Purification, biochemical properties and active sites of N-acetyl-β-d-hexosaminidases from human seminal plasma

Abstract

Two isoenzymes of N-acetyl-beta-D-glucosaminidase (EC 3.2.1.30) (Hex A and Hex B) from human seminal plasma were purified to homogeneity with specific activities of 26 and 60 units/mg of protein respectively. N-Acetyl-beta-D-glucosaminidase activity was inseparable from N-acetyl-beta-D-galactosaminidase activity in both Hex A and Hex B by various conventional chromatographic procedures. Although Km values of N-acetyl-beta-glucosaminidase activity of Hex A and Hex B were similar (1.33 mM), those of N-acetyl-beta-galactosaminidase activity were 0.14 mM for Hex A and 0.40 mM for Hex B. However, pH optima and temperature optima were identical for N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities of both isoenzymes; Hex A was far more heat-sensitive than Hex B. Thiol-reactive compounds such as silver salts, mercuric salts, p-chloromercuribenzoate and thimerosal strongly inhibited the N-acetyl-beta-glucosaminidase activities of both isoenzymes. GSH protected the enzyme activities from inactivation caused by these reagents, confirming the presence of thiol groups at the active centres. Inhibitions of N-acetyl-beta-glucosaminidase activities of both isoenzymes by metal salts and organic anions were comparable; acetate and arsenite were effective inhibitors for both isoenzymes. In contrast, inhibitions of N-acetyl-beta-glucosaminidase activities of the two isoenzymes by iodoacetic acid, iodoacetamide and ethylmaleimide were not comparable; Hex B was more susceptible to inhibition by these agents at 20 mM concentration. The N-acetyl-beta-glucosaminidase activities of both isoenzymes are strongly inhibited, in decreasing order, by N-acetyl-galactosamine, mannosamine, disaccharic acid lactone, N-acetylglucosamine and gluconolactone. The Ki values of the N-acetyl-beta-glucosaminidase and N-acetyl-beta-galactosaminidase activities for N-acetylhexosamines and results from mixed-substrate kinetics indicated that the activities for the two substrates are located at different sites in Hex A and at the same site in Hex B. The Mr values of Hex A and Hex B were determined to be 195,000 and 210,000 respectively by gel filtration through Sephadex G-200. SDS/polyacrylamide-gel electrophoresis revealed that Hex A and Hex B are each composed of four subunits corresponding to Mr about 50,000 each. No further polypeptide chain was obtained after reduction and alkylation of Hex A and Hex B with 10 mM-dithiothreitol and 10 mM-iodoacetamide.

The activity of N-acetyl-β-hexosaminidase in boar seminal plasma is linked with semen quality and its suitability for cryopreservation

“…Nacetyl-b-hexosaminidase is found not only inside the acrosome but also on the surface of the sperm plasma membrane [26]. It is also present in cytoplasmic droplets of immature spermatozoa [27].…”

“…A fraction with estimated molecular weight of 90 kDa was also reported regardless of electrophoretic conditions. The differences in the molecular weight of native enzymes could result from the presence of four to eight additional polypeptide chains in a b-HEX molecule of boar seminal plasma [26]. The enzyme shows affinity for Zn 2þ ions; therefore, it can affect sperm plasma membrane.…”

Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila

“…The inhibiting effect of Cu 2+ may suggest that cysteine residues are implicated in the catalytic function of the enzymes. Additional experiments using Ag + , Cd 2+ or Hg 2+ ions, p-chloromercuribenzoate or N-ethylmaleimide 29 in combination with mutational studies on the neuraminidases may help to fully understand the catalytic role of potentially affected thiol groups. Almost complete inhibition of Am0705 and Am2085 was observed by Co 2+ , whereas Am0707 and Am1757 could maintain more than 60% of their enzymatic activities.…”

Acrosomal Enzymes

“…Since these two enzymatic activities have not been separated biochemically, the enzyme is commonly known as P-hexosaminidase (Hex). The regionalization of its activity in mammalian epididymis, suggests a physiological role for the enzyme in the maturation of sperm as they through the epididymis andlor in the fertilization process (Kapoor and Gupta, 1986).…”