Esterases in mycobacteria

Esterases ofMycobacterium phlei isolated by means of Sephadex G-100 chromatography could be temporarily activated by adding calcium ions. This activation could not be brought about in crude enzyme preparations from cells or in crude extracts from the culture filtrate. It was demonstrated that compounds (or a compound) without the esterase activity isolated after the separation of crude enzyme preparations on Sephadex G-100 (peak C) are responsible for the above difference. It was found that the suppression by compounds from the “C peak” of the temporary activation of esterases with calcium ions depends most probably on the ratio of these compounds to the quantity of the enzyme rather than on their concentration. In addition, the compounds of the C peak themselves were found to activate esterases. The activation was lower than the temporary activation with calcium ions. The possible mechanisms of the temporary activation of esterases and the importance of these findings, from the point of view of regulation of the activity of esterases during submerged cultivation ofMycobacterium phlei, are discussed.     

Relationship between the composition of the regulatory complex of esterases and the age ofMycobacterium phlei culture

During submerged cultivation ofMycobacterium phlei a mixture of macromolecular compounds ia released into the medium. Concentrated filtrates of cultures of different ages were separated on Sephadex G-25 fine and polyanions were found to predominate in the young culture. During further days of fermentation the proportion of polycations significantly increases. The results are discussed with respect to the regulatory complex of esterases.     

Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.     

Drosophila lactate dehydrogenase: Molecular and genetic aspects

(LDH) obtained from larvae of Drosophila melanogaster was purified to homogeneity by affinity chromatography on oxamate-Sepharose. The purification procedure is simple to operate and gives a homogeneous preparation in a good yield (34.86%) after only two steps. Utilizing the homogeneous LDH preparation, an attempt was made to characterize the LDH molecule. Thus, it was found that the N-terminal amino acid is isoleucine, and the enzyme is tetrameric and composed of four identical subunits of apparent molecular weight 38,000, suggesting that it is controlled by a single gene. Homogeneous LDH preparations exhibit one band on neutral acrylamide gels when the substrate is either dl-lactic acid or l-(+)-lactate. The optimum temperature is 45°C for the purified enzyme and 40°C for the crude homogenate. The K _m values for pyruvate and NADH are 0.154 and 0.027mm, respectively, while the K _m values for lactate and NAD are 29.4 and 1.33mm, respectively. A discontinuity in the E _a slope was observed at a transition temperature of 30°C. The E _a value between 20 and 30°C was calculated as 12.06 kcal/mol, while between 30 and 45°C the E _a value was 4.01 kcal/mol. This evidence, together with other observations reported in the literature, suggests that the LDHs of invertebrates and vertebrates have arisen by divergent evolution from a common ancestral gene.     

Polysaccharide production and the possible occurrence of GDP-d-mannose dehydrogenase inAzotobacter vinelandii

During the growth ofAzotobacter vinelandii in batch culture in Burk's 2% glucose medium supplemented with 50mg EDTA per litre, water-insoluble capsular polysaccharide material accumulated in cultures prior to the appearance of water-soluble polysaccharide in the culture medium. On isolation, hydrolysis and chromatography, both these polysaccharides were observed to be composed of carbohydrate monomers having the same chromatographic mobilities as glucose, rhamnose, guluronic acid and mannuronic acid. The activity of GDP-d-mannose dehydrogenase recorded in crude cell-free extracts fromAzotobacter vinelandii, when these polysaccharides were produced, may indicate a close similarity between the biosynthetic pathway of alginate synthesis in marine Phaeophyceae and this soil microorganism.     

Pseudomonas aeruginosa contains an inducible methylcitrate synthase

Assaying for enzymes of propionyl-CoA catabolism in crude extracts ofPseudomonas aeruginosa was complicated by the presence of a short-chain acyl-CoA hydrolase. Interference by the hydrolase was avoided by using permeabilized whole cells. Cells treated this way exhibited low or undetectable levels of propionyl-CoA carboxylase, propionyl-CoA dehydrogenase, and α-hydroxyglutarate synthase, enzymes initiating propionyl-CoA catabolism in many prokaryotes, but did contain high levels of methylcitrate synthase, an enzyme of the methylcitrate cycle for propionyl-CoA catabolism. Gel filtration experiments revealed a peak of methylcitrate synthase distinct from the citrate synthase of the bacterium. The enzyme was induced by growth on propionate and on propanol and heptanoate, precursors of propionate. These results suggest thatP. aeruginosa may utilize the methylcitrate cycle to metabolize propionate, a pathway heretofore only described in the yeastCandida lipolytica.     

Esterases in mycobacteria

Esterases ofMycobacterium phlei (acetic ester acetyl hydrolase E.C.3.1.6 and carboxylic esterhydrolase E.C.3.1.1.1.) obtained after separation on Sephadex G-100 can be temporarily, for a short time interval, activated by adding calcium ions. The activation of esterases isolated from cells was non-repeteable, whereas the temporary activation of esterases from the culture filtrate could be repeated by increasing concentrations of calcium ions. However, the value of activation gradually decreased. Similarly with calcium ions strontium ions were also effective, however, higher concentrations were required and the activation was non-repeatable. Magnesium ions were practically without any effect. Possible mechanisms of the temporary activation of esterases ofMycobacterium phlei are discussed.     

Deacylation of acetylated amino acids by the esterase fromMycobacterium phlei

In addition to typical acetyl esters, acetic ester aoetyl-hydrolase fromMycobacterium phlei can degrade aliphatic acetylated amino acids under suitable conditions.     

Snake venom phosphodiesterase: A zinc metalloenzyme

Snake venom phosphodiesterase fromCrotalus adamanteus can be purified by blue sepharose chromatography followed by gel exclusion chromatography on Sephadex G-100. The enzyme is judged to be homogeneous by SDS gel electrophoresis. Atomic absorption spectrometry indicates a stoichiometry of 1.07 g-atom of zinc per mole of purified enzyme. The enzyme is inhibited by a wide variety of structurally different metal binding agents, e.g., 1,10-phenanthroline, thioglycolic acid,l-cysteine, 8-hydroxy-5-quinoline sulfonic acid, EDTA, and dipicolinic acid. The results of both the chelator inhibition and the atomic absorption analysis indicate that snake venom phosphodiesterase is a zinc metalloenzyme. Snake venom phosphodiesterase shares a number of mechanistic features in common with the nucleotidyl transferases. All of these enzymes contain zinc, are activated by magnesium, and catalyze α-β phosphoryl bond cleavage. Mechanistic studies of phosphodiesterase may therefore be helpful in understanding the mechanism of the hydrolytic step catalyzed by all of these enzymes.     

Genetic variation in the activity of the histidine catabolic enzymes between inbred strains of mice: A structural locus for a cytosol histidine aminotransferase isozyme (Hat-1)

Variation in activity of the main histidine catabolic enzymes (histidase, urocanase, and aminotransferase) has been surveyed using inbred strains of mice (C57BL, DBA, Peru, SM, and SWR). Some variation was found in the activity of all enzymes, but only in the case of cytosolic histidine aminotransferase was it greater than twofold (SM 3.3-fold greater than C57BL). The divergent strains for the activity of this enzyme were crossed and the F ₁ 's were backcrossed; the segregation analysis indicated a single locus with additively acting alleles (designated Hat-1: a allele SM, b allele C57BL). Cytosolic histidine aminotransferase differed in heat stability between SM and C57BL, indicating that Hat-1 is a structural locus. The conflict in the biochemical literature (Morris et al., 1973; Noguchi et al., 1976a, b) over the number and subcellular distribution of the histidine aminotransferase isozymes is partly resolved by the acquisition of a variant at the Hat-1 locus. Hat-1 affects the cytosolic form but not the mitochondrial form of the enzyme. Purification and analysis of the isozymes of histidine aminotransferase from livers of C57BL and SM mice will further clarify the situation.     

Enzyme preparations fromAspergillus flavus for structural studies of the peach gum polysaccharide

Extra- and intracellular glycanohydrolases were isolated fromAspergillus flavus and partially characterized. Both preparations exhibited β-galactosidase activity. Gel chromatography of the extracellular enzyme preparation on Sephadex revealed one protein fraction containing β-galactosidase activity and a second one exhibiting mainly β-xylosidase activity. Electrophoresis in starch gel and disc electrophoresis in polyacrylamide gel showed that the preparation obtained from the cultivation broth contained five protein fractions, whereas two protein fractions could be detected in the intracellular preparation. Hydrolysis of a partially degraded polysaccharide of peach gum by the above preparations yieldedd-galactose as the main product and traces ofd-mannose,l-arabinose,d-xylose and a number of oligosaccharides.     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

Structure-based rational design and introduction of arginines on the surface of an alkaline α-amylase from Alkalimonas amylolytica for improved thermostability

In this study, the thermostability of an alkaline α-amylase from Alkalimonas amylolytica was significantly improved through structure-based rational and the introduction of multiple arginines (Arg) on the protein surface. Based on an analysis of the tertiary structure, seven residues (glutamine (Gln) 166, Gln 169, serine (Ser) 270, lysine (Lys) 315, Gln 327, asparagine (Asn) 346, and Asn 423) were selected as engineering targets and individually replaced with arginine. Five of the seven single-mutated enzymes—S270R, K315R, Q327R, N346R, and N423R—showed enhanced thermostability. Multiple arginines were subsequently introduced on the protein surface, and the quintuple-mutated enzyme S270R/K315R/Q327R/N346R/N423R showed a 6.4-fold improvement in half-life at 60 and a 5.4 °C increase in melting temperature (T _m) compared with those of wild-type enzyme. Concomitantly, the optimal temperature, optimal pH, and catalytic efficiency of this mutated enzyme also improved. The mutated enzyme displayed a large shift in optimal pH from 9.5 to 11.0. In addition, the optimum temperature increased from 50 to 55 °C, and the catalytic efficiency (k _cat/K _m) increased from 1.8?×?10⁴ to 3.6?×?10⁴ L/(g?·?min). The intramolecular interactions of mutated enzymes that contributed to increased thermostability were examined through comparative analysis of the model structures of wild-type and mutated enzymes. The thermostable mutated enzymes generated in this study have potential applications in the textile industry.     

Investigation of the properties of alcohol dehydrogenase from bean,rape, wheat and broad bean

Preparations of alcohol dehydrogenase (ADH) were prepared from germinating seeds by a procedure including fractionation with ammonium sulphate, desalting on a column of Sephadex G-25 and chromatography on DEAE-cellulose, the specific activity of ADH was in the case ofPhaseolus 31 times,Brassica 43 times,Triticum 47 times, andVicia 212 times higher than that of crude extract. The enzymes were homogeneous when filtrated on Sephadex G-200. Molecular weight of all the four studied ADH was approximately 63 000. Some kinetic properties as K_m for ethanol as substrate, substrate specificity towards different alcohols, and the effect of some intermediates of sugar metabolism and of some inhibitors on the activity of the enzymes were also followed. The results obtained are discussed with respect to possible mechanism of action of the plant ADH.     

Pleiotropic and other genetic effects influencing the activities of brain and liver enzymes in congenic lines of C57BL/6J mice with defined electrophoretic variant markers

A single genetic factor may affect the realization of several enzymes. To investigate the extent of pattern pleiotropy in the mouse, the activities of 28 enzymes in livers and brains from an inbred stock of C57BL/6J Nctr and five F₁ stocks heterozygous for known electrophoretic variants were measured. Five congenic backcross stocks of C57BL/6J, each homozygous for one or more electrophoretic markers, were mated with C57BL/6J Nctr to construct the heterozygous variant F₁ stocks. One of the five F₁ stocks had no enzyme activities significantly different from those of C57BL/6J Nctr, while two had one enzyme, one had four enzymes, and another had six enzymes with activities that were significantly different from those of C57BL/6J Nctr. The latter two F₁ stocks with multiple activity differences were those having the largest proportion of their genome of donor origin. Two of the F₁ stocks were different from each other for one enzyme, and two were different for another enzyme. These differences and the relationship of these enzyme activities to the variant genes suggest that several genetic factors may affect an enzyme's realization.     

Quantitative Extraction of Adenosine Triphosphate from Cultivable and Host-Grown Microbes: Calculation of Adenosine Triphosphate Pools

Existing data on adenosine triphosphate (ATP) pools in microbes are deficient for two reasons: (i) incomplete extractions of ATP, and (ii) the failure to take into account that the adverse effects of extracting procedures on standard ATP exert analogous effects on the ATP released from bacterial cells. Methods for correcting observed yields and calculating ATP pools have been demonstrated. Three bacterial species were used in the studies on extraction of ATP: Escherichia coli, Mycobacterium phlei, and Mycobacterium lepraemurium. Perchloric acid and n-butanol were disqualified because of their failure to extract total bacterial ATP even from E. coli and because of inconvenient procedures. The new extraction procedure had minimal effects on standard ATP, liberated 100% of the ATP pools from the three representative species of microbes, and caused no ionic imbalance or quenching of bioluminescence. This method involves vortexing of cell suspensions for 10 s with 23% chloroform (vol/vol), heating at 98 C for the required time (E. coli, 3 min; M. phlei, 5 min; M. lepraemurium, 10 min) and then 1 min at 98 C with vacuum to dry the samples. Heat or chloroform alone may suffice for some microbes and release total ATP from plant and animal cells.     

Acid-soluble chromosomal proteins in maize root and callus cells and after rhizogenesis induction in callus tissues

Callus tissues originating fromZea mays root meristem, induced for rhizogenesis callus, meristematic and differentiated maize root cells for isolation of nuclei and acid-soluble chromosomal proteins were used. Cytological investigations proved that rhizogenesis begins with the formation of meristematic centres, followed by root differentiation about 5–12 days after the treatment with α-naphtalene acetic acid (NAA). When applying electrophoresis in 15% polyacrylamide gel, differences between the electrophoretic profiles of acid-soluble chromosomal proteins, isolated from root cells and from callus tissues, were established. The main differences concern histone H1 and probably H4. There are no differences between electrophoretic patterns of acid-soluble chromosomal proteins of nonorganized callus and callus induced for rhizogenesis. The possible explanation of these results is discussed.     

A new subspecies of the ayu,Plecoglossus altivelis, (Plecoglossidae) from the Ryukyu Islands

A new subspecies of the ayu,Plecoglossus altivelis ryukyucnsis, is described on the basis of specimens from Amami-oshima and Okinawa Islands, the Ryukyu Islands, Japan. This new subspecies is distinguished fromP. altivelis altivelis by the fewer numbers of pectoral fin rays, longitudinal scales and scales above and below the lateral line, and also by the unique electrophoretic mobilities of several enzymes.     

Studies of some ways in which carrot mosaic virus can be transmitted

The transmission of carrot mosaic virus (CMV) by the crude sap to 11 varieties of plants from 4 families was demonstrated. From these plants the virus could be transferred back to a healthy carrot cultivated from the seed in isolation. The incubation time required for the appearance of the symptoms of CMV was 7–20 days. The plants on which mosaic or spot symptoms appeared on the leaves after transfer by the sap at temperatures below 15°C remains habitually healthy after the transfer of virus at higher temperatures. The results of the mechanical inoculation of CMV by the crude infectious sap to young carrots cultivated from seeds differentiated this virus fromApium virus 1, which after mechanical inoculation causes chlorosis of the youngest carrot leaves in contrast to CMV. A further differentiation of CMV fromApium virus 1 is shown by the fact that CMV can be transferred only to the familyDaucaceae. It differs in this fromApium virus 1 which is transferred exclusively to this family (Köhler, Klinkowski 1954). CMV is differentiated fromCucumis virus 1/Doolittle Smith by some different host plants.