Phytase from Wheat Bran

myo-Inositol mono-, di-, tri-, tetra-, and pentaphosphate were prepared by enzymic hydrolysis of myo-inositol hexaphosphate with a 1,500-fold purified phytase preparation from wheat bran and the subsequent Dowex 1 column chromatography. Relative initial rates of hydrolysis of these inositol phosphates by phytase were nearly the same each other and the activation energy of hydrolysis was about 11,000 cal. per mole for all these substrates. Km values did not vary widely with the substrates. The hydrolysis of inositol phosphates proceeded in a complicated way, except inositol monophosphate, where the reaction was of the first order. The enzyme hydrolyzed the substrates in the manner that removed phosphate group of them one by one. When mixed substrate was used the enzyme showed a preferential attack on the highest member of the phosphates present. From the mixed substrate test, it was concluded that wheat bran phytase is a single enzyme.     

Method for determining nitrogen-containing cationic surface-active agents using butyrylcholinesterase

The biochemical method for determination of cetyltrimethyl ammonium or cetylpyridinium, both being nitrogenated cationic surfactants, has been devised by using horse blood serum butyrylcholinesterase as analytical reagent. The method streams from the fact that surfactants tested are inhibitors of butyrylcholinesterase hydrolysis of butyrylcholin, a cationic substrate, but in this case they activate enzymatic hydrolysis of 1-naphthylacetate, a neutral substrate. Presence two opposite effects enlarges reliability to identifications. Use the sensitive fluorimetric method to registrations of activation of hydrolysis a substrate 1-naphtylacetate vastly to reduce the threshold of determination of surfactants above.     

Correlation of thrombin-induced glycoprotein V hydrolysis and platelet activation

To assess the possibility that hydrolysis of the platelet surface thrombin substrate, glycoprotein V, is a necessary step in thrombin-induced platelet activation, thrombin-catalyzed hydrolysis of glycoprotein V was correlated with thrombin-induced platelet activation. Hydrolysis of tritium-labeled glycoprotein V on washed human platelets was measured by the appearance of a labeled supernatant fragment, and platelet activation was measured as secretion of ATP. Hydrolysis of glycoprotein V was linear with respect to both thrombin concentration and time of incubation. The extent of platelet activation was correlated with the rate of hydrolysis but not with the amount hydrolyzed. Maximum platelet activation could be obtained with thrombin treatments resulting in hydrolysis of as little as 4% of glycoprotein V per min. Glycoprotein V was partially removed from platelets by pretreatment with either platelet calcium-dependent protease or chymotrypsin. The rate of thrombin-catalyzed hydrolysis of the remaining glycoprotein V from these pretreated platelets was as little as 1.5% the rate from control platelets, but there was no impairment of the extent of platelet activation. Thus, these protease-pretreated platelets compared with control platelets showed a different correlation of glycoprotein V hydrolysis with platelet activation. Glycoprotein V was also partially removed by pretreatment of prostacyclin-inhibited platelets with thrombin. After removal of thrombin and prostacyclin, these platelets were desensitized to subsequent activation by thrombin. Incubation of desensitized platelets with nonsaturating levels of thrombin led to less than 25% of the activation seen with control platelets but to a slightly greater hydrolysis of glycoprotein V. Thus, the desensitization to thrombin was not due to loss of ability of the activating thrombin to hydrolyze glycoprotein V. These results do not exclude a role for glycoprotein V as a component of the platelet thrombin receptor, but they indicate that there is no simple relationship between thrombin-induced hydrolysis of glycoprotein V and platelet activation.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed proteolysis

The effects of ATP, vanadate, and molybdate on cathepsin D-catalyzed hydrolysis of proteins and peptides were examined. Hydrolysis of bovine serum albumin, hemoglobin, parathyroid hormone, and a synthetic octapeptide was activated by ATP. Degradation of the protein substrates all had similar ATP concentration dependence, but the magnitude of the activation varied. Kinetic constants for ATP activation were obtained with a synthetic substrate. ATP increased kcat from 0.4 to 2 s-1 but did not change KM. Kact for ATP was 800 microM. Studies with pepstatin-Sepharose confirm that ATP does not alter the substrate binding site on cathepsin D. Pepsin, a homologous aspartate protease, was not activated by ATP. It was also found that vanadate and molybdate inhibit cathepsin D-catalyzed proteolysis. However, this inhibition was dramatically dependent on substrate concentration and was eliminated at high substrate. Hydrolysis of the synthetic peptide was not inhibited at concentrations of molybdate below 50 microM, and above this concentration the peptide precipitated. Protein substrates were also found to precipitate in the presence of molybdate. The ATP dependence of the enzyme was not altered by molybdate or vanadate. These results suggest that inhibition by vanadate and molybdate is related to interactions with the substrate rather than with cathepsin D. It is concluded that ATP activation of cathepsin D may play a physiological role in regulation of proteolysis in lysosomes, but that vanadate and molybdate inhibition of lysosomal proteolysis does not establish ATP dependence.     

Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.     

ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome

The 26S proteasome degrades polyubiquitinated proteins by an energy-dependent mechanism. Here we define multiple roles for ATP in 26S proteasome function. ATP binding is necessary and sufficient for assembly of 26S proteasome from 20S proteasome and PA700/19S subcomplexes and for proteasome activation. Proteasome assembly and activation may require distinct ATP binding events. The 26S proteasome degrades nonubiquitylated, unstructured proteins without ATP hydrolysis, indicating that substrate translocation per se does not require the energy of hydrolysis. Nonubiquitylated folded proteins and certain polyubiquitylated folded proteins were refractory to proteolysis. The latter were deubiquitylated by an ATP-independent mechanism. Other folded as well as unstructured polyubiquitylated proteins required ATP hydrolysis for proteolysis and deubiquitylation. Thus, ATP hydrolysis is not used solely for substrate unfolding. These results indicate that 26S proteasome-catalyzed degradation of polyubiquitylated proteins involves mechanistic coupling of several processes and that such coupling imposes an energy requirement not apparent for any isolated process.     

Effect of apolipoprotein C-II on the temperature dependence of lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholines. A hydrophobic model for the mechanism

The lipoprotein lipase-catalyzed hydrolysis of diacylphosphatidylcholines (PC) in mixed micelles of Triton X-100/PC was studied as a function of temperature in the presence and absence of apolipoprotein C-II (apo-C-II), the activator protein for lipoprotein lipase. Dilauroyl-, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylcholine (di-C12-PC, di-C14-PC, di-C16-PC, and di-C18-PC, respectively) were used as substrates. No systematic relationship between substrate fatty acyl chain length and either the rates of the activation energies for hydrolysis in the presence or absence of apo-C-II was observed. However, there was a linear relationship between fatty acyl chain length and both the logarithm of the activation factor (the ratio of enzyme activity with apo-C-II to that without apo-C-II) and the difference in activation energy in the presence and absence of apo-C-II. These relationships were not the result of an alteration in the physical form of the substrate, since a mixture of di-C14-PC and di-C16-PC gave activation factors for each PC which were the same as those obtained for each individual lipid. From the temperature dependence of the activation factor, thermodynamic functions of the apo-C-II-induced change in the reaction pathway were calculated. The free energy of activation decreased linearly with increasing chain length as the result of a linear increase in activation entropy which more than offset the unfavorable increase in activation enthalpy. We propose that the apo-C-II-mediated increase in the rate of the lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine is associated with transfer of a fatty acyl chain of the substrate or product to a more hydrophobic environment within the transition state complex.     

Unusual kinetic behavior of Endothia parasitica protease in hydrolysis of small peptides

Endothia parasitica protease hydrolyzes l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide at the peptide bond. l-Phenylalanyl-l-leticine amide, N-carbobenzoxy-l-leucyl-l-phenylalanine amide, N-carbobenzoxy-l-leucyl-l-pheml-alanine, N-carbobenzoxy-l-phenylalanyl-l-valine amide, and l-leucyl-β-naphthyl-amide are not hydrolyzed. In contrast to the kinetics of hydrolysis of casein and oxidized B-chain of insulin and activation of trypsinogen by Endothia parasitica protease which are normal, reaction progress curves for hydrolysis of l-leucyl-l-leucine amide and l-leucyl-l-phenylalanine amide are sigrnoidal. Initially, the reaction rates were of the order of 0.5–2.5% of the maximum rates eventually attained. With increasing time of incubation the reaction rates became faster and faster until maximum rates were achieved. This abnormal behavior was not eliminated by recrystallization of substrate or by incubation of enzyme alone or with products of the reaction prior to addition of substrate. Addition of a new aliquot of substrate, vizl-leucyl-l-leucine amide, to the reaction prior to complete hydrolysis of all of a previous aliquot of the same substrate, or reactions containing a mixture of oxidized B chain of insulin and l-leucyl-l-leucine amide, gave normal reaction progress curves. The duration of abnormal behavior before a maximum rate was attained was a function of enzyme concentration and temperature but not of substrate concentration even though substrate was in less than saturating amounts. The reaction data follow second-order autocatalytic kinetics with respect to enzyme concentration. It is proposed that most of the enzyme is in an inactive form in absence of substrate but is rapidly converted to the active form on combination with a good substrate such as trypsinogen, casein, or oxidized B chain of insulin. However, with a poor substrate such as l-leucyl-l-leucine amide, conversion to active enzyme is mediated through formation of an active enzyme-inactive enzyme complex followed by combination with substrate and hydrolysis.     

A simple method for the determination of individual rate constants for substrate hydrolysis by serine proteases

A simple method is presented for the determination of individual rate constants for substrate hydrolysis by serine proteases and other enzymes with similar catalytic mechanism. The method does not require solvent perturbation like viscosity changes, or solvent isotope effects, that often compromise nonspecifically the activity of substrate and enzyme. The rates of substrate diffusion into the active site (k1), substrate dissociation (k-1), acylation (k2), and deacylation (k3) in the accepted mechanism of substrate hydrolysis by serine proteases are derived from the temperature dependence of the Michaelis-Menten parameters kcat/Km and kcat. The method also yields the activation energies for these molecular events. Application to wild-type and mutant thrombins reveals how the various steps of the catalytic mechanism are affected by Na+-binding and site-directed mutations of the important residues Y225 in the Na+ binding environment and L99 in the S2 specificity site. Extension of this method to other proteases should enable the derivation of detailed information on the kinetic and energetic determinants of protease function.     

Characterization of acidic and neutral sphingomyelinase activities in crude extracts of HL-60 cells

The enzymatic activities of acidic and neutral sphingomyelinases (aSMase and nSMase) in crude extracts of HL-60 cells prepared by short ultrasonic irradiation (sonicates) were characterized. It was found that although both have similar Km and Vmax (approximately 0.2 mM and approximately 3.5 nmol/mg per h, respectively), the two activities differ in many other aspects, including the following: (1) the aSMase activity has higher stability at 37 degrees C; (2) the aSMase is much less sensitive to Triton X-100 ( > 5 mM), compared with < or = 0.4 mM for the nSMase; (3) the nSMase, but not the aSMase, can discriminate between the natural bovine sphingomyelin substrate and the fluorescent substrate lissamine rhodamine dodecanoyl sphingosyl phosphocholine, suggesting that nSMase has higher substrate specificity. TNFalpha, which upon incubation with the HL-60 cells induces cellular SM hydrolysis, does not affect Km or Vmax of the nSMase in HL-60 sonicates. This suggests that TNFalpha may operate through translocation of either the enzyme or the substrate, thereby enhancing substrate availability and rate of hydrolysis, and not through enzyme activation. The relevance of these studies to the sphingomyelin cycle is discussed.     

Allosteric activation of cGMP-specific,cGMP-binding phosphodiesterase (PDE5) by cGMP

The effects of cGMP binding on the catalytic activity of cGMP-specific, cGMP-binding phosphodiesterase (PDE5) are unclear because cGMP interacts with both allosteric and catalytic sites specifically. We studied the effects of cGMP on the hydrolysis of a fluorescent substrate analogue, 2'-O-anthraniloyl cGMP, by PDE5 partially purified from rat cerebella. The preparation contained PDE5 as the major cGMP-PDE activity and was not contaminated with cAMP- or cGMP-dependent protein kinases. The Hill coefficients for hydrolysis of the analogue substrate were around 1.0 in the presence of cGMP at concentrations <0.3 microM, while they increased to 1.5 at cGMP concentrations >1 microM, suggesting allosteric activation by cGMP at concentrations close to the bulk binding constant of the enzyme. Consistent with an allosteric activation, increasing concentrations of cGMP enhanced the hydrolysis rate of fixed concentrations of 2'-O-anthraniloyl cGMP, which overcame competition between the two substrates. Such activation was not observed with cAMP, cyclic inosine 3',5'-monophosphate, or 2'-O-monobutyl cGMP, indicating specificity of cGMP. These results demonstrate that cGMP is a specific and allosteric activator of PDE5, and suggest that in cells containing PDE5, such as cerebellar Purkinje cells, intracellular cGMP concentrations may be regulated autonomously through effects of cGMP on PDE5.     

Substrate activation of porcine pancreatic kallikrein by N alpha derivatives of arginine 4-nitroanilides

Hydrolysis of several N alpha-substituted L-arginine 4-nitroanilides with porcine pancreatic kallikrein was studied under different conditions of pH, temperature, and salt concentration. At high substrate concentrations a deviation from Michaelis-Menten kinetics was observed with a significant increase in the hydrolysis rates of almost all substrates. Kinetic data were analyzed on the assumption that porcine pancreatic kallikrein presents an additional binding site with lower affinity for the substrate. Binding to this auxiliary site gives rise to a modulated enzyme species which can hydrolyze an additional molecule of the substrate through a second catalytic pathway. The values of both Michaelis-Menten and catalytic rate constants were higher for the modulated species than for the free enzyme, suggesting a mechanism of enzyme activation by substrate. Kinetic data indicated similar substrate requirements for binding at the primary and auxiliary sites of the enzyme. Tris(hydroxymethyl)aminomethane hydrochloride and NaCl were shown to alter the kinetic parameters of the hydrolysis of N alpha-acetyl-L-Phe-L-Arg 4-nitroanilide by porcine pancreatic kallikrein but not the enzyme activation pattern (ratio of the catalytic constants for the activated and the free enzyme forms). Similar observations were made when the hydrolysis of D-Val-L-Leu-L-Arg 4-nitroanilide was studied under different pH and temperature conditions.     

Kinetic properties of wild-type and altered recombinant amidases by the use of ion-selective electrode assay method

A novel assay method was investigated for wild-type and recombinant mutant amidases (EC 3.5.1.4) from Pseudomonas aeruginosa by ammonium ion-selective electrode (ISE). The initial velocity is proportional to the enzyme concentration by using the wild-type enzyme. The specific activities of the purified amidase were found to be 88.2 and 104.2 U mg protein(-1) for the linked assay and ISE methods, respectively. The kinetic constants--Vmax, Km, and Kcat--determined by Michaelis-Menten plot were 101.13 U mg protein(-1), 1.12x10(-2) M, and 64.04 s(-1), respectively, for acrylamide as the substrate. On the other hand, the lower limit of detection and range of linearity of enzyme concentration were found to be 10.8 and 10.8 to 500 ng, respectively, for the linked assay method and 15.0 and 15.0 to 15,000 ng, respectively, for the ISE method. Hydroxylamine was found to act as an uncompetitive activator of hydrolysis reaction catalyzed by amidase given that there is an increase in Vmax and Km when acetamide was used as the substrate. However, the effect of hydroxylamine on the hydrolysis reaction was dependent on the type of amidase and substrate involved in the reaction mixture. The degrees of activation (epsilon(a)) of the wild-type and mutant (T103I and C91A) enzymes were found to be 2.54, 12.63, and 4.33, respectively, for acetamide as the substrate. However, hydroxylamine did not activate the reaction catalyzed by wild-type and altered (C91A and W138G) amidases by using acrylamide and acetamide, respectively, as the substrate. The activating effect of hydroxylamine on the hydrolysis of acetamide, acrylamide, and p-nitrophenylacetamide can be explained by the fact that additional formation of ammonium ions occurred due to the transferase activity of amidases. However, the activating effect of hydroxylamine on the hydrolysis of p-nitroacetanilide may be due to a change in conformation of enzyme molecule. Therefore, the use of ISE permitted the study of the kinetic properties of wild-type and mutant amidases because it was possible to measure initial velocity of the enzyme-catalyzed reaction in real time.     

D-dihexanoylphosphatidylcholine is not a pure competitive inhibitor of phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine

The inhibition of Crotalus adamanteus phospholipase A2 hydrolysis of L-dihexanoylphosphatidylcholine by D-dihexanoylphosphatidylcholine was investigated with inhibitor and substrate in the monomeric concentration range. The results showed that the D-enantiomer acts as a partial (not pure) competitive inhibitor of the enzyme. These results suggest that an ESI complex exists, in which hydrolysis of the substrate still occurs. Thus, binding of the D-enantiomer to the enzyme decreases the affinity for the substrate by a factor, alpha, while Vmax is unaffected. The value of alpha was determined to be 4.70 +/- 0.14. These findings complicate the use of D-phosphatidylcholines in mixed micelles with the L-enantiomer as a possible method to investigate the mechanism of interfacial activation of this enzyme.     

Effects of fatty acids on activity of cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver

Effects of fatty acids, prostaglandins, and phospholipids on the activity of purified cGMP-stimulated cyclic nucleotide phosphodiesterase from calf liver were investigated. Prostaglandins A2, E1, E2, F1 alpha, and F2 alpha, thromboxane B2, and most phospholipids were without effect; lysophosphatidylcholine was a potent inhibitor. Several saturated fatty acids (carbon chain length 14-24), at concentrations up to 1 mM, had little or no effect on hydrolysis of 0.5 microM [3H]cGMP or 0.5 microM [3H]cAMP with or without 1 microM cGMP. In general, unsaturated fatty acids were inhibitory, except for myristoleic and palmitoleic acids which increased hydrolysis of 0.5 microM [3H]cAMP. The extent of inhibition by cis-isomers correlated with the number of double bonds. Increasing concentrations of palmitoleic acid from 10 to 100 microM increased hydrolysis of [3H]cAMP with maximal activation (60%) at 100 microM; higher concentrations were inhibitory. Palmitoleic acid inhibited cGMP hydrolysis and cGMP-stimulated cAMP hydrolysis with IC50 values of 110 and 75 microM, respectively. Inhibitory effects of palmitoleic acid were completely or partially prevented by equimolar alpha-tocopherol. Palmitelaidic acid, the trans isomer, had only slightly inhibitory effects. The effects of palmitoleic acid (100 microM) were dependent on substrate concentration. Activation was maximal with 1 microM [3H]cAMP and was reduced with increasing substrate; with greater than 10 microM cAMP, palmitoleic had no effect. Inhibition of cGMP hydrolysis was maximal at 2.5 microM cGMP and was reduced with increasing cGMP; at greater than 100 microM cGMP palmitoleic acid increased hydrolysis slightly. Palmitoleic acid did not affect apparent Km or Vmax for cAMP hydrolysis, but increased the apparent Km (from 17 to 60 microM) and Vmax for cGMP hydrolysis with little or no effect on the Hill coefficient for either substrate. These results suggest that certain hydrophobic domains play an important role in modifying the catalytic specificity of the cGMP-stimulated phosphodiesterase for cAMP and cGMP.     

Stimulation of the thiol-dependent ADP-ribosyltransferase and NAD glycohydrolase activities of Bordetella pertussis toxin by adenine nucleotides, phospholipids, and detergents

Pertussis toxin catalyzed ADP-ribosylation of the guanyl nucleotide binding protein transducin was stimulated by adenine nucleotide and either phospholipids or detergents. To determine the sites of action of these agents, their effects were examined on the transducin-independent NAD glycohydrolase activity. Toxin-catalyzed NAD hydrolysis was increased synergistically by ATP and detergents or phospholipids; the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) was more effective than the nonionic detergent Triton X-100 greater than lysophosphatidylcholine greater than phosphatidylcholine. The A0.5 for ATP in the presence of CHAPS was 2.6 microM; significantly higher concentrations of ATP were required for maximal activation in the presence of cholate or lysophosphatidylcholine. In CHAPS, NAD hydrolysis was enhanced by ATP greater than ADP greater than AMP greater than adenosine; ATP was more effective than MgATP or the nonhydrolyzable analogue adenyl-5'-yl imidodiphosphate. GTP and guanyl-5'-yl imidodiphosphate were less active than the corresponding adenine nucleotides. Activity in the presence of CHAPS and ATP was almost completely dependent on dithiothreitol; the A0.5 for dithiothreitol was significantly decreased by CHAPS alone and, to a greater extent, by CHAPS and ATP. To determine the site of action of ATP, CHAPS, and dithiothreitol, the enzymatic (S1) and binding components (B oligomer) were resolved by chromatography. The purified S1 subunit catalyzed the dithiothreitol-dependent hydrolysis of NAD; activity was enhanced by CHAPS but not ATP. The studies are consistent with the conclusion that adenine nucleotides, dithiothreitol, and CHAPS act on the toxin itself rather than on the substrate; adenine nucleotides appear to be involved in the activation of toxin but not the isolated catalytic unit.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Transient kinetics of a cGMP-dependent cGMP-specific phosphodiesterase from Dictyostelium discoideum

Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in approximately 30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme is activated about fourfold by low cGMP concentrations. The phosphodiesterase has two distinct cGMP-binding sites: a catalytic site and an activator site. cAMP does not bind to either site; inosine 3',5'-monophosphate (cIMP) binds only to the catalytic site, whereas 8-bromoguanosine 3',5'-monophosphate (c-b8-GMP) preferentially binds to the activator site. For detailed kinetical measurements we have used [3H]cIMP as the substrate and c-b8-GMP as the activator. c-b8-GMP activated the hydrolysis of [3H]cIMP by reducing the Km, whereas the Vmax was not altered. The hydrolysis of [3H]cIMP was measured at 5-s intervals by using a new method for the separation of 5'-nucleotides from cyclic nucleotides. The hydrolysis of [3H]cIMP by nonactivated enzyme or by preactivated enzyme was linear with time, which indicates that a steady state is reached at the catalytic site within 5 s after addition of the substrate. In contrast, the hydrolysis of [3H]cIMP immediately after activation by 0.1 microM c-b8-GMP was not linear with time, but increased in a quasi-exponential manner with a time constant of 21 s. This suggests that a steady state at the activator site is only reached in 30-45 s after addition of the activator. The on-rate of activation (k1) was 3 X 10(5) M-1s-1 for c-b8-GMP and 1.4 X 10(5) M-1s-1 for cGMP. The off-rate of activation (k-1) was 0.03 s-1 for both c-b8-GMP and cGMP. The significance of these kinetic constants for the chemoattractant-mediated cGMP response in vivo is discussed.     

Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.