Purification of the glutamate decarboxylase from human brain]

A method of purifying the glutamate decarboxylase from human brain is described. The enzyme was purified 8 000 fold in regard to the initial homogenate and appears homogenous by electrophoresis, both in denaturing and non-denaturing conditions. The molecular weight of the native enzyme and its subunits indicate that GAD from human brain is formed by two similar if non identical polypeptide chains. The Km for glutamate and pyridoxal phosphate found for the human enzyme, respectively 1,2.10(-3) M and 0,13.10(-6) M, are close to the Km found for the Mouse enzyme.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Summary Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000×g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closley related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.We would like to thank Miss S. Fukushima for technical assistance.     

Action of iodine on the tomato pectinesterase

Summary The organophosphates and carbamates did not inhibit the isolated tomato pectinesterase. Therefore this enzyme cannot be defined as serine-type esterase. The enzyme is inhibited by iodine and the inhibition (irreversible and non-competitive) is dependent on the degree of enzyme purification.     

Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

Summary The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu²⁺ and Fe²⁺. The rate of H₂O₂ formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na⁺ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.     

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.     

Immunological studies on the key enzyme of arginine biosynthesis in Pseudomonas aeruginosa.

A method to manufacture specific antisera with a minute amount of prue enzyme is presented. The influence of antibodies on activity and inhibition of an allosterically regulated enzyme was studied.     

Purification of calf thymus adenosine diphosphate ribose polymerase]

Calf thymus poly ADPR polymerase has been purified to electrophoretic homogenity. The enzyme has a molecular weight of 120,000 +/- 10,000 dalton. The substrate affinity is very high (apparent Km 82.5 micrometer). The presence of exogenous DNA does not appear to be a requisite for enzymatic activity of the purified enzyme.     

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).     

Age-dependent response of lactate dehydrogenase of pituitary of rat to testosterone

Summary The effects of time and various doses of testosterone on the responsiveness of lactate dehydrogenase of pituitary of 7-, 38- and 78-week-old rats were studied. The activity of the enzyme increases in 78-week-old rats. Castration decreases the enzyme activity at all ages. Maximum increase in the enzyme activity is seen with 50 and 100 g of testosterone 4 h after administration of hormone to castrated rats. No further time and dose-dependent effect is observed. The magnitude of increase for the enzyme is higher at the age of 38 weeks and decreases in 78-week-old rats.To whom all correspondence should be addressed. The authors are grateful to Professor M.S. Kanungo, Biochemistry Laboratory, Department of Zoology, Banaras Hindu University for the suggestions and encouragement. K.V.G. thanks the PL-480 for a research fellowship.     

What’s new in the renin-angiotensin system?

Angiotensin-converting enzyme 2 (ACE2) is a recently discovered homologue of the key enzyme of the renin-angiotensin system, the angiotensin-converting enzyme. The ACE2 enzyme is mainly expressed in cardiac blood vessels and tubular epithelia of the kidneys. Together with ACE2's unique metallocarboxypeptidase activity, the restricted tissue distribution suggests a distinctive physiological function in blood pressure, blood flow and fluid regulation. The ace2 gene was mapped to quantitative trait loci affecting susceptibility to hypertension in rats. Furthermore, ACE2 appears to be a negative regulator of ACE in the heart. ACE2 messenger RNA and protein levels are substantially regulated in the kidney of diabetic and pregnant rats. The mechanism of ACE2 function and its physiologic significance are not yet fully understood; however, as ACE2 differs in its specificity and physiological role from ACE, this opens a new potential venue for drug discovery aimed at cardiovascular disease, hypertension and diabetic complications.     

On the distribution of plasma L-asparaginase

The guinea-pig, Cavia porcellus, is unusual in possessing plasma L-asparaginase, an enzyme with anti-tumor activity, 21 additional species have been examined as to the presence of this enzyme: the results confirm and extend its remarkably limited species distribution.     

Regional distribution of thiamin pyrophosphokinase in rat brain

The highest specific activity of thiamin pyrophosphokinase was found in the cerebellum, and lower activity in cerebral cortex and midbrain. The regional difference in the enzyme activity was similar to that in thiamin content and the influx rate in rat brain, suggesting that the enzyme is involved in the thiamin transport.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Affinity chromatography of glucose dehydrogenase

Porcine liver beta-D-glucose dehydrogenase, a multi-functional protein, has been purified to apparent homogeneity. The enzyme has been separated from the endoplasmic reticulum using Triton X-114 and further purified using NAD to release glucose dehydrogenase from a NADP-linked sepharose column. The purified enzyme is capable of producing both NADH and NADPH in vivo as indicated by kinetic studies.     

Administration of D-alanine did not cause increase of D-amino acid oxidase activity in mice.

D-amino acid oxidase (DAAO) activity was not altered in the liver and kidney by oral administration of D-alanine to adult mice. The enzyme was apparently not induced by the enteric microflora either, since the enzyme activity in the liver and kidney of germ-free mice was not different from that of specific-pathogen-free mice. The times of appearance of DAAO activity and of free D-amino acids in the kidney were elucidated using suckling mice. DAAO activity started to increase 7 days after birth, and reached almost the adult level by 28 days. The content of free neutral D-amino acids also increased with age, in a similar fashion. A possible conclusion is that the enzyme activity normally increases during this period, to eliminate the free D-amino acids which have increased with age in the suckling mice. Consequently, the administration of D-alanine had no further effect in increasing enzyme activity.     

Regional distribution of thiamin pyrophosphokinase in rat brain

Summary The highest specific activity of thiamin pyrophosphokinase was found in the cerebellum, and lower activity in cerebral cortex and midbrain. The regional difference in the enzyme activity was similar to that in thiamin content and the influx rate in rat brain, suggesting that the enzyme is involved in the thiamin transport.