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A low-molecular-weight and a high-molecular-weight 1,4-β-glucan glucanohydrolase (Cχ enzyme) have been isolated from a commercial cellulase preparation derived from culture filtrates of the fungus THchoderma vMde. The purification method for the isolation of the low-molecular-weight enzyme is a three-step procedure including chromatography on Bio-Gel P-10, chromatography on a dipolar adsorbent (arginine-fiepharose 6 B) and isoelectric focusing. The starting material for the isolation of the high-molecular-weight enzyme was pre-fractionated by chromatography on Bio-Gel P-10, by DEAE-Sephadex chromatography and by SE-Sephadex chromatography as described previously by us. Further fractionation of this material was achieved by affinity chromatography and repeated isoelectric focusing. Free zone electrophoresis of the low-molecular-weight enzyme indicated a homogeneous protein. The high-molecular-weight enzyme was homogeneous in sedimentation equilibrium analysis. The molecular weights of the enzymes were 12 500 and 50 000 ± 2000 respectively. The former value was determined by chromatography on a calibrated column of Bio-Gel P-100 and the latter value by sedimentation equilibrium analysis. The low-molecular-weight enzyme was isoelectric at pH 4.60 (10°C) and contained 21% carbohydrate. The corresponding values for the high-molecular-weight enzyme were pH 3.39 and 12%. Both enzymes were active in releasing free fibers from filter-paper. The low-molecular-weight enzyme was estimated to be about twice as effective as the high-molecular-weight enzyme in this regard. [ABSTRACT FROM AUTHOR]

An investigation has been made of the acid soluble protein fractions of wool that has been reduced with benzylmercaptane and treated with methyliodide. When this modified wool was shaken in dilute formic acid (50 v/v) at room temperature for 1 hour, 7% of a protein material was dissolved. After dialysis and subsequent lyophylisation the protein material was isolated, and shown to be heterogeneous by paper electrophoresis. Fractionation of this protein on Sephadex G-75 in dilute acetic acid gave more than six components of relatively high content of tyrosine and phenylalanine. One of these fractions, obtained in good yield, was purified by rechromatography. For further separation the protein fraction was subjected to ion exchange chromatography on Dowex 1 (×2) columns. By stepwise elution with buffers of decreasing pH-values, containing acetic acid and 8 M urea, four subfractions were obtained. The amino acid compositions of reduced wool, of the protein obtained by formic acid extraction, of the fraction after gel filtration on Sephadex G-75 and of one subfraction on Dowex 1 (×2) are given. In all three fractions an increase in glycine, tyrosine and phenylalanine content was observed with relatively low amounts of basic and acidic amino acids. The subfraction obtained after the final step of separation on Dowex 1 (×2) was further characterised by endgroup determinations. Dnp­glycine was the only N-terminal amino acid with traces of Dnp­alanine as contaminant. Hydrazinolysis resulted in tyrosine on the C-terminus. After digestion of the subfraction by trypsin and chymotrypsin a number of peptides with different chain lengths was obtained, indicating that, tyrosyl residues had accumulated and that larger arginyl­peptides were present. Sedimentation measurements by ultracentrifugation indicated that the subfraction had a weight average of apparent molecular weight in the range of 9,000 to 13,000. This is in agreement with the molecular weight of 16,400 derived from the amount of Dnp­glycine and the calculated one from the amino acid composition. The analytical results show that the subfraction represents a single polypeptide chain containing only one N-terminal residue and about hundred aminoacids of which about fifty are glycine, tyrosine and phenylalanine. [ABSTRACT FROM AUTHOR]

Flavanone synthase from irradiated cell suspension cultures of parsley was purified to apparent homogeneity. Molecular weights of about 77000 for the enzyme and about 42000 for the subunits were determined respectively by sedimentation-equilibrium measurements and disc-gel electrophoresis in the presence of dodecyl sulfate. A specific antiserum was prepared for the enzyme and was used in an assay for flavanone synthase mRNA activity in partially purified RNA preparations. The apparent molecular size of flavanone synthase mRNA was estimated by sucrose gradient centrif- ugation and gel electrophoresis under partially denaturing conditions. Values of about 17S and Mr = 0.62 × 106 were obtained. The fractionation patterns suggested that flavanone synthase mRNA was homogeneous in size. All together, the results support the idea that the enzyme is composed of two subunits which are probably identical. Amino acid analysis and a microbial assay were carried out to test the possible occurrence of cysteamine, β-alanine, and pantothenate in the enzyme. The results were negative, indicating the absence of pantetheine or a similar residue. The possible similarity in mechanism between flavanone synthase and 3-oxoacyl-(acyl carrier protein) synthase is discussed. [ABSTRACT FROM AUTHOR]