Immobilization of lactate dehydrogenase on polyacrylamide beads

Pig muscle lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) was covalently immobilized on polyacrylamide beads containing carboxylic functional groups activated by water-soluble carbodiimide. The effects of immobilization on the catalytic properties and stability of the lactate dehydrogenase were studied. There was no shift in the pH optimum of the immobilized enzyme compared to that of the soluble one. The apparent optimum temperature of the soluble enzyme was 65 degrees C, while that of the immobilized enzyme was between 50 and 65 degrees C. The apparent Km values of the immobilized enzyme with pyruvate and NADH substrates were higher than those of the soluble enzyme. As a result of immobilization, enhanced stabilities were found against heat treatment, changes in pH, and urea denaturation.     

The effect of neutral and charged micelles on the acid-base dissociation of the local anesthetic tetracaine

The influence of surfactant micelles on the acid-base dissociation of the charged tertiary amino group of the local anesthetic, tetracaine, has been investigated. From measurements of tetracaine fluorescence as a function of bulk pH, apparent $\text{p}\text{K}$ values of 6.88, 7.58 and 9.92 were found in the presence of cationic, neutral and anionic micelles, respectively, in 10 mM NaCl. These values are considerably displaced with respect to the $\text{p}\text{K}$ in aqueous solution which is 8.26. Such large shifts can be attributed to the effect of the surface polarity and electrical potential on the dissociation behavior of the anesthetic bound to micelles. It can be expected that the acid-base dissociation of a local anesthetic adsorbed to nerve fibers will also be affected by the properties of the membrane surface. Thus, it is suggested that the influence of the interfacial region on the $\text{p}\text{K}$ of surface-bound molecules should not be disregarded when estimating the proportion of charged and uncharged forms of local anesthetics interacting with axonal membranes.     

Nitrogen Source Regulates Glutamate Dehydrogenase NADP Synthesis in Neurospora crassa

Neurospora crassa glutamate dehydrogenase-NADP (EC 1.3.1.3) has a higher activity when mycelium is grown on ammonium or nitrate as nitrogen source than when grown on glutamate or glutamine. Quantitative immunoelectrophoresis established that, under all conditions, enzyme activity corresponded to enzyme concentration. Isotope incorporation studies demonstrated that the nitrogen source exerts its regulation at the level of de novo enzyme synthesis.     

Homology and repair of UV-irradiated plasmid DNA in haemophilus influenzae

UV-irradiated plasmid pNov1 containing a cloned fragment of chromosomal DNA could be repaired by excision, but plasmid p2265 without homology to the chromosome could not. Establishment of pNov1 was more UV resistant in Rec⁻ than in Rec⁺ cells.     

Synthesis and hydrolysis of 1,3-beta-xylosidic linkages by endo-1,4-beta-xylanase of Cryptococcus albidus

Purified extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) of the yeast Cryptococcus albidus was found to catalyze not only the known 1,4-beta-transfer, but an alternative transglycosylation reaction leading to the formation of 1,3-beta-glycosidic linkages. From a mixture of products of beta-xylanase degradation of phenyl beta-D-xylopyranoside three xylooligosaccharide fractions, differing chromatographically from the 1,4-beta-linked products, were isolated by preparative paper chromatography. Their structure was elucidated by mass spectrometry, 13C-NMR spectroscopy and enzymic hydrolysis by beta-xylanase and beta-xylosidase. The isomeric xylotriose was identified as 3-O-beta-D-xylopyranosyl-4-O-beta-D-xylopyranosyl-D-xylose. The fraction of isomeric tetrasaccharides was found to be represented mainly by 4-O-beta-D-xylopyranosyl-3-O-beta-D-xylopyranosyl-4-O-beta-D-xylopyranosyl- D-xylose. The xylooligosaccharides containing one 1,3-beta-linkage were also produced on the enzyme treatment of 1,4-beta-xylotriose and 1,4-beta-xylan. When treated with the enzyme responsible for their synthesis, the isomeric xylooligosaccharides were hydrolyzed at the 1,3-beta-linkage, despite the fact the enzyme does not attack 1,3-beta-xylan. The results are interpreted in the relation to the characterized four-subsite substrate-binding site of the enzyme.     

Trypanosoma cruzi: 4-aminopyrazolopyrimidine in the treatment of experimental Chagas' disease

An allopurinol metabolite, 4-aminopyrazolopyrimidine, was tested on two different strains of mice (NMRI-IVIC and C57Bl/6J) that had been infected 4 days earlier with the virulent Ya strain of Trypanosoma cruzi. Low doses of 4-aminopyrazolopyrimidine (0.125-0.500 mg/kg body wt/day) for 10 days induced a significant reduction in parasitemia (direct counts and subinoculation experiments) and increased survival time (without any evidence of toxicity) compared with untreated animals. When tested in vitro, 4-aminopyrazolopyrimidine was sixfold more active than allopurinol as a trypanostatic drug. The low therapeutic doses of 4-aminopyrazolopyrimidine suggest that this drug may be useful in the treatment of acute Chagas' disease.     

Growth rate,sugar consumption,chlortetracycline production and anhydrotetracycline oxygenase activity inStreptomyces aureofaciens

The relationship between the activity of ATC oxygenase, CTC production and growth rate was investigated in a low-producing strain ofStreptomyces aureofaciens, closely related to the wildtype strain, and in a higher-producing variant. Different growth rates were achieved by using glucose, fructose and sucrose as carbon sources. Activity of the enzyme and CTC yield in both strains were inversely proportional to the rate of sugar utilization but in the higherproducing variant sugar utilization was slower than in the low-producing strain. The expression of ATC oxygenase was less sensitive to “catabolite repression” in the higherproduc ing strain. BT, a stimulator of CTC production, markedly inhibited growth of the higher-producing variant in a medium with slowly utilized sugars (fructose and sucrose) but had little effect on growth of the lowproducing strain. It also increased the level of ATC oxygenase in both strains under all experimental conditions. It could be established that there was no obligatory relationship between the increase of antibiotic synthesis and the increase of enzyme activity.     

Effect of the polysaccharide ofAgrobacterium radiobacter on the growth of plants and occurrence of damping-off in sugar beet

Agrobacterium radiobacter, strain B6 (a strain isolated in this laboratory, which limited the occurrence of damping-off of sugar beet and influenced growth of plants in hot-house and field experiments) was found to produce an acidic exopolysaccharide in a mineral medium with various carbon sources. Hydrolyzates of the polysaccharide contained glucose, galactose, glycerol, succinic acid and pyruvic acid, whose quantitative content varied according to the carbon source used. The polysaccharide isolated from the medium containing glucose exhibited the highest physiological activity. Seeds germinated best and sugar beet roots were found to grow most rapidly in a medium containing 0.2 % (W/W) of the polysaccharide. The roots exposed for 3 d in this medium grew 2.7-fold as compared with non-treated plants. Higher sumbers of microorganisms were detected on the surface of roots treated with the polysaccharide. Growth of roots was also stimulated when immersing the seeds (30 min) in a 0.2 –0.4 % solution of this polysaccharide. After a two-fold treatment the roots were less damaged by the fungusPythium ultimum. Plants from seeds treated with the polysaccharide grew in the field soil more rapidly than the non-treated plants but worse than after bacterization of the seeds byA. radiobacter B6 and were only partially protected against the damping-off of sugar beet.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.