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Biomedical subjects

L W Fleming

Publications and source records attributed to L W Fleming.

62 records · Page 4Linked to original sources
PubMed1967

Purification and characterization of yeast beta-glucosidases.

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.

Chromatography, Gel↗
PubMed1967

Evidence for multiple molecular forms of yeast beta-glucosidase in a hybrid yeast.

A mixture of beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) eluted from diethylaminoethyl cellulose in two peaks, whereas the enzyme from a hybrid, S. fragilis x S. dobzhanskii (Y-42), eluted in a single broad peak. The highest Y-42 activity fractions eluted at a sodium chloride molarity which was intermediate to the molarities at which most of the Y-18 and Y-19 activity was eluted. In cellulose polyacetate strips, Y-42 enzyme migrated as a diffuse band which spanned the distances migrated by the enzymes from the parent yeast strains. Antisera against either Y-18 or Y-19 enzyme precipitated 80 to 90% of Y-42 enzyme activity. When Y-42 enzyme was dissociated by heat or urea and reacted with parental antiserum, a concomitant increase in the opposite parental activity was demonstrable in both precipitation and complement-fixation (CF) tests. Urea-dissociated beta-glucosidases were resolvable by sucrose-gradient centrifugation into multiple bands displaying specific CF activity. When the enzymes were exposed to 4 m urea for 12 min, particles of approximately 110,000 molecular weight were obtained. By extending the exposure time to 40 min, and incorporating 0.5 m urea in the gradients, smaller particles were detected with molecular weights ranging from 18,000 to 23,000. Attempts to regenerate enzyme activity after dissociation with urea were only moderately successful. Results suggested that a slightly acidic environment favored reassociation, as did the presence of 2-mercaptoethanol. Residual urea also seemed important. It is proposed that the structural genes for both Y-18 and Y-19 enzyme are present in Y-42 cells with either independent or closely interacting regulatory mechanisms. Since synthesis of the two parental-type polypeptides may be unequal, the availability of enzyme subunits for subsequent polymerization in the cell cytoplasm might be equalized at the polysome level. Random association of subunits would produce a binomial distribution of true hybrid enzyme molecules.

Centrifugation, Density Gradient↗