Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the branching enzyme

A branching enzyme was extracted from the mycelia of Neurospora crassa and was purified to electrophoretic homogeneity by procedures including DEAE-Sephacel column chromatography, 6-aminohexyl-Sepharose 4B column chromatography and gel filtration on Toyopearl HW-55S. The final yield of the branching enzyme activity was 15.1%, and the final purified enzyme preparation showed a specific activity of 702 units per mg of protein. The molecular weight of this enzyme was estimated to be 80,000 by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel. The amino acid composition and the carbohydrate content of this enzyme were analyzed. The isoelectric point of this enzyme determined by polyacrylamide gel isoelectrofocusing was 5.6. The branching activity of the enzyme was confirmed by its action on amylopectin as well as by the combined action of this enzyme and N. crassa glycogen synthase. The action of this enzyme on amylopectin decreased the wavelength of the absorption maximum of the glucan-iodine complex, and increased the amount of the short unit chains of the debranched product. The product obtained by the combined action yielded beta-limit dextrin upon hydrolysis with beta-amylase. No multiplicity was found for the branching activity either by chromatography or by electrophoresis.     

Amino acid sequences of the tryptic peptides from carboxymethylated L-asparaginase from Escherichia coli.

S-Carboxymethylated L-asparaginase was digested with trypsin and the resulting peptides were isolated by using gel filtration, ion exchange column chromatography and paper chromatography. Among the peptides thus isolated, 27 peptides were considered not to overlap and the sum of the amino acids from these 27 peptides is in good agreement with amino acid composition of the enzyme. The amino acid sequences of the peptides were determined by fragmentation with various enzymes and subtractive Edman degradation.     

Studies of immunosuppression by cobra venom factor. I. On early IgG and IgM responses to sheep erythrocytes and DNP-protein conjugates.

The immunosuppressive activity of CVF was evaluated in mice immunized with sheep erythrocytes and dinitrophenylated proteins. Serum antibody levels to these immunogens were estimated in activity units and on a weight basis for IgG. IgM as well as IgG antibody responses were diminished in mice pretreated with CVF. However, when soluble immunogens were incorporated in CFA the suppressive effect associated with CVF was inapparent. It is suggested that C depletion per se may not fully account for the observed immunosuppression. The latter may result not only from the depression of C3 levels but also from the biologic activities of C cleavage products some of which modulate the secretory functions and state of activation of macrophages.     

STIMULATION OF BRAIN ACETYL-CoA HYDROLASE BY ATP AND ATP ANALOGUES

The effects of ATP and ATP analogues on the brain acetyl-CoA hydrolase (EC 3.1.2.1) were studied. The enzyme was stimulated reversibly by ATP-Mg²⁺ the presence of Mg²⁺ being absolutely required for the stimulation. The stimulatory effect of ATP was highly specific since adenine nucleotides other than ATP had no stimulatory effects and nucleoside triphosphates other than ATP stimulated the enzyme much less than ATP in the following order: ATP > ITP CTP, UTP GTP. A phosphate modified analogue of ATP, AMP-PNP had a similar stimulatory effect to that of ATP. Other ATP analogues such as AMP-PCP and AMPCPP showed less stimulatory effect than ATP. The order of the stimulatory effects of these ATP analogues was: ATP > AMP-PNP > AMP-PCP > AMPCPP. The concentrations needed for half-maximal stimulation of ATP, AMP-PNP and AMP-PCP were approx 0.11 mm , 0.22 mm, and 0.22 mm , respectively. Double reciprocal plots demonstrated that ATP as well as AMP-PNP produced a significant decrease in the apparent K_m, value for acetyl-CoA and an increase in V_max indicating that these nucleotides increased the affinity for acetyl-CoA through binding at a site other than the catalytic site. The data described above suggest that the rate of hydrolysis of acetyl-CoA may be regulated by the concentration of ATP in the micro-environment of the enzyme.     

Separation of two different heavy meromyosins. Evidence for the presence of myosin isozymes in rabbit skeletal muscle.

Heavy meromyosin prepared from rabbit skeletal myosin by chymotryptic digestion was separated into two different heavy meromyosins by Sepharose 4B-6 aminohexyl PPi column chromatography. SDS-gel electrophoresis of one fraction of heavy meromyosin, which was eluted with 75 mM ammonium acetate, showed that it contained the small polypeptide chains, g3 and g2, as well as the large chains. The other fraction of heavy meromyosin, which was eluted with 85 mM ammonium acetate, contained g1 and g2. We concluded that the two heavy meromyosins arose from two different populations (isozymes) of myosin. No significant difference in Ca2+-ATPase activity was detected between the two heavy meromyosins.     

Reversible inhibition of adenylate cyclase activity of rat brain caudate nucleus by oxidized glutathione

Basal and dopamine-stimulated adenylate cyclase (EC 4.6.1 1.) activities were strongly inhibited by GSSG, but not by GSH. Adenylate cyclase that had been inactivated by GSSG was reactivated by incubation with various sulfhydryl compounds including GSH. Formation of mixed disulfides by reaction between GSSG and protein-SH groups increased on incubation with GSSG and returned to the normal level on subsequent incubation with DTT.     

Acetolysis of Leuconostoc mesenteroides NRRL B-1299 Dextran. Isolation and characterization of tetrasacharrides containing secondary linkages from the borate-insoluble fraction

Fractionation of the deacetylated acetolyzate of the borate-insoluble fraction of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 gave five tetrasaccharide fractions, isolated after chromatography on charcoal—Celite, paper chromatography, and paper electrophoresis. Examination of partial acid hydrolyzates of the tetrasaccharide fractions and their corresponding alditols, the relation between the logarithm of their partition functions (α') and molecular size, and methylation studies, showed them to be (a) 2³-α-d-glucosyl-nigerotriose (1), (b) a mixture of 6-α-nigerotriosyl-d-glucose (2) and 6¹-α-d-glucosyl-nigerotriose (3) and/or 6²-α-d-glucosyl-nigerotriose (4), (c) a mixture of 2¹-α-nigerosyl-isomaltose (5) and 3²-α-isomaltosyl-kojibiose (6) and/or 6²-α-nigerosyl-kojibiose (7), (d) 2-α-nigerotriosyl-d-glucose (8) and (e) nigerotetraose (9).     

Studies on urinary kallikreins. I. Purification and characterization of human urinary kallikreins.

Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A-50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2 and -3 were estimated to be 2.7 X 10(4), 2.7 X 10(4), and 2.9 X 10(4), respectively, by gel filtration on a Sephadex G-100 column. The optimum pH's of HUK-1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to by 1.17 X 10(4), 5.1 X 10(3), and 1.45 X 10(4) cal per mole, respectively. The Km values were estimated toward N-alpha-tosyl-L-arginine methyl ester (TAME), N-alpha-benozyl-L-arginine ethyl ester (BAEE), and N-alpha-benozyl-L-arginine methyl ester (BAME).     

Voltage-dependent capacitance as a probe for albumin adsorption onto a solid surface

The process of adsorption of bovine serum albumin onto a platinum electrode was monitored through the measurement of a nonlinear electrochemical property. The principle of the new method is that a sinusoidal voltage source is applied to a test solution and the waveform of the output current is analyzed by Fourier transformation. It was found that the intensities of the higher harmonics in the Fourier transformation change depending on the concentration of albumin and with time. From the higher harmonics, voltage dependence of the capacitance was quantitatively evaluated. The change of the state of albumin adsorbed onto the platinum plate was also monitored from the pattern of 'crack' of adsorbed albumin by using scanning electron microscopy. These results were discussed in relation to the mechanism of bimodal adsorption of albumin.