Studies on Human β-d-N-Acetylhexosaminidases: II. KINETIC AND STRUCTURAL PROPERTIES

Abstract

Abstract β-d-N-Acetylhexosaminidase A and B purified to homogeneity from human placenta have pH optima of 4.4 using 4-methylumbelliferyl-β-d-N-acetylgalactosaminide as substrate. The Km of both hexosaminidase A and hexosaminidase B for this synthetic substrate was found to be 0.5 mm and heat of activation for both the enzymes was 10,500 cal. No effect of any divalent ions studied was observed except for a 25% decrease in the enzyme activity of both hexosaminidase A and B in the presence of 5 mm cupric ion. p-Hydroxymercuribenzoate, 1 mm, almost completely inhibited both hexosaminidase A and hexosaminidase B activity. N-Ethylmaleimide, arsenate, and iodoacetate had no effect on the hexosaminidases. No inhibition of hexosaminidase A or hexosaminidase B was observed in the presence of 30 mm concentration of various anions except for acetate which brought about 75% inhibition of both hexosaminidase A and hexosaminidase B activity. Using Sephadex G-200 filtration, the molecular weight of hexosaminidase A and B was estimated at 140,000. However, using the sedimentation equilibrium technique the molecular weight was found to be 100,000 ± 3,000 and 101,400 ± 4,700 for hexosaminidase A and B, respectively. After S-carboxymethylation, the subunit molecular weight of hexosaminidase A and B was determined using various concentrations of polyacrylamide in disc electrophoresis in the presence of sodium dodecyl sulfate. Hexosaminidase B dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000, whereas hexosaminidase A dissociated into one major subunit corresponding to a molecular weight of 17,000 to 18,000 and two more protein bands corresponding to about 35,000 and to 55,000. Similar results were obtained when hexosaminidase A and B were dissociated into subunits by treatment with guanidine hydrochloride or by treatment with maleic anhydride in the presence of sodium dodecyl sulfate and the subunits separated by polyacrylamide gel electrophoresis. In urea-starch gel electrophoresis hexosaminidase A dissociated into three major bands and hexosaminidase B into two bands. In urea-starch gel the electrophoretic mobility of one of the bands of dissociated hexosaminidase A and hexosaminidase B seemed to be very similar or identical. Both hexosaminidase A and hexosaminidase B have blocked NH2-terminal groups. The COOH-terminal amino acids in hexosaminidase A was found to be serine and in hexosaminidase B was found to be aspartic acid or asparagine. The amino acid composition of hexosaminidase A and hexosaminidase B was determined and found to differ.