Purification of Two ß-N-Acetyl-d-glucosaminidases from Beef Spleen
Jacob A. Verpoorte 1
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1 From the Department of Biochemistry, Dalhousie University, Halifax, Canada
Two enzymes (A and B), each with ß-N-acetyl-d-glucosaminidase and ß-N-acetyl-d-galactosaminidase activity, were obtained from beef spleen extracts. Final purifications: Enzyme A, 310-fold; B, 260-fold. Purity was confirmed by gel electrophoresis and ultracentrifugation. Only A was bound by DEAE-cellulose, at pH 7.0. The two enzymes had identical weight-average and z-average molecular weights: the latter was unchanged by addition of guanidine hydrochloride or dithiothreitol, but decreased greatly when both were present, indicating more than one peptide chain in each enzyme. Amino acid compositions were similar, but more sialic acid and neutral carbohydrate were present in A than in B. Optical rotatory and circular dichroism spectra also were similar: no Cotton effects were observed at 250 to 300 nm, and rotations and ellipticities were very low at wave lengths <250 nm. Enzyme structure could not be deduced by fitting with calculated curves. Km values were identical but Vmax was slightly greater in A; changes in pH affected Km and Vmax of both enzymes.
Kinetic studies showed that the two p-nitrophenyl substrates tested compete for the active sites on both enzymes. Stimulation by bovine serum albumin enhanced Vmax but did not affect Km. Both enzymes were strongly inhibited by various metal ions; neither was affected by sulfhydryl reagents, but activity vanished during treatment with dithiothreitol.
Submitted on March 16, 1972
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