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Biomedical subjects

M G Shepherd

Publications and source records attributed to M G Shepherd.

At least 91 records · Page 5Linked to original sources
PubMed1980

Germ tube induction in Candida albicans.

A reproducible and simple system for the production of germ tubes from yeast cells of Candida albicans using glucose and glutamine as substrates has been described. During germ tube formation there was a doubling of the dry weight but the number of cells remained constant. Although the DNA content did not change for the first 4 h of germ tube formation, the RNA content more than doubled. The DNA and RNA content of C. albicans blastospores are 4.5 x 10(-15) g per cell and 48 x 10(-15) g per cell respectively. Nystatin, phenethyl alcohol, 2,4-dinitrophenol, azaserine, salicylhydroxamic acid, and 5-fluorocytosine were all effective inhibitors of germ tube formation. Cysteine, potassium cyanide, and polyoxin D did not prevent germination. The incorporation of both uracil and leucine occurred rapidly during germ tube formation. The inhibitors of RNA synthesis, actinomycin D. cordycepin, and daunomycin prevented germination and inhibited uracil incorporation. The translational inhibitors, trichodermin, aurin tricarboxylic acid, puromycin, and cyloheximide were effective in inhibiting both germ tube formation and leucine incorporation.

Candida albicans↗
PubMed1978

Inactivation and modification of lactate oxidase with fluorodinitrobenzene.

1. Dinitrophenylation of 2 +/- 0.2mol of residues/mol of enzyme-bound FMN resulted in the complete inactivation of the flavoenzyme L-lactate oxidase. 2. Hydrolysates of the inactivated enzyme contained 1mol each of Nim-Dnp-histidine (abbreviation: Dnp-,2,4-dinitrophenyl-; Nim indicates that either of the N atoms in the imidazole ring is substituted) and epsilon-Dnp-lysine/mol of FMN. 3. Competitive inhibitors decreased the extent of inactivation to a 10% loss of activity, and dinitrophenylation was decreased from 2 to approx. 0.5mol/mol of FMN. Only Nim-Dnp-histidine was detected in the hydrolysates. 4. Although the dinitrophenylated enzyme did not possess enzyme activitiy, L-lactate reduced approx. 50% of the enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme formed a complex with bisulphite. 6. The modified enzyme (2mol of Dnp/mol of FMN) was unable to bind substrate analogues and competitive inhibitors.

Amino Acids↗
PubMed1978

Substrate specificity and mode of action of a cellulase from Aspergillus niger.

The mode of action and substrate specificity of a cellulase purified from Aspergillus niger were examined. The enzyme showed little capacity to hydrolyse highly ordered cellulose, but readily attacked soluble cellulose derivatives and amorphous alkali-swollen cellulose. Activity towards barley glucan and lichenin was greater than with CM-cellulose. Low activity was detected with CM-pachyman (a substituted beta-1,3-glucose polymer) and xylan. Activity towards yeast glucan, mannan, ethlene glycol chitin, glycol chitosan, laminarin, polygalacturonic acid and pectin could not be demonstrated. Cellobiose and p-nitrophenyl beta-D-glucoside were not hydrolysed, whereas the rate of hydrolysis of the higher members of the reduced cellulodextrins increased with chain length. The central bonds of cellotetraosylsorbitol and cellopentaosylsorbitol were the preferred points of clevage. Kinetic data indicated that the specificity region of the cellulase is five glucose units in length. The evidence indicates that the cellulase is an endoglucanase.

Aspergillus niger↗
PubMed1978

The alternate respiratory pathway of Candida albicans.

Candida albicans contains a cryptic cyanide and antimycin A insensitive respiratory system. This alternate oxidase was found (i) at all growth rates from mu = 0.05 to 0.26 in a chemostat culture and (ii) in both mycelial and yeast forms of the organism. Neither chloramphenicol nor cycloheximide prevented the expression of the alternate oxidase. Salicylhydroxamic acid was a potent inhibitor of the cyanide insensitive respiration. The respiration of mitochondria grown in the presence of antimycin A was not inhibited by cyanide or antimycin A but was inhibited by salicylhydroxamic acid.

Antifungal Agents↗
PubMed1977

Chemical modification of cellulase from Aspergillus niger.

N-Bromosuccinimide completely inactivated the cellulase, and titration experiments showed that oxidation of one tryptophan residue per cellulase molecule coincided with 100% inactivation. CM-cellulose protected the enzyme from inactivation by N-bromosuccinimide. The cellulase was inhibited by active benzyl halides, and reaction with 2-hydroxy-5-nitrobenzyl bromide resulted in the incorporation of 2.3 hydroxy-5-nitrobenzyl groups per enzyme molecule; one tryptophan residue was shown to be essential for activity. Diazocarbonyl compounds in the presence of Cu2+ ions inhibited the enzyme. The pH-dependence of inactivation was consistent with the reaction occurring with a protonated carboxyl group. Carbodi-imide inhibited the cellulase, and kinetic analysis indicated that there was an average of 1 mol of carbodi-imide binding to the cellulase during inactivation. Treatment of the cellulase with diethyl pyrocarbonate resulted in the modification of two out of the four histidine residues present in the cellulase. The modified enzyme retained 40% of its original activity. Inhibition of cellulase activity by the metal ions Ag+ and Hg2+ was ascribed to interaction with tryptophan residues, rather than with thiol groups.

Aspergillus niger↗
PubMed1977

The structure of L-lactate oxidase from Mycobacterium smegmatis.

1. An improved purification was developed for L-lactate oxidase from Mycobacterium smegmatis. 2. The mol.wt. of the native enzyme by a sedimentation-equilibrium analysis was 345 000, and other ultracentrifuge methods gave values in the range 345 000-350 000. 3. An amino acid analysis, determinations of protein and flavin, a sedimentation-velocity analysis and an approach to equilibrium analysis gave values for the subunit mol.wt. in the range 43 500-47 000. 4. It was concluded that L-lactate oxidase contains eight subunits of mol.wt. 43 500. 5. Cross-linking of the subunits with dimethyl suberimidate and electron-microscopy studies were consistent with an octameric structure.

Amino Acids↗
PubMed1977

Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue.

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75-1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [(14)C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the (14)C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

Binding, Competitive↗
PubMed1977

Purification and properties of a cellulase from Aspergillus niger.

A cellulolytic enzyme was isolated from a commercial cellulase preparation form Aspergillus niger. A yield of about 50mg of enzyme was obtained per 100g of commerial cellulase. The isolated enzyme was homogeneous in the ultracentrifuge at pH 4.0 and 8.0, and in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but showed one major and two minor bands in disc gel electrophoresis. No carbohydrate was associated with the protein. Amino acid analysis revealed that the enzyme was rich in acidic and aromatic amino acids. Data from the amino acid composition and dodecyl sulphate/polyacrylamide-gel electrophoresis indicated a molecular weight of 26000. The purified enzyme was active towards CM-cellulose, but no activity towards either cellobiose or p-nitrophenyl beta-D-glucoside was detected under the assay conditions used. The pH optimum for the enzyme was pH 3.8-4.0, and it was stable at 25 degrees C over the range pH 1-9; maximum activity (at pH 4.0) was obtained at 45 degrees C. The cellulase was more stable to heat treatment at pH 8.0 than at 4.0. Kinetic studies gave pK values between 4.2 and 5.3 for groups involved in the enzyme-substrate complex.

Amino Acids↗
PubMed1977

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.

Adenosine Triphosphate↗
PubMed1976

The production and growth characteristics of yeast and mycelial forms of Candida albicans in continuous culture.

The growth characteristics of Candida albicans CM145,348 have been examined under aerobic conditions in continuous culture. At different steady states the environment was controlled with respect to the concentrations of dissolved oxygen, carbon and nitrogen, the pH, and the temperature. Dry matter, substrate concentration, yield, specific oxygen uptake, specific carbon dioxide release and respiration quotient were examined as a function of the dilution rate. The morphology depended on the carbon source. Maltose produced a mycelial morphology, whereas with lactate a yeast culture was obtained. With fructose or glucose as a carbon source a mixed morphology of yeast, pseudo-mycelial and mycelial forms was produced. A larger number of different growth conditions were examined in batch culture but a mixed morphology was always obtained.

Aerobiosis↗
PubMed1976

Mannitol production in fungi during glucose catabolism.

The levels of phosphofructokinase (EC 2.7.1.11) and mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) have been determined in a number of Mucor and Penicillium species. Mannitol-1-phosphate dehydrogenase was found in only one species of mucor, Mucor rouxii, and this with a specific activity much lower than that found in Penicillium species. All of the fungi tested in the Ascomycetes class exhibited mannitol-1-phosphate dehydrogenase activity. Interference from both mannitol-1-phosphate dehydrogenase and NADH oxidase (EC 1.6.99.5) caused some difficulty initially in detecting phosphofructokinase in Penicillium species; the Penicillium phosphofructokinase is very unstable. Penicillium notatum accumulates mannitol intracellularly; detection of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase (EC 3.1.3.22) activity in cell-free extracts indicates that the mannitol is formed from glucose via fructose-6-phosphate and mannitol-1-phosphate; no direct reduction of fructose to mannitol could be detected. The mannitol-1-phosphate dehydrogenase was specific for mannitol-1-phosphate and fructose-6-phosphate; NADP+(H) could not replace NAD+(H). The phosphatase (EC3.1.3.22) exhibited a distinct preference for mannitol-1-phosphate as substrate; all other substrates tested exhibited less than 25% of the activity observed with mannitol-1-phosphate.

Alcohol Oxidoreductases↗
PubMed1975

Preparation of the lactate oxidase apoenzyme and studies on the binding of flavin mononucleotide to the apoenzyme.

1. Lactate oxidase from Mycobacterium smegmatis is completely resolved into free flavin and apoenzyme by treatment with acid (NH4)2SO4. 2. Reconstitution involves rapid binding of FMN, but the recovery of enzyme activity was slower and appeared to be biphasic. 3. The preparation of the holoenzyme obtained differs from the native enzyme in specific activity, extinction coefficients and mobility on disc-gel electrophoresis. 4. Dialysis of this reconstituted enzyme in 0.1 M-sodium phosphate buffer, pH 7.0, at 0 degrees C for 1 week yields a preparation which closely resembles the native enzyme.

Alcohol Oxidoreductases↗
PubMed1974

Intracellular protein breakdown in thermophilic and mesophilic fungi.

1. The rate of protein breakdown was determined on growing and non-growing cultures of thermophilic and mesophilic fungi. 2. In growing cells protein breakdown was negligible. 3. In non-growing cells the breakdown rate of total protein varied between 5.2%/h and 6.7%/h. These values were found to be dependent on both the temperature of the protein breakdown assay and the temperature of growth of the organism. 4. The rate of breakdown of soluble protein in thermophilic fungi was 9-15%/h whereas the rate in mesophilic fungi for the soluble protein fraction was only 4%/h.

Aspergillus↗
PubMed1972

Purification and properties of Penicillium glucose 6-phosphate dehydrogenase.

1. Glucose 6-phosphate dehydrogenase was isolated and partially purified from a thermophilic fungus, Penicillium duponti, and a mesophilic fungus, Penicillium notatum. 2. The molecular weight of the P. duponti enzyme was found to be 120000+/-10000 by gelfiltration and sucrose-density-gradient-centrifugation techniques. No NADP(+)- or glucose 6-phosphate-induced change in molecular weight could be demonstrated. 3. Glucose 6-phosphate dehydrogenase from the thermophilic fungus was more heat-stable than that from the mesophile. Glucose 6-phosphate, but not NADP(+), protected the enzyme from both the thermophile and the mesophile from thermal inactivation. 4. The K(m) values determined for glucose 6-phosphate dehydrogenase from the thermophile P. duponti were 4.3x10(-5)m-NADP(+) and 1.6x10(-4)m-glucose 6-phosphate; for the enzyme from the mesophile P. notatum the values were 6.2x10(-5)m-NADP(+) and 2.5x10(-4)m-glucose 6-phosphate. 5. Inhibition by NADPH was competitive with respect to both NADP(+) and glucose 6-phosphate for both the P. duponti and P. notatum enzymes. The inhibition pattern indicated a rapid-equilibrium random mechanism, which may or may not involve a dead-end enzyme-NADP(+)-6-phosphogluconolactone complex; however, a compulsory-order mechanism that is consistent with all the results is proposed. 6. The activation energies for the P. duponti and P. notatum glucose 6-phosphate dehydrogenases were 40.2 and 41.4kJ.mol(-1) (9.6 and 9.9kcal.mol(-1)) respectively. 7. Palmitoyl-CoA inhibited P. duponti glucose 6-phosphate dehydrogenase and gave an inhibition constant of 5x10(-6)m. 8. Penicillium glucose 6-phosphate dehydrogenase had a high degree of substrate and coenzyme specificity.

Centrifugation, Density Gradient↗