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Raffinose metabolism in escherichia coli k 12 purification and properties of a new alpha galactosidase ec specified by a transmissible plasmid

, : Raffinose metabolism in escherichia coli k 12 purification and properties of a new alpha galactosidase ec specified by a transmissible plasmid. European Journal of Biochemistry 67(1): 95-104

The utilization by E. coli K12 of raffinose as sole C source depends on a new raffinose transport system, an invertase [EC] and an .alpha.-galactosidase [EC] specified by the Raf-plasmid D1021. The .alpha.-galactosidase was purified to homogeneity from a mutant strain which synthesizes the enzyme constitutively. .alpha.-Galactosidase hydrolyzes p-nitrophenyl-.alpha.-D-galactoside (Km 0.14 mM), methyl-.alpha.-D-galactoside (Km 30 mM), melibiose (Km 3.2 mM) and raffinose (Km 60 mM). Its activity is strongly inhibited by Ag+, p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of 1 form of .alpha.-galactosidase with an isoelectric point of 5.1. The purified enzyme has an sw,20 [sedimentation coefficient] value of 11.7 .+-. 0.3 S and a MW of 329,000 .+-. 4000; this value is not reduced at high dilutions. When examined by dodecylsulfate gel electrophoresis, purified .alpha.-galactosidase yields a single subunit band of MW 82,000 suggesting that the intact enzyme consists of 4 subunits. Amino acid analysis indicates the presence of .apprx. 712 amino acid residues per 1/4 molecule including 8 half-cystine residues. No carbohydrate moiety was detected. High resolution EM micrographs and Markham rotation of .alpha.-galactosidase show enzyme molecules of .apprx. 11 .times. 11 nm containing 4 globular subunits in a tetragonal arrangement. The plasmid-coded .alpha.-galactosidase differs from the homologous E. coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance. It can thus be used as a marker enzyme suitable for the in vivo detection of Raf-plasmids.

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