Galactosyltransferase of Neurospora

Summary An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 xg. The enzyme assayed in the 100,000 xg supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.Acknowledgment. This work was supported by a grant from the Research Corporation.     

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.     

On the metabolism of prostaglandins by rat brain homogenate

Summary Using radio-immuno assays for prostaglandins and prostaglandin metabolites, three prostaglandin metabolizing enzymes were found in the 100,000×g supernatant of rat brain, 15-hydroxy-prostaglandin-dehydrogenase, ¹³ and prostaglandin E-9-keto-reductase. Specific activity of the latter enzyme was highest in striatum and midbrain and lowest in cortex, cerebellum and spinal cord.This work was supported by the Deutsche Forschungsgemeinschaft (SFB No. 70).     

Dipeptidyl aminopeptidase in neonatal rat brain regions

Summary Changes in the activity of dipeptidyl aminopeptidase in rat pituitary gland and various brain regions were examined at 3, 5 and 20 weeks of age. The enzyme activity per g tissue wet weight in pituitary gland was the highest of all tissues studied. Subcellular distribution of the activity was also studied. The highest enzyme activity was found in a crude mitochondrial fraction including synaptosomes.     

Effect of topically applied phenobarbital on O-dealkylase activity in mouse skin

Summary Topical application of phenobarbital to mice produces an increase in cutaneous microsomal proteins and apparently also of cytochrome P450, but results in a kinetically noncompetitive inhibition of p-nitrophenethole O-dealkylase activity in preparations of 10,000×g supernatant from skin.     

Effect of benzimidazole on nicotinamide adenine dinucleotide phosphate phosphomonoesterase activity in wheat leaves.

Nicotinamide adenine dinucleotide phosphate phosphomonoesterase was isolated and partially purified from wheat (Triticum aestivum L. var. Selkirk) leaves. The enzyme had KNADP value of 1.4 X 10(-4) M and a pH optimum of 5.9. In vitro activity of this enzyme was unaffected by precursors of NAD (nicotinamide and nicotinic acid) or cytokinins (kinetin and benzimidazole). However, when detached wheat leaves were treated with solutions of these compounds, the precursors lowered the specific activity while the cytokinins enhanced the activity. It is suggested that spatial separation and compartmentation of the enzyme and its substrate NADP account for the similar effect of benzimidazole on both.     

Evidence of protein kinase activity in 2 murine oncornaviruses]

A protein kinase activity has been detected in two strains of murine Oncornaviruses, MSV/MLV and EFV. This activity phosphorylates not only endogenous viral proteins but also exogenous substrates (histones and phosvitin). The stimulation of enzyme activity by detergents along with the increase of specific activity in viruses treated with trypsin during purification suggest that the enzyme is located in the viral particle.     

The polyphosphate synthetase of Saccharomyces cerevisiae]

The polyphosphate-synthetase, isolated from a homogenate of phosphate starved cells, catalyses the synthesis of linear polyphosphates from orthophosphate. It is localized in the membrane fraction which deposits between 400 and 1000 X g; its optimal pH is 7.1; its KM toward orthophosphate is 4.0 X 10(-4) M; ATP stimulates the reaction. The enzyme synthezises especially polyphosphates with short chain length.     

The absence of gangliosides in a higher plant

Summary We report attempts to isolate and purify sialic acid-containing glycolipids (gangliosides) from etiolated hypocotyls of soybean (Glycine max) using methods developed for rat liver. The maximum amounts of ganglioside sialic acid present was found to be less than. 0.021 nmole/g fresh weight or less than 1:100,000 the amounts present in rat liver. We conclude that this tissue lacks gangliosides.     

Purification of 20α-hydroxysteroid dehydrogenase from human erythrocytes

Summary 20-hydroxysteroid dehydrogenase was found in the supernatant after hemolysis of human erythrocytes, and the enzyme was isolated from the membrane-free hemolysate on a DEAE-cellulose column. The NADPH-generating system was essential for the reaction.Acknowledgments. We wish to thank Dr A. Tomoda, Department of Biochemistry, Kanazawa University School of Medicine, and Dr T. Yubisui, Department of Biochemistry, Medical College of Oita, for their advice and useful discussions.     

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.     

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.     

Model of the interaction between trypsin and alpha 2-macroglobulin of porcine serum]

A comparative study of the dissociation into subunits of Porcine alpha2 M, either native or bound to trypsin (Tn), has been carried out in order to determine the modifications of the alpha2 M structure due to the formation of the Tn-alpha2 M complex. Analytical ultra-centrifugation at pH 3.5 shows that the dissociation is smaller when alpha 2 M is bound to trypsin. Electrophoresis in 4% polyacrylamide gels, in presence of 0.1% SDS, of alpha2 M and Tn-alpha2 M incubated in 1% SDS leads to the same conclusion; the enzyme must stabilize the quaternay structure of alpha2 M. In presence of SDS + beta-mercaptoethanol, only a molecular weight (M.W.) 200,000 band is revealed in electrophoresis pattern of native alpha2 M. In the case of reduced Tn-alpha2 M, some other bands of M.W. 100,000, 50,000, 30,000 appear. When trypsin is inactivated by TLCK 100,000 M.W. band is present, accompanied by the 200,000 M.W. band whose intensity is function of the alpha2 M concentration. The 100,000 M.W. band appears therefore characteristic of the formation of the complex which must imply a proteolytic cleavage in the middle of the 100,000 polypeptidic chain of alpha2 M. A model of the complex is proposed in which the enzyme forms a proteic bridge between the two halves of the alpha2 M molecule.     

An alkaline protease in the delayed hypersensitivity skin lesions in the guinea-pigs.

An alkaline hemoglobinolytic protease was extracted from the delayed hypersensitivity skin lesions induced by bovine gamma-globulin as an antigen in the guinea-pig. The enzyme was heat-labile and inhibited by thiol-blocking reagents. The mol.wt. was more than 100,000 and optimal pH around 9.     

Monoclonal antibodies to L-asparaginase

Five hybridomas secreting monoclonal antibody to E. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G1 (1 clone), Ig G2 (2 clones) and Ig G3 (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (Kd) ranging between 2.5 X 10(-9) M and 6.3 X 10(-10) M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Effect of lymphokines produced in a grafted system on production of prostaglandins by macrophages]

Mice peritoneal macrophages cultured in vitro release a prostaglandin-like activity as evaluated by a bio-assay using Rat stomach fundus. The prostaglandin release is greatly increased when macrophages are incubated with the supernatant of mixed lymphocyte cultures between recipient and donor of a skin allograft. This phenomenon was found in all strains of Mice tested except C3H/HeJ Mice, a strain already known for its defective responsiveness to bacterial lipopolysaccharide (LPS).     

Lactate dehydrogenase activity in cell subpopulations of 7,12-dimethylbenz(a)anthracene-induced mammary tumors

Heterogeneity of cells in a solid tumor prevents direct assessment of enzyme activity of cell subpopulations by conventional homogenization techniques. Lactate dehydrogenase (LDH) activity before and after ovariectomy and following estrogen supplementation in density-defined cell subpopulations from DMBA-induced mammary tumors was determined. Different levels of LDH activity were found in different cell subpopulations. After ovariectomy the level of LDH activity declined. Restoration of the circulatory estrogen level resulted in increased enzyme activity. The highest level of LDH was found in a band which consisted mainly of poorly differentiated cells. This cell subpopulation also tended to be more responsive to endocrine manipulation.     

Zinc deficiency and lung converting enzyme activity in rats.

Angiotensin converting enzyme activity was found to be significantly decreased in the isolated perfused lung from zinc-deficient rats when compared with that of controls. Addition of zinc ion to the superfusion medium did not cause a recovery in this decreased activity of the enzyme. It is postulated that zinc deficiency probably produces a structural change in the lung angiotensin converting enzyme.