Purification and characterization of mouse alcohol dehydrogenase from two inbred strains that differ in total liver enzyme activity

Alcohol dehydrogenase activity in mouse liver homogenate-supernatants is 1.7 times greater in the C57BL/10 strain than in the BALB/c strain, regardless of whether activity is expressed in units per gram liver, total liver, or milligram DNA. The K _m values for ethanol and NAD⁺, approximately 0.4 and 0.03mm, respectively, of enzyme purified from both strains are similar. Moreover, the K _i for NADH, 1 µm, the pH optimum for ethanol oxidation, 10.5, and the V _max for ethanol oxidation, 160 min^–1, for ADH from the C57BL/10 and BALB/c strains are similar. Therefore, the difference in ADH activity in the two strains cannot be due to differences in the catalytic properties of the enzyme. The electrophoretic and isoelectric focusing patterns and two-dimensional tryptic peptide maps of the purified enzyme from both strains are identical. Thus the amino acid sequences of enzyme from C57BL/10 and BALB/c mice must also be identical or very similar. The difference in ADH activity in the two strains is most likely the result of genetic differences in the content of ADH protein in liver.Supported by NIAAA Grant AA 04307.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Initial studies of the use of aminopeptidases for the differentiation ofBacillus sphaericus strains

Summary Analysis of aminopeptidase activity in commercially available enzyme panels (SAP-II and SAP-III) indicated qualitative differences among the eight strains ofBacillus sphaericus tested. These differences involved specific substrates utilized, the importance of sampling time, as well as differences between whole cells and cell-free extracts. Further detailed analysis of four of thel-amino acid--naphthylamide substrates using polyacrylamide gel electrophoresis (PAGE) demonstrated a series of allelic differences in the multilocus patterns observed. The resulting phenogram constructed from the enzyme and PAGE information showed a marked separation of the noninsecticidal strains from the insecticidal strains (58% similarity). In addition, there was a separation within the insecticidal strains of strain SSII-1, phage type 2 from strain 2297, phage type 4, and of both of these strains from the remaining highly insecticidal members of phage type 3 (strains 1593, 2013-4, 2362, 2500).     

Nucleotide Sequence Variation Does Not Relate to Differences in Kinetic Properties of Neutral Trehalase from the Insect Pathogenic Fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

Genetic variability in a putative virulence factor, the neutral trehalase (Ntl) gene, was examined in strains of the insect pathogenic fungi Metarhizium anisopliae and Metarhizium flavoviride by restriction fragment length polymorphism (RFLP). The Ntl gene was sequenced from four of these strains that showed dissimilar RFLP patterns. Enzyme kinetic experiments were also performed on the partially purified neutral trehalase in order to assess whether nucleotide changes in these strains related to differences in enzyme catalytic function (i.e., K _m , V _max, and K _cat). Finally, the Metarhizium strains were assessed in bioassays against waxworm larvae in order to relate nucleotide variation with Ntl enzyme kinetics and insect virulence. The greatest RFLP variation was observed with Rsa1. M. flavoviride was found to be most dissimilar in RFLP patterns when compared with the M. anisopliae strains. RFLP patterns for Ntl were diagnostic markers for previously studied genetic groups of M. anisopliae. Comparisons of Ntl sequences showed that the introns were found to be more variable (6.2%) than the exons (3.1%). Comparisons of the translated nucleotide codons showed high levels (91%) of synonymous sequence variation between strains. Another fraction of the remaining mutations was neutral, resulting in amino acid substitutions with similar functions. The neutral trehalase was partially purified by preparative isoelectric focus, revealing a single band of enzyme activity as assessed by analytical isoelectric focusing (pI ca. 5). Kinetic properties of the neutral trehalases revealed no differences between the M. anisopliae strains, while the M. flavovoride had a lower K _cat/K _m . However, there was lower virulence in one strain that showed Ntl enzyme kinetic properties that were similar to the other strains, suggesting that factors other than neutral trehalase may be responsible for delimiting virulence in this insect pathogenic fungi. Although there is nucleotide variation in genes involved in pathogenicity, this variation is mostly neutral in nature, and there is strong stabilizing selection to maintain enzyme function.     

Variation in Drosophila acetylcholinesterase

We examined the Canton-S strain of Drosophila melanogaster for electrophoretic variation of the enzyme acetylcholinesterase. The pattern of bands obtained (stained with acetylthiocholine) depended on age, sex, and tissue (i.e., head vs. body part and hemolymph). However, through mixing experiments, it was concluded that most of these apparent differences were due to modification of the enzyme by unknown substances located in the fly's body. The electrophoretic pattern of head acetylcholinesterase was altered so that it became characteristic of the body which was present during extraction. For example, when heads of D. melanogaster were homogenized in an extract from D. lebanonensis bodies, the characteristic AChse bands of melanogaster were absent and instead the bands of lebanonensis were found. it was found that extraction of adult heads in 0.1 M potassium phosphate buffer alone or with a 2-min exposure to 1 mg/ml trypsin at 20 C gave the most reproducible results, independent of age and sex. Using these conditions, 25 strains of D. melanogaster and 30 strains of D. pseudoobscura were examined without finding any reproducible electrophoretic variant of acetylcholinesterase. In addition, 53 strains from 39 other Drosophila species produced a total of only six electrophoretic forms of the head enzyme. Additional electromorphs were found when whole flies were used, but these were not studied in detail because of the possibility that they could be due to postextraction modification.This study was supported by the National Research Council of Canada, Grant No. A0629 and A0235.     

Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation

Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.     

Effect of Sodium Taurocholate on the In Vitro Growth of Lactobacilli

Abstract The effect on growth of a conjugated bile salt (sodium taurocholate) at physiological concentration was determined using cultures of Lactobacillus strains of murine origin. The bile salt stimulated the growth of one strain, did not affect the growth of another, but inhibited the growth of strains that produced relatively large amounts of the enzyme bile salt hydrolase. Comparison of the growth of isogenic strains that differed in the ability to produce bile salt hydrolase demonstrated that inhibition of growth was due to the accumulation of cholic acid in the culture medium as a result of the enzyme activity. Received: 15 January 1996; Revised: 26 March 1996; Accepted: 29 March 1996     

Studies on the extracellular p-nitrophenyl β-d-cellobiosidase activity of a thermophilic streptomycete

Summary The activity of ten actinomycete strains against p-nitrophenyl -d-cellobioside (pNPC) was investigated. Intra- and extracellular activities were detected in all strains, although activities were found to vary between strains. Extracellular pNPC activity, detected in the thermophilic Streptomycete EC22 was six times greater than that of the next best strain. Culture supernatant studies on Streptomyces strain EC22 revealed that pNPC activity was optimal at 65° C and between pH 6.0 and pH 8.0, although this temperature value represented a compromise between increased reaction rate and thermal denaturation. At 65° C, the half-life of activity against pNPC was 150 min. The K _m value of pNPC activity was found to be 3.33 mm. Enzyme activity was subject to competitive inhibition by both glucose and cellobiose with substrate inhibition constant (K _i) values of 2.2 mm and 1.4 mm respectively. Fast protein liquid chromatograph (FPLC) of culture supernatants from EC22 using anion exchange chromatography revealed the presence of two proteins with pNPC activity. This result was confirmed using gel-filtration chromatography which estimated the molecular mass of the two proteins to be approximately 48 and 33 kDa, with the specific activity of the 48 kDa enzyme being eight times greater than that of the 33 kDa protein. Offprint requests to: A. S. Ball     

Genotypic diversity in Oenococcus oeni by high-density microarray comparative genome hybridization and whole genome sequencing

Many bacteria display substantial intra-specific genomic diversity that produces significant phenotypic variation between strains of the same species. Understanding the genetic basis of these strain-specific phenotypes is especially important for industrial microorganisms where these characters match individual strains to specific industrial processes. Oenococcus oeni, a bacterium used during winemaking, is one such industrial species where large numbers of strains show significant differences in commercially important industrial phenotypes. To ascertain the basis of these phenotypic differences, the genomic content of ten wine strains of O. oeni were mapped by array-based comparative genome hybridization (aCGH). These strains comprised a genomically diverse group in which large sections of the reference genome were often absent from individual strains. To place the aCGH results in context, whole genome sequence was obtained for one of these strains and compared with two previously sequenced, unrelated strains. While the three strains shared a core group of conserved ORFs, up to 10% of the coding potential of any one strain was specific to that isolate. The genome of O. oeni is therefore likely to be much larger than that present in any single strain and it is these strain-specific regions that are likely to be responsible for differences in industrial phenotypes.     

Single Molecule Assays of β-Galactosidase from Two Wild-type Strains of <Emphasis Type="Italic">E. coli</Emphasis>: Effects of Protease Inhibitors on Microheterogeneity and Different Relative Activities with Differing Substrates

β-Galactosidase was induced in E. coli wild type strains ATCC 8677 and 35321 in the presence of various protease inhibitors. Single enzyme molecule assays were performed using a capillary electrophoresis based protocol. The presence of the protease inhibitors had a minimal effect on the average and distribution of single molecule activities. Two novel capillary electrophoresis based single enzyme molecule assays for β-galactosidase were developed using DDAO-β-d-galactopyranoside and fluorescein-β-d-digalactopyranoside as substrates. Double incubations were performed on individual enzyme molecules to demonstrate the reproducibility of the assays. Assays performed on β-galactosidase from strains 8677 and 35321 demonstrated that the relative activities of the enzyme for the different substrates differed between the strains. Sequencing showed that these two strains differ in their primary sequence by a single amino acid substitution in position 280, which is in the region of the active site.     

Regulation of aspartokinase activity in non-fermentative,marine eubacteria

Summary Cell-free extracts of gram-negative, non-fermentative, marine eubacteria were assayed for aspartokinase activity. The organisms tested included polarly flagellated species and groups which had GC contents in their DNAs of 46 to 64 moles % (Alteromonas, Pseudomonas) as well as species which had peritrichous flagellation and moles % GC contents of 53 to 68 (Alcaligenes). The results of these studies suggested that in all the strains tested, aspartokinase activity was catalyzed by a single enzyme. On the basis of the effect ofl-threonine,l-lysine,l-methionine, andl-isoleucine on activity, five different types of aspartokinases (designated I through V) were delineated. In aspartokinase types I through IV,l-threonine andl-lysine inhibited activity by means of a concerted feedback inhibition; in type V, activity was inhibited byl-threonine but unaffected byl-lysine. In types I, III, and IV,l-threonine andl-lysine alone were inhibitory, while in type II these effectors had virtually no effect on activity when tested singly. Three distinct responses were observed in the presence of two other end products of the aspartate pathway,l-methionine andl-isoleucine. In types I and II, these two amino acids usually stimulated activity and overcame the inhibition byl-threonine andl-lysine; in types IV and V,l-methionine andl-isoleucine had no effect; and in type III these amino acids inhibited activity. The results of this study indicate that the aspartokinases of a number of species and groups of marine bacteria have similarities and differences which should be of use in making future taxonomic groupings.     

The influence of host plant genotype on variation in population densities of the gorse thrips,Sericothrips staphylinus (Thysanoptera: Thripidae), and its consideration in relation to release strategies

Large variations in population densities of Sericothrips staphylinus were observed between gorse bushes at a field site in an earlier Tasmanian study. A replicated potted plant experiment was conducted to assess whether genetic variation between gorse bushes could affect densities of S. staphylinus. There were significant differences in the number of S. staphylinus recovered from the individual bushes sampled within sites and individual bushes across all sites. This provides evidence that genetic variation between gorse bushes is at least partly responsible for the observed variability in field populations. The result validates the current release strategy used for the field establishment of S. staphylinus in south-eastern Australia. This strategy involves making multiple small releases (ca. 250 adults per bush per site) instead of a small number of large releases (ca. 1000 per bush per site), thereby reducing variation in numbers due to genetic affects. Genetic variation between individual plants of the same species and associated effects on herbivore densities should be considered when devising release strategies for biological control agents.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Patterns of puffing activity during the third larval instar and the prepupal period of two different strains of D. melanogaster (Oregon and vg6) are compared. The variation in puffing activity observed is both quantitative (involving the mean size or timing of activity of individual puffs) and qualitative. The pattern of activity of 64% of the puffs is the same in the two strains, 12% show strain differences in puff size and 19% in the time of their activity. One puff (64C) is active only in one of the strains (vg6). In genetic experiments this puff segregates normally and the puff locus has been mapped genetically to a site coincident with, or at least very close to, the cytogenetic position of the puff. In heterozygotes the puff is homozygous only when the maternal and paternal homologues are synapsed. When the homologues are asynapsed only the homologue from the vg6 parent is puffed at 64C. With the exeption of some strains closely related to vg6 no other strain of D. melanogaster has been found to possess puffing activity at 64C. In vg6/In(3LR)C165 heterozygotes 64C forms a heterozygous puff even when the homologues are synapsed. In the discussion consideration is given to the various factors that control puff size.     

Improved production and properties of β-glucosidase influenced by 2-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose in the culture medium of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Increased production, secretion, and activity of β-glucosidase in the filamentous fungus Termitomyces clypeatus was achieved in presence of the glycosylation inhibitor 2-deoxy-d-glucose (0.05%, w/v) during submerged fermentation. Enzyme activity increased to 163 U/mL by adding mannose (2 mg/mL) to the medium. Such a high enzyme activity has not been achieved without mutation or genetic manipulation. The K_m and V_max of the enzyme in culture medium were determined to be 0.092 mM and 35.54 U/mg, respectively, with p-nitrophenyl β-d-glucopyranoside as substrate, confirming its high catalytic activity. The enzyme displayed optimum activity at pH 5.4 and 45°C. The enzyme was fairly stable between acidic to alkaline pH and retained about 75 ∼ 65% residual activities between pH 4 and 10.6 and demonstrated full activity at 45°C for 3 days. The enzyme was also stable in the presence of Zn²⁺ and Mg²⁺ and 80% of the residual activity was observed in the presence of Mn²⁺, Ca²⁺, K⁺, Cu²⁺, EDTA, and sodium azide. Around 70% of the activity was retained in the presence of 2 M guanidium HCl and 3 M urea, whereas the activity was 5 and 2 times higher in the presence of 4 mM beta-mercaptoethanol and 50 mM DTT, respectively. The enzyme obtained from the culture filtrate showed potential cellulose saccharifying ability which increased further when supplemented with commercial cellulase. Thus, this enzyme could be used without any additional downstream processing for commercial cellulase preparation and production of bioethanol or for other biotechnological applications.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase in<Emphasis Type="Italic"> Enterobacter aerogenes</Emphasis> expressing<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin for efficient oxygen uptake

This study is the first utilizing Vitreoscilla hemoglobin in a heterologous bacterium, Enterobacter aerogenes, to determine the effect of such a highly efficient oxygen-uptake system on the production of l-asparaginase, an enzyme that has attracted considerable attention due to its anti-tumor activity. Here, we show that the Vitreoscilla hemoglobin expressing strain has from 10-fold to more than two orders of magnitude lower l-asparaginase activity than the wild type or the control without the Vitreoscilla hemoglobin gene under different aeration conditions. Aeration and agitation were also determining factors for enzyme production. The enzyme activity was reduced considerably under both full aerobic and anaerobic conditions, while the highest enzyme activity was determined in cultures under low aeration and low agitation. Also, the effect of different concentrations of glucose on enzyme production showed catabolic repression. Glucose at 1% caused almost total inhibition of enzyme activity, while at 0.1% it showed a slightly stimulatory effect on enzyme production, compared with glucose-free medium.     

Benzoylarginine peptidase and iminopeptidase profiles ofTreponema denticola strains isolated from the human periodontal pocket

Seven clinical isolates and the ATCC strain 35405 ofTreponema denticola, obtained from human periodontal pockets, were studied for peptidase activity with several chromogenic compounds as substrates. The cell sonicates of all strains hydrolyzed phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-arginine (a collagenase substrate), azocasein, and the 2-naphthylamines ofl-proline,l-hydroxyproline,l-pyrrolidine, and benzoyl-l-arginine, but the rates of hydrolysis varied considerably from strain to strain. Fast protein liquid chromatography on gel and anion exchange columns revealed further biochemical differences between the strains. The ATCC strain consistently produced several proline iminopeptidases, whereas four of the clinical isolates yielded high and three yielded low iminopeptidase activity. The ATCC strain and six clinical isolates displayed high benzoylarginine peptidase activity. The use ofN-l-prolyl-2-naphthylamine as substrate revealed more differences between the strains than other substrates. The substrate specificity of the enzymes discovered suggests that they may be important for the nutrition of the organism or in the protection of the organism against chemical defense factors present in the gingival pocket.     

Protein polymorphism and genetic divergence in slow loris (genusNycticebus)

In this study, protein electrophoresis was assayed to detect genetic variation in GenusNycticebus. A total of 29 samples (2N. coucang and 27N. pygmaeus) were analyzed for 42 genetic loci. In the 27 samples ofN. pygmaeus, 4 loci were observed to be polymerphic. Therefore, the estimatedP value (proportion of polymorphic loci) is 0.095, theA value (average number of alleles each locus) is 1.045, and theH value (mean individual heterozygosity) is 0.040. After comparing theH ofN. pygmaeus with those of other primates reported, we found that the protein variation inN. pygmaeus is slightly lower than the average level. Additionally, we also observed obivious allele difference betweenN. pygmaeus andN. coucang. There are no shared alleles between these two species in eight loci. TheNei's genetic distance between them was calculated as 0.2541, which falls in the spectrum of genetic difference between species in primates.     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Regulation of homoserine dehydrogenase inAzotobacter species

The regulation of homoserine dehydrogenase activity was studied in nineAzotobacter strains belonging to five different species. In all the species the enzyme is subject to feedback inhibition byl-threonine andl-isoleucine, the first being much more active as inhibitor. The inhibition byl-threonine is noncompetitive with respect to NADPH and of mixed type with respect to aspartate-Β-semialdehyde; the inhibition byl-isoleucine is noncompetitive with respect to both substrates. The synthesis of homoserine dehydrogenase inAzotobacter chroococcum I.P. is somewhat repressed by 1mm l-methionine and 5mm l-isoleucine. In all the strains examined either NADPH or NADH can serve as cofactors for this activity, though the ratio of activity with the two pyridine nucleotides (NADPH/NADH) shows higher values (3.3–3.8) in the speciesmacrocytogenes andinsignis than in thechroococcum, beijerinckii andvinelandii group (1.5–1.6). The pattern of control of this enzyme in the genusAzotobacter is discussed in relation to other bacterial homoserine dehydrogenases. We are grateful to Dr. G. N. Cohen, Service de Physiologie Microbienne, Institut Pasteur, Paris, for helpful discussions and encouragements.     

α-l-Fucosidase in human fibroblasts. I. The enzyme activity polymorphism

Low plasma α-l-fucosidase activity is a recessive polymorphic trait observed in 8% of the normal population. The molecular basis of this polymorphism remains unclear and its expression is tissue specific. As the low-activity (variant) phenotype is expressedin vitro in cultured human fibroblasts, this cell type was chosen to study the enzyme activity polymorphism. Fibroblast cell lines derived from individuals with low plasma fucosidase activity (variants) have less than 30% of the fucosidase activity of fibroblast cell lines established from individuals with high plasma fucosidase activity (nonvariants). No qualitative differences in the synthesis, processing, and extracellular release of newly made α-l-fucosidase could be demonstrated among variant and nonvariant cell strains. Cells pulsed with³H-leucine for 10 min produce a 51-kDa protein which is rapidly processed to a 55-kDa intermediate. The latter is converted to a mature 59-kDa intracellular and a 61-kDa extracellular end product, in both variant and nonvariant fibroblast cell lines. Variant and nonvariant fibroblast cell lines also release relatively equal amounts of fucosidase into the extracellular medium. Therefore, differences in processing or extracellular release of fucosidase between variants and nonvariants are not the basic mechanism of this tissue-specific activity polymorphism. We are indebted to the “Belgische Vereniging voor Strijd tegen Mucoviscidose” for financial support.