Isolation of two inactive fragments of a Rhizopus sp. glucoamylase: relationship among three forms of the enzyme and the isolated fragments.
Two inactive fragments of glucoamylase [EC 3.2.1.3] from a Rhizopus sp. were isolated from the glucoamylase fraction obtained by CM-Sephadex chromatography in the previous purification of the glucoamylase (T. Takahashi et al. (1978) J. Biochem. 84, 1183-1194); the fraction contained at most 2.5% of the fragments, besides a mixture (97.5%) of three glucoamylases, designated as Gluc1 (M.W. 74,000), Gluc2 (M.W. 58,600), and Gluc3 (M.W. 61,400) in order of content. The two isolated fragments, named fragments H and L in order of size, were found to be homogeneous in polyacrylamide gel electrophoresis, isoelectric focusing, and ultracentrifugation analysis. The molecular weights of fragments H and L were 16,700 and 14,400, respectively. Fragment H had alanine as its N-terminal residue although fragment L was heterogeneous at the N-terminal amino acid. The N-terminal amino acids of Gluc1, Gluc2, and Gluc3 were found to be alanine, glutamic acid, and lysine, respectively; the three enzymes had the same C-terminal amino acid sequence of -Ser-Ala . OH. Immunodiffusion demonstrated that both fragments cross-reacted with Gluc1 and Gluc3 but not with Gluc2, although the three enzymes had common antigenicity. These results, together with the amino acid and sugar compositions of the fragments, indicate that the two fragments were derived from the N-terminal glycopeptide moiety of Gluc1 by the action of a proteolytic enzyme(s) with concomitant formation of Gluc2; Gluc3 also seems to be produced by proteolytic modification of Gluc1 with loss of its N-terminal peptide moiety.
D000596:Amino Acids / Q000032:analysis; D000936:Antigen-Antibody Complex; D002241:Carbohydrates / Q000032:analysis; D005087:Glucan 1,4-alpha-Glucosidase / Q000302:isolation & purification*; D005959:Glucosidases / Q000302:isolation & purification*; D007106:Immune Sera; D005779:Immunodiffusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D012233:Rhizopus / Q000201:enzymology*
D000596:Amino Acids; D000936:Antigen-Antibody Complex; D002241:Carbohydrates; D007106:Immune Sera; D010446:Peptide Fragments; D005959:Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase