Glucoamylase electrophoresis

Fig. (27). Cel electrophoresis of glucoamylase in filtrate and purified glucoamylase, enzyme gel unstained and coupled with anti-glucoamylase antibodies.

The purification was by chromatography on DEAE cellulose. Electrophoresis of the purified enzyme and the original filtrate are shown in Fig. (27) [71]. Rabbits were immunized with the pure glucoamylase [72]. Antibodies in the serum were purified by affinity chromatography. The affinity pattern for glucoamylase purification is shown in Fig. (28). Electrofocusing and assay by the coupled method for antibodies is shown in Fig. (29). The glucoamylase antibodies consist of 8 isomers [73],... 

Fig. 3.—Gel electrophoresis and agar diffusion. 1 = gel stained for protein 2 = gel embedded in agar 3 = precipitin band 4 = glucoamylase antigen.

Little is known of the structure, the nature of the active site, or the physical properties of the glucoamylases. The purified enzyme firom A. niger has been reported to contain at least two isozymes, which are readily separated by electrophoresis, - and gommc-amylase has been separated into two components, one of which has an optimal pH of 4, and the other, of about 6.5. 

The separation by polyacrylamide gel electrophoresis and subsequent analysis of the components of guinea pig intestinal brush border membrane revealed the presence of inter alia enzyme complexes , a-D-glucosidase-glucoamylase, a-D-glucosidase-sucrose, and a-D-glucohydrolase-glucoamylase. Glucoamylase activity has been solubilized from rat intestinal mucosa (see p. 406). ... 

Glucoamylase from an Endomycopsis species has been purified to homogeneity on disc electrophoresis in polyacrylamide gel, isoelectric focusing, and ultra-... 

The data from these experiments did not give a clear-cut picture as to the origin of the extracellular amylase activity. Some autolysls did take place during the stationary phase, but the excretion of protein was clearly growth related. The liberation of glucose (Figures 1 and 2) Indicated that glucoamylase was part of the amylase activity. Preliminary experiments (unpublished) with gel electrophoresis have shown more than one protein with amylase activity. 

An acid a-D-glucosidase in human liver has been purified by adsorption onto Sephadex, followed by desorption with methyl a-D-glucopyranoside, a competitive inhibitor.The enzyme (p/4.58) was shown to be homogeneous by ultracentrifugation and disc gel electrophoresis. Guanidinium hydrochloride, but not urea, dissociated the enzyme. The glucoamylase activity, but not the maltase activity, was inhibited by methyl a-D-glucopyranoside. 

A partially purified glucoamylase from A. niger has been characterized by biochemical, physicochemical, and optical methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the enzyme consists of two principal components (mol. wts. 6.30 x 10 and 5.75 X 10 ). Small proportions of dissociated and aggregated species are also present, but the size of the monomer (3.0 x 10 ) was deduced from sedimentation studies, etc. Chemical modification of the enzyme indicated that tyrosyl residues are located at the active site. The tertiary structure of the molecule contains 15—25% of a-helix, as well as /9-structure and disordered segments. 

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