Functional consequences of treating turkey liver fructose-1,6-bisphosphatase with penicillin G

Summary Treatment of turkey liver fructose-1,6-bisphosphatase with penicillin G progressively inactivated the enzyme and desensitized the enzyme toward high substrate inhibition. The treatment also led to reduced sensitivity to AMP inhibition and the loss of cooperative interaction among AMP-binding sites. These altered properties were not reversed by dialysis, but were prevented when treatment with penicillin G was performed in the presence of substrate.This work was in part supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA) and in part by Faculty Research Grant from Southern School of Pharmacy, Mercer University, Atlanta (Georgia, USA).     

Irreversible inactivation of yeast glucose-6-P dehydrogenase by penicillin G

Summary Yeast glucose-6-P dehydrogenase is irreversibly inactivated by penicillin G. Kinetic data show that 1 molecule of penicillin G reacts with each active unit when the enzyme is inactivated The rate of inactivation increases greatly with increasing pH. This irreversible inactivation by penicillin G is largely prevented by pyridoxal-P, a reversible inactivator of this enzyme. Prior treatment of penicillin G with penicillinase totally abolishes its ability to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resouces, NIH (USA).     

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.     

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.     

Einfluß von Penicillin auf die Wurzelkultur(Zea mays) Nachweis von β-Indolylessigsäure (Heteroauxin) im Handelspenicillin

Summary (1) Pure Penicillin Sodium G (150 U/cm³) gives no inhibition in the growth-rate ofmays-roots in sterile organe culture.(2) Commercial penicillin (Penicillin Sodium Squibb, 5 U/cm³) inhibits the growth-rate ofmays-roots in a fairly high degree, due to its content of indole acetic acid. We have been able to separate the substance from the yellow dyes of commercial penicillin by chromatographic adsorption and to identify it colorimetrically as indole acetic acid.     

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.     

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.     

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.     

Purification and properties of ornithine aminotransferase from rat brain

Summary Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, K_m, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.Acknowledgments. D.R.D. is thankful to U.G.C., India, for the award of a fellowship under the special assistance programme. Present address: Department of Pediatrics and Communicable Diseases, F2815, Box 066, C.S. Mott Children's Hospital, University of Michigan, Ann Arbor, MI 48109, USA.     

Inhibition of sarcolemmal Na,K, Mg ATPase from the guinea pig heart is not compatible with a homogeneous population of non-interacting ouabain receptors

Summary The inhibition of sarcolemmal Na, K, Mg ATPase from the guinea pig heart by ouabain was evaluated with a coupled enzyme assay. Models of negative cooperativity and of two independent receptors fitted the inhibition data equally well. The analysis was not compatible with a homogeneous population of non-interacting ouabain receptors.Acknowledgments. The technical assistance of P. Mayr and G. Ruhland is gratefully acknowledged.     

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.     

Purification and properties of ornithine aminotransferase from rat brain

Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gamma gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, Km, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.     

Guinea pig liver cytosol has an L-threonine deaminase activity distinct from L-serine deaminase]

Using sodium sulfate precipitation, "Sephadex G200" gel filtration and polyacrylamide gel electrophoresis, a L-threonine desaminase was demonstrated in the Guinea-Pig liver cytosol. This enzyme was separated from the guinea pig liver L-serine desaminase possessing an auxiliary activity on L-threonine substrate described by us in a previous work. The optimals for pH (7,1) and temperature (+ 55 degrees C) and the apparent molecular weight (134,000 + 20,000) were established.     

Induction of IgG1 and IgE responses to protein-conjugated and unconjugated beta-lactam antibiotics in the mouse--efficacy of Freund's complete adjuvant

One or two injections two weeks apart of protein-conjugated penicillin G, cephalothin or cefmetazole emulsified with Freund's complete adjuvant were quite effective in producing anti-antibiotic antibodies of the IgE as well as of the IgG1 class in mice. Long-lasting and boostable production of both antibody classes was also obtained against unconjugated cephalothin or cefmetazole, though the positivity depended on the mouse strain.     

Potentiation of the inhibition of xanthine oxidase by a combination of 6-mercaptopurine and 6-thioguanine

Summary Carcinostatic agents, 6-mercaptopurine and 6-thioguanine inhibited the in vitro and in vivo activity of the enzyme xanthine oxidase (xanthine-oxygen oxidoreductase E.C. 1.2.3.2.). Simultaneous, addition of a mixture of the 2 antimetabolites produced a synergistic effect on the inhibition of the enzyme activity.We thank the University Grants Commission, New Delhi, for their financial assistance and the Dean, M.G.M. Medical College Indore, for providing necessary facilities.     

Induction of IgG1 and IgE responses to protein-conjugated and unconjugated β-lactam antibiotics in the mouse-efficacy of Freund's complete adjuvant

Summary One or two injections two weeks apart of protein-conjugated penicillin G, cephalothin or cefmetazole emulsified with Freund's complete adjuvant were quite effective in producing anti-antibiotic antibodies of the IgE as well as of the IgG₁ class in mice. Long-lasting and boostable production of both antibody classes was also obtained against unconjugated cephalothin or cefmetazole, though the positivity depended on the mouse strain.     

A propos d'antibiotiques insolubles

Summary The authors show the antibiotic activity of water-insoluble salts of hydrosoluble antibiotics in the case of penicillin G (salts of iron, copper, silver, gold, and uranium) and streptomycin (oleate).     

Heparin inhibition of the antithrombin activity of alpha-2-macroglobulin]

The interaction between thrombin and alpha-2-macroglobulin was studied on human purified materials, either in the presence or in the absence of heparin, by kinetic analysis of thrombin inhibition and polyacrylamide gel electrophoresis. In the absence of heparin, binding of thrombin to alpha-2-macroglobulin, shown by electrophoresis, leads to the loss of the coagulant property of the enzyme. In the presence of heparin the rate of inhibition of thrombin clotting activity by alpha-2-macroglobulin is strongly decreased. Heparin binds to thrombin, impairing the formation of thrombin-alpha-2-macroglobulin complex. These data show that heparin paradoxically protects thrombin from inhibition by alpha-2-macroglobulin whereas it increases the enzyme inhibition by antithrombin III. Such a phenomenon could be of practical interest for treatment of thrombosis in patients with high plasma level of alpha-2-macroglobulin and low level of antithrombin III, such as occurs in the nephrotic syndrome.