Publications

(11 Cell extracts and extracellular culture fluids of species of the yeast genus Schizosaccharomyces exhibited exo-beta-(1 leads to 3)- and exo-beta-(1 leads to 6)-glucanase (EC 3.2.1.-) activities. (2) Using a combination of Sephadex G-100 and DEAE-cellulose chromatography, the exo-beta-(1 leads to 3)-glucanases from the cell extracts and culture fluid of Schizosaccharomyces japonicus var. versatilis were purified extensively. The enzymes from either location exhibited similar purification and other properties. (3) The purified enzymes hydrolysed the beta-(1 leads to 6)-glucosidic linkage in addition to the beta-(1 leads to 3) linkage. Heat denaturation, inhibition and electrophoretic studies indicated that both hydrolytic activities were properties of a single protein. Laminarin and pustulan hydrolysis followed Michaelis-Menten kinetics. The Km and V for laminarin hydrolysis were 6.25 mg/ml and 350 mumol of glucose released/min/mg protein, and for pustulan they were 166 mg/ml and 52 mumol of glucose released/min/mg protein. (4) The exo-beta-glucanase was assigned a molecular weight of 43 000. (5) the purified enzyme failed to hydrolyse isolated cell walls from either baker's yeast or Schizosaccharomyces pombe or to induce protoplast formation from intact cells of S. japonicus var. versatilis or Saccharomyces cerevisiae.

Kinetic analyses of monoanion inhibition and 15Cl nuclear magnetic resonance at 5.88 MHz were employed to study monoanion interactions with the zinc metalloenzyme, renal dipeptidase. The enzyme-catalyzed hydrolysis of glycyldehydrophenylalanine exhibited competitive inhibition when the reaction rate was determined in the presence of the monovalent anions fluoride, chloride, bromide, iodide, azide, nitrate, or thiocyanate or upon the addition of the divalent anion, sulfate. Competitive inhibition was produced by these anions. One anion was bound per enzyme molecule, and except in the case of fluoride all of the anions appeared to bind at the same site. Cyanide ion produced a much more effective inhibition of renal dipeptidase than the other monoanions, and it was shown that two cyanide ions were bound per enzyme molecule. An investigation of the effect of pH upon monoanion inhibition suggested that the anion inhibitors bind to the group with a pK of approximately 7.8. Complete dissociation of this group (approximately pH 8.4) eliminates the inhibitory effect of anions. The 35Cl line broadening produced by renal dipeptidase in 0.5 M NaCl solutions was 100 times more effective than that produced by equivalent concentrations of aquozinc(II). The line broadening was dependent upon the concentration of the metalloenzyme and independent of the frequency of the exciting radiation. When zinc ion was removed from the metalloenzyme by dialysis or when chloride was titrated from the metalloenzyme by cyanide, line broadening was decreased. Treatment of renal dipeptidase with saturating concentrations of the competitive inhibitor, guanosine triphosphate, in the presence of 0.5 M NaCl also produced a significant decrease in the 35Cl line width. The 35Cl line broadening produced by renal dipeptidase was shown to decrease with increasing pH through the range pH 5.8-10.8. This line-width variation with pH appeared to result from the titration of a site on the metalloprotein with an approximate pK of 7.4. Temperature studies of 35Cl line broadening by the metalloenzyme in the presence of chloride and cyanide inhibitors suggest that the fast exchange process pertains and that the dominant relaxation mechanism is quadrupolar in nature.