Effect of various factors on the activity of trehalase from the larvae ofSesamia inferens walker (Insecta)

Summary Trehalase from the salivary glands and the midgut ofSesamia inferens showed optimum activity at pH 5.8, and at temperatures of 50 and 60°C respectively. The increase in the incubation period, enzyme concentration, and substrate concentration respectively increased the end-product, the hydrolysis, and the rate of hydrolysis of the substrate. Dialysis did not affect, tryptophan accelerated, and other amino acids and end-product inhibited the enzyme activity.The author wishes to express his thanks to ProfessorR. Rakshpal for his constructive criticism, and to University Grants Commission, India, for awarding a Junior Research Fellowship.     

Determination of the kinetic parameters of enzyme inhibition of the K-type mechanism: application to the control of alpha-glucosidase activity of honeybee hemolymph by glucose]

The alpha-glucosidasic activity of emerging honeybees haemolymph is submitted to a feed-back inhibition by glucose, according to a mechanism of the "K" type (competitive). The "resulting affinity-constant" (measured in the presence of the enzyme both with substrate and inhibitor) is linear function of the inhibitor concentration. The affinity constants between enzyme and pure substrate on one hand, and between enzyme and pure inhibitor on the other hand, were determined by means of this relation, which led to respectively equivalent values after determinations under in vitro or in vivo inhibitions.     

A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet

Summary A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.     

A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet

A strong fibrinolytic activity was demonstrated in the vegetable cheese Natto, which is a typical soybean food eaten in Japan. The average activity was calculated at about 40 CU (plasmin units)/g wet weight. This novel fibrinolytic enzyme, named nattokinase, was easily extracted with saline. The mol. wt and pI were about 20,000 and 8.6, respectively. Nattokinase not only digested fibrin but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251), which was more sensitive to the enzyme than other substrates tried. Diisopropyl fluorophosphate and 2,2,2-trichloro-1-hydroxyethyl-o,o-dimethylphosphate strongly inhibited this fibrinolytic enzyme.     

Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase

Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.     

Debriding ability of a novel multi-enzyme preparation isolated from Antarctic krill (Euphausia superba)

Summary The wound-debriding activity of various types of proteolytic enzymes and proteases from Antarctic krill (multi-enzyme system consisting of both endo- and exopeptidases) was evaluated. The results, based on the enzymatically acieved weight reduction of a necrotic animal material (excised rat skin) in vitro, clearly showed that the multi-enzyme system (krill) had a higher degrading activity than the single enzyme preparation, or that with only a few enzymes. The debriding effect of the krill enzymes was markedly related to the enzyme concentration, resulting in 70–100% substrate degradation after 24 h. The digesting capacity of trypsin reached about 50%, but an increase in concentration of this enzyme did not substantially influence its overall activity. The effect of streptokinase-streptodornase, collagenase and plasmin-desoxyribonuclease was weak (10–20% digested).     

Mixed "n" type inhibition of alpha-glucosidase activity by glucose in hemolymph of honeybee prenymphs]

Under in vitro inhibition of alpha-glucosidasic activity by glucose in hemolymph of Bee prenymphas, the reaction order (n) (predetermined according to the initial natural glycemia) decreases with increasing inhibitor concentration and the affinity constant between enzyme and substrate undergoes lower variations than in other cases where (n) does not change. The study of inhibited molecular forms suggests the possibility of a gradual negative-cooperativity mechanism concerning a dimeric form of the enzyme.     

Die Bedeutung der Mitochondrienstruktur für die Zitronensäuresynthese

Summary Rat liver mitochondria proved to need a structural integrity for optimal citric acid synthesis. Structural impairment leads to an inhibition of the synthetic activity of the mitochondria, the degree of inhibition being inversely related to substrate concentration; it causes, however, no inactivation of the condensing enzyme. The dependence of this reaction upon the structural conditions is the consequence of an interaction between the two mitochondrial enzymes acetyl-CoA deacylase and condensing enzyme.     

Effect of the ionic strength on the kinetic properties of the mitochondrial L-malate dehydrogenase

Increase of the ionic strength inhibits the catalytic activity of the mitochondrial MDH, reduces substrate inhibition and decreases the affinity of the substrates for the enzyme.     

On the possible mode of action of neurohormones on cholinesterase activity in the ventral nerve cord of scorpion,Heterometrus fulvipes

Summary It is shown that K_m of ChE is not affected by the neurohormones but V_max is increased and decreased in presence of acceleratory and inhibitory neurohormones respectively. Hence it is suggested that the neurohormones might modulate the enzyme activity by altering the maximal velocities (V_max) rather than affecting the enzyme affinity (K_m) towards the substrate.Acknowledgment. The present study was carried out at S. V. University, Tirupati, A. P. India, and the authors are highly beholden to Prof. K. S. Swami, Head of the Department of Zoology, S. V. University, for his kind help and encouragement. Thanks are also due to the University Grants Commission, New Delhi, India, for placing one of them (N. V.) as a National Research Associate which made the present work possible.     

Inhibition of rat liver transglutaminase by nucleotides

Summary Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Inhibition of rat liver transglutaminase by nucleotides.

Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Effect of the ionic strength on the kinetic properties of the mitochondrial L-malate dehydrogenase

Summary Increase of the ionic strength inhibits the catalytic activity of the mitochondrial MDH, reduces substrate inhibition and decreases the affinity of substrates for the enzyme.This work was supported by a grant from the Conselho Nacional de Desenvolvimento Tecnológico (CNPq), Brazil.     

Quantitative determination of the activity of acid peptidases of industrial origin]

Reagent ninhydrine-Cd++, reacts with free alpha and epsilon amino groups of proteins. Horse-heart apomyoglobin was subjected to exhaustive succinylation, rendering the product non reactive to ninhydrine. The succinylglobin was submitted to enzyme digestion at pH 2.0, 4.0, 4.7 and 6.0. The commercially available enzymes contain mainly pepsin-like and chymosin-like enzymes. The enzymatic digests of succinyl-globin contain new free alpha-amino groups reacting with ninhydrin. Enzymatic digestion was performed under various condition (ratio E/S, pH). The results were compared to those obtained with synthetic substrate: PRO-HIS-LEU-SER-PHE(NO2)-NLEU-ALA-LEU-OME. The price of the synthetic substrate used, was more than 100 times the cost of succinyl-globin, thus the use of this substrate is a valuable tool for the quantitative estimation of peptidase activity in commercially available (pepsin, chymosin-like) enzymes.     

Trypsin activity in the hepatopancreas ofMacrobrachium lamarrei (Crustacea: Decapoda)

Summary Trypsin from the hepatopancreas ofMacrobrachium lamarrei showed optimum activity at pH 7.5 and temperature 45°C. The enzyme activity increased with the increase in incubation period and enzyme concentration. Michaelis constant of the enzyme was 2.38×10^–2 M.Acknowledgments. The authors are thankful to University Grants Commission, India for the award of a Junior Research Fellowship.     

Trypsin activity in the midgut ofSarcophaga ruficornis andMusca domestica (Diptera: Insecta)

Summary The pH and temperature for the optimum activity of trypsin from the midgut ofSarcophaga ruficornis andMusca domestica was 7.5 and 8.0 respectively and 50°C. The enzymic activity increased with the increase in incubation period and enzyme concentration.Thanks are due to Prof.R. Rakshpal for guidance.     

Angiotensin I converting enzyme activity in pulmonary tissue of fetal and newborn rabbits

Summary Angiotensin I converting enzyme in pulmonary tissue of fetal and newborn rabbits was measured using Hip-His-Leu as substrate. Enzyme activity was detected in the late fetal period, increased gradually until birth and increased markedly after birth. Enzyme activity reached adult levels on the 2nd and 3rd day after birth. This observations suggests that the metabolic activity of the lung for angiotensin develops suddenly at the time of derivery.This work was supported in part by grant No. 187032 from Ministry of Education.     

A126 in the active site and TI167/168 in the TI loop are essential determinants of the substrate specificity of PTEN

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P₂ and PI(3,4,5)P₃. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P₂, PI(3,4)P₂, and PI(3,4,5)P₃. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTEN_CiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTEN_CiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P₃, but not toward PI(4,5)P₂. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P₂ and PI(3,4,5)P₃ of PTEN_CiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P₃ in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN’s substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.     

Is the basal activity of rat stomach histidine decarboxylase affected by antrectomy?

The serum gastrin concentration and the gastric histidine decarboxylase activity are high in freely fed, unoperated rats but low in antrectomized rats. Following food deprivation the serum gastrin level and the enzyme activity are reduced simultaneously in the unoperated rats. After fasting for 36-48 h - but not before - the enzyme activity drops to the same low levels as in antrectomized rats.     

Identification of type A monoamine oxidase in mouse and rabbit liver mitochondria

Summary Type A monoamine oxidase was identified in liver mitochondria of mouse and rabbit. 5-Hydroxytryptamine was a common substrate for type A and type B monoamine oxidase in these enzyme preparations where its concentration was 1.0 mM.