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).     

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

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 perfomed in the presence of substrate.     

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.     

Inactivation of yeast glucose-6-P dehydrogenase by aspirin

Summary Glucose-6-P dehydrogenase is irreversibly inactivated by treatment with Na salts of aspirin. Kinetic data show that 1 molecule of aspirin reacts with each active unit when the enzyme is inactivated. The rate of inactivation is enhanced with increasing pH but is reduced in the presence of glucose-6-P or NADP⁺. Na salicylate fails to inactivate the enzyme.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

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.     

Effect of light adaptation on electrical responses of the retinas of four species of bats

Summary The levels of light adaptation at which the retinas of 4 species of microchiropteran bats became unable to generate electroretinograms were progressively ordered. The order correlated well with light preferences based on activity patterns of the 4 species. These results suggest that the ability of the retina to function in ambient light may govern some natural behaviors of these bats.This work was supported in part by grant No. EY 01228 from the U.S.P.H.S. and was facilitated by equipment provided by Bob Hope Fight for Sight Award No. G522 and a grant from Research to Prevent Blindness, Inc. Portions of this work were presented at the Fifth International Bat Research Conference, Albuquerque, New Mexico, USA, August, 1978.     

Cell specific response of cardiac poly ADP-R and DNA synthesis to circulatory stress

Summary Aortic coarctation induces a large increase in poly ADP-R synthetase activity in non-cardiocyte nuclei, and in cardiocyte nuclei inhibition occures, suggesting a differentiation dependent regulation of polymer metabolism. In noncardiocyte nuclei DNA and poly ADP-R (polyadenosine diphosphoribose) synthesis exhibit positive correlation.Ackowledgment. This work was supported in part by the United States Air Force Office of Scientific Research (F-49620-81-C-007) and in part by a Program Project Grant of the United States Public Health Service (HL-24056). G. Jackowski is the recipient of a Postdoctoral Scholarship of the Canadian Heart Association and E. Kun is a Research Career Awardee of the United State Public Health Service.     

Modulation of 3-hydroxy-3-methyl glutaryl CoA reductase by 2,3-diphosphoglyceric acid

Summary Rat liver microsomal 3-hydroxy-3-methylgularyl CoA (HMG-CoA) reductase was activated by 50% at a concentration of 0.4 mM 2,3-diphosphoglyceric acid (DPG) and by 11-fold at 10 mM DPG. DPG also prevented the inactivation of HMG-CoA reductase by ATP and Mg⁺⁺. Rat liver microsomal HMG-CoA reductase prepared in the presence of 1 mM DPG was significantly more active than when prepared in the absence of DPG. Activation of the enzyme by DPG and protection of the enzyme against inhibition by ATP and Mg⁺⁺ by DPG were also observed with solubilized HMG-CoA reductase.This work was supported by Research Award # 697 G2-1 from the American Heart Association, Greater Los Angeles Affiliate, and by grant # 1R01 HL22672 from the National Institutes of Health. We thank M. Brun and M. Curtis for their excellent technical assistance.     

Inhibition of liver fructose 1,6-bisphosphatase activity by Zn2+: Reversal by imidazole pyruvate

Summary Imidazole pyruvate was found to be a very potent natural chelating agent in reversing the inhibition of liver fructose 1,6-bisphosphatase activity by Zn²⁺. This metabolite may play a physiological role in gluconeogenesis.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH, USA.     

Effect of pancreatic polypeptide on DNA-synthesis in the pancreas

Summary Bovine pancreatic polypeptide increases DNA-synthesis in the rat pancreas; no effect is observed in stomach (oxyntic area), duodenum or liver. BPP neither augments or inhibits the trophic action of cholecystokinin.Acknowledgment. Pure pancreatic polypeptide was donated by Dr. R. E. Chance (Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Ind, USA). G. R. Greenberg is supported by a Fellowship of the Medical Research Council of Canada.     

Suppressed collagenolytic activity in polymorphonuclear leucocytes from diabetic humans

Summary Extracts of polymorphonuclear leucocytes (PMNL) from diabetic human exhibited less collagenolytic activity than extracts from normoglycemic control subjects. Partially purified control extracts produced ^A and ^B collagen breakdown products of the type generated by mammalian collagenase; the diabetic preparation produced decreased amounts of the same products. The diabetic PMNLs may synthesize abnormally low levels of collagenase or contain inactive forms of this enzyme.Supported by a grant (No. DE-03987) from the National Institute of Dental Research (N.I.H.), USA. This study forms part of the Ph.D. thesis of G.A. Nicoll.Acknowledgments. The authors wish to thank Ms Salema Karim and Mr F.R. Singh for excellent technical assistance.     

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.     

Laccase production byPolyporus sanguineus under different nutritional and environmental conditions

Summary Laccase production was higher in malt extract medium than in lignin, andPolyporus sanguineus appears to be better thanPolyporus versicolor andTrametes hirsuta (syn.Polyporus hirsutus) for enzyme production. Phenolic compounds, of which resorcinol was the most active, induced enzyme production; while sugars repressed it. A temperature of 37°C, pH 3 and indulin AT at a concentration of 0.2% gave the best enzyme yield.A part of Ph. D. thesis of D.S. Arora.Acknowledgments. We thank Westvaco Chemical Division, USA, and Dr P.S. Rehill, Forest Research Institute, Dehradun, India for providing lignin samples and the fungal strain, respectively. D.S. Arora further thanks University Grants Commission, New Delhi for financial assistance.     

Effect of cyclophosphamide on development of reticulum cell sarcoma in SJL/J mice

Summary Studies to determine the effect of cyclophosphamide (CY) on the development of reticulum cell sarcoma (RCS) in SJL/J mice indicated a dependence on the duration of the test period. Age also appeared a factor of importance. Thus, a comparison of tumor incidences at 52 weeks of age showed maximal inhibition when CY was administered at 40 weeks, minimal inhibition when the drug was given at 30 weeks, and intermediate inhibition when treatment was initiated at 10 and 20 weeks. Consistent with these findings, long-term treatment of 40-week-old SJL/J mice with low doses of CY resulted in an increase in the mean survival time and in a reduction in the incidence of RCS. This work was supported in part by a University College Faculty Research Grant.     

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 neural tube closure by ionophore A23187 in chick embryos

Summary Ionophore A23187 inhibited closure of the chick neural tube through its effects on cytoskeletal components.This study was supported in part by grants from the Research Council and the Charles and Johanna Busch Memorial Fund of Rutgers University. Ionophore A23187 was provided through the courtesy of Dr R.L. Hamill, Lilly Research Laboratories, Indianapolis, Indiana, USA.     

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.     

Potentiation by crude kallikrein of the myotropic effect of angiotensin I in the isolated rabbit aortic strip

Summary Crude kallikrein (Padutin^®), but not pure kallikrein, when preincubated with angiotensin I caused a potentiation of the myotropic effect of decapeptide on the isolated continuously superfused rabbit aortic strip. Addition of converting enzyme inhibitor, SQ 20881, to the medium inhibited this potentiation. The potentiation by crude kallikrein of the myotropic effect of angiotensin I is probably due to the conversion of decapeptide to octapeptide angiotensin II. This study indicates that Padutin is not a pure kallikrein preparation and probably contains a kininase fraction which causes the conversion of angiotensin I.The authors are greatful to Prof. G. L. Haberland, Bayer AG, Elberfeld (BRD), for his generous gift of pure Kallikrein^® KZC 1/75; to Bayer, Leverkusen (BRD), for Padutin^® and to Squibb, New Jersey, USA, for SQ 20881. This work is supported in part by Eczacibai Research Foundation, Levent, Istanbul, Turkey. The technical assistance of Mr. M. Kabaçam is greatly appreciated.