Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus - PubMed
Purification and characterization of a developmentally regulated carboxypeptidase from Mucor racemosus
M E DiSanto et al. J Bacteriol. 1992 Jan.
Abstract
A developmentally regulated carboxypeptidase was purified from hyphae of the dimorphic fungus Mucor racemosus. The enzyme, designated carboxypeptidase 3 (CP3), has been purified greater than 900-fold to homogeneity and characterized. The carboxypeptidase migrated as a single electrophoretic band in isoelectric focusing polyacrylamide gel electrophoresis (PAGE), with an isoelectric point of pH 4.4. The apparent molecular mass of the native enzyme was estimated by gel filtration to be 52 kDa. Sodium dodecyl sulfate (SDS)-PAGE under nonreducing conditions revealed the presence of a single polypeptide of 51 kDa. SDS-PAGE of CP3 reacted with 2-mercaptoethanol revealed the presence of two polypeptides of 31 and 18 kDa, indicating a dimer structure (alpha 1 beta 1) of the enzyme with disulfide-linked subunits. By using [1,3-3H]diisopropylfluorophosphate as an active-site labeling reagent, it was determined that the catalytic site resides on the small subunit of the carboxypeptidase. With N-carboben zoxy-L-phenylalanyl-L-leucine (N-CBZ-Phe-Leu) as the substrate, the Km, kcat, and Vmax values were 1.7 x 10(-4) M, 490 s-1, and 588 mumol of Leu released per min per mg of protein, respectively. CP3 was determined to be a serine protease, since its catalytic activity was blocked by the serine protease inhibitors diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and 3,4-dichloroi Socoumarin (DCI). The enzyme was strongly inhibited by the mercurial compound p-chloromercuribenzoate. The carboxypeptidase readily hydrolyzed peptides with aliphatic or aromatic side chains, whereas most of the peptides which contained glycine in the penultimate position did not serve as substrates for the enzyme. Although CP3 activity was undetectable in Mucor yeast cells, antisera revealed the presence of the enzyme in the yeast form of the fungus. The partial amino acid sequence of the carboxypeptidase was determined.
Similar articles
-
Isolation and characterization of carboxypeptidase III from germinating triticale grains.
Drzymała A, Bielawski W. Drzymała A, et al. Acta Biochim Biophys Sin (Shanghai). 2009 Jan;41(1):69-78. doi: 10.1093/abbs/gmn008. Acta Biochim Biophys Sin (Shanghai). 2009. PMID: 19129952
-
Drzymała A, Prabucka B, Bielawski W. Drzymała A, et al. Plant Physiol Biochem. 2012 Sep;58:195-204. doi: 10.1016/j.plaphy.2012.06.025. Epub 2012 Jul 13. Plant Physiol Biochem. 2012. PMID: 22831920
-
Cheng TC, Ramakrishnan V, Chan SI. Cheng TC, et al. Protein Sci. 1999 Nov;8(11):2474-86. doi: 10.1110/ps.8.11.2474. Protein Sci. 1999. PMID: 10595552 Free PMC article.
-
Production, purification, and properties of serine carboxypeptidase from Paecilomyces carneus.
Umetsu H, Hishinuma K, Wake H, Ichishima E. Umetsu H, et al. Curr Microbiol. 1996 Jul;33(1):44-8. doi: 10.1007/s002849900072. Curr Microbiol. 1996. PMID: 8661688
-
St Leger RJ, Bidochka MJ, Roberts DW. St Leger RJ, et al. Arch Biochem Biophys. 1994 Nov 1;314(2):392-8. doi: 10.1006/abbi.1994.1458. Arch Biochem Biophys. 1994. PMID: 7979380
Cited by
-
Purification and characterization of a new type of serine carboxypeptidase from Monascus purpureus.
Liu F, Tachibana S, Taira T, Ishihara M, Yasuda M. Liu F, et al. J Ind Microbiol Biotechnol. 2004 Jan;31(1):23-8. doi: 10.1007/s10295-004-0107-z. Epub 2004 Jan 29. J Ind Microbiol Biotechnol. 2004. PMID: 14749965
-
Tabuchi M, Iwaihara O, Ohtani Y, Ohuchi N, Sakurai J, Morita T, Iwahara S, Takegawa K. Tabuchi M, et al. J Bacteriol. 1997 Jul;179(13):4179-89. doi: 10.1128/jb.179.13.4179-4189.1997. J Bacteriol. 1997. PMID: 9209031 Free PMC article.
-
Debittering effect of Actinomucor elegans peptidases on soybean protein hydrolysates.
Li L, Yang ZY, Yang XQ, Zhang GH, Tang SZ, Chen F. Li L, et al. J Ind Microbiol Biotechnol. 2008 Jan;35(1):41-7. doi: 10.1007/s10295-007-0264-y. Epub 2007 Oct 18. J Ind Microbiol Biotechnol. 2008. PMID: 17943333
-
Liu F, Tachibana S, Taira T, Ishihara M, Kato F, Yasuda M. Liu F, et al. J Ind Microbiol Biotechnol. 2004 Dec;31(12):572-80. doi: 10.1007/s10295-004-0190-1. Epub 2004 Dec 9. J Ind Microbiol Biotechnol. 2004. PMID: 15592905
-
Electrophoretic karyotype of Mucor circinelloides.
Nagy A, Vágvölgyi C, Balla E, Ferenczy L. Nagy A, et al. Curr Genet. 1994 Jul;26(1):45-8. doi: 10.1007/BF00326303. Curr Genet. 1994. PMID: 7954895
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources