by ADP from butanedione modification was 5.0 +/- 1.3 per subunit.     

The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.     

Inhibition of pyruvate dehydrogenase and pyruvate dehydrogenase phosphate phosphatase by glyoxylate

The overall reaction catalyzed by the pyruvate dehydrogenase complex from rat epididymal fat tissue is inhibited by glyoxylate at concentrations greater than 10 μm. The inhibition is competitive with respect to pyruvate; K_i was found to be 80 μm. Qualitatively similar results were observed using pyruvate dehydrogenase from rat liver, kidney, and heart. Glyoxylate also inhibits the pyruvate dehydrogenase phosphate phosphatase from rat epididymal fat, with the inhibition being readily detectable using 50 μm glyoxylate. These effects of glyoxylate are largely reversed by millimolar concentrations of thiols (especially cysteine) because such compounds form relatively stable adducts with glyoxylate. Presumably these inhibitions by low levels of glyoxylate had not been previously observed, because others have used high concentrations of thiols in pyruvate dehydrogenase assays. Since the inhibitory effects are seen with suspected physiological concentrations, it seems likely that glyoxylate partially controls the activity of pyruvate dehydrogenase in vivo.     

Binding of the rat liver 7-8 S dexamethasone receptor to deoxyribonucleic acid

The 7-8 S form of the [3H]dexamethasone (9 alpha-fluoro-11 beta,17,21-trihydroxy-16 alpha-methylpregna-1,4-diene-3, 20-dione) receptor from rat liver cytosol can be converted to the 3-4 S form by RNase treatment or high salt, suggesting a salt-sensitive association between the receptor protein and RNA. In DNA-cellulose column assays, the gradient-purified 3-4 S form bound DNA more efficiently than the 7-8 S form, though the 7-8 S form was also capable of binding to DNA-cellulose to a significant extent. Activated 7-8 S dexamethasone receptor could be released from its association with soluble DNA by treatment with DNase I. Sucrose gradient analysis showed that the released receptor sedimented as the 7-8 S form and was sensitive to RNase treatment, which induced a conversion to the 3-4 S form. Activated RNase-generated 3-4 S receptor again displayed a higher degree of binding to soluble DNA and was recovered in the 3-4 S form following DNase extraction. The fact that the 3-4 S form bound immobilized or soluble DNA more efficiently suggests that the associated RNA of the 7-8 S form interferes directly or indirectly with the receptor association with DNA. The observation that the receptor binds to DNA in its 7-8 S form suggests that the receptor complex is capable of binding RNA and DNA concurrently.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.     

Kinetics of the inactivation of Escherichia coli glutamate apodecarboxylase by phenylglyoxal.

The possible interaction of the phosphate moiety of pyridoxal phosphate with a guanidinium group in glutamate apodecarboxylase was investigated. The holoenzyme is not inactivated significantly by incubation with butanedione, glyoxal, methylglyoxal, or phenylglyoxal. However, the apoenzyme is inactivated by these arginine reagents in time-dependent processes. Phenylgloxal inactivates the apoenzyme most rapidly. The inactivation follows pseudo-first-order kinetics at high phenylglyoxal to apoenzyme ratios. The rate of inactivation is proportional to phenylglyoxal concentration, increases with increasing pH, and is also dependent on the type of buffer present. The rate of inactivation of the apoenzyme by phenylglyoxal is fastest in bicarbonate — carbonate buffer and increases with increasing bicarbonate — carbonate concentration. Phosphate, which inhibits the binding of pyridoxal phosphate to the apoenzyme, protects the apodecarboxylase against inactivation by phenylglyoxal. When the apodecarboxylase is inactivated with [¹⁴C]phenylglyoxal, approximately 1.6 mol of [¹⁴C]phenylglyoxal is incorporated per mol subunit. The phenylglyoxal is thought to modify an arginyl residue at or near the pyridoxal phosphate binding site of glutamate apodecarboxylase.     

Stimulation of monovalent ion fluxes and DNA synthesis in 3T3 cells by melittin and vasopressin is not mediated by phospholipid deacylation

Melittin at subtoxic concentrations stimulates monovalent ion fluxes and, together with insulin, synergistically increases DNA synthesis, but has little effect on phospholipase activity. The Na⁺ ionophore monensin increases Na-K pump activity without affecting phospholipase activity; whereas, the divalent cation ionophore A23187 has reciprocal effects. Finally, vasopressin and insulin potently stimulate ion fluxes and DNA synthesis but have no effect on phospholipase activity. Thus, the polypeptide mitogens melittin, vasopressin, and insulin alter membrane function and subsequently stimulate DNA synthesis in quiescent 3T3 cells by mechanisms independent of phospholipid deacylation.     

Effect of estradiol and progesterone on glucosamine 6-phosphate synthase in the uterus of rat

Injection of estradiol to ovariectomised rats caused significant increase in the activity of glucosamine 6-phosphate synthase of uterus. Progesterone did not cause any increase in the activity of the enzyme; however, it antagomised the effect of estradiol. It was observed that the enzyme is predominantly localised in the endometrium of uterus.     

Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine

S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.     

Characteristics of the nucleotide phosphohydrolases of hamster embryo cells

The characteristics and distribution of nucleotide phosphohydrolases of hamster embryo cells were examined. The hydrolysis of ATP and ADP by monolayers of hamster embryo cells was stimulated by Mg²⁺ and Ca²⁺ ions. In contrast, the hydrolysis of AMP was not increased by these ions. These observations suggest that the enzyme hydrolyzing AMP (ecto-AMPase) is different from the enzymes hydrolyzing ATP and ADP. About 70–90% of the total activity of the nucleotide phosphohydrolases of hamster embryo cells was localized on the surface of the cell membrane. These ecto-enzymes hydrolyzed a variety of nucleotides at rates comparable to those observed for the hydrolysis of AMP, ADP, and ATP. These findings indicated that the ecto-enzymes of hamster embryo cells have a low substrate specificity. The activities of the ecto-enzymes of tumor cells induced in the hamster by Rous sarcoma virus were greatly diminished or abolished in comparison to those of normal hamster embryo cells. This suggests that the ecto-nucleotide phosphohydrolases of hamster embryo cells may be suitable biochemical markers of transformation.