PURIFICATION AND CHARACTERIZATION OF N - ACETYL - D-GLUCOSAMINIDASE FROM BOVINE KIDNEY

Abstract

The present work was designed to investigate physicochemical properties of N-acetyl-I3-D-glucosaminidase purified from bovine kidney. The enzyme was purified by ammonium sulphate fractionation, ion exchange chromatography on DEAEceliulose and gel filtration chromatography on Sephadex G-200. The subunit (or apparent) molecular weight was estimated by SDS-PAGEto be 40,000 daltons (40kDa) and 18,000 daltons (18kDa) for the a and /3 subunits (two bands) respectively of NAGase A isoenzyme. NAGase B, on SDS-PAGEproduces a single band corresponding to a subunit molecular weight of 18,000 daltons (18kDa), suggesting that NAGaseA may be a heterodimer of a /3subunits while NAGaseB may be a homodimer of /3/3subunits. The Michaelis-Menten constant, Km, of the enzyme for p-nitrophenyl-Nacetyl-/ 3-D-glucosaminide (substrate) was 0.625mM while the maximum velocity, Vmax,was 0.913 nrnole/rnl/rnin for the NAGase A isoenzyme and 5.0nmole per ml/min for the NAGaseB isoenzyme. The activity of the enzyme was not significantly altered by the addition of 20mM NH4C1,(NH4)2S04,K2S04, ZnS04, Na2S04, MgS04 , NaCll, ZnCh, CaCh and MgCh while addition of 20mM CuCI2and CUS04significantly inhibited the enzyme activity. The inhibitor constant, Ki, of the enzyme was 0.09 and 1.22 mM for Nacetyl-/ 3-D galactosamine and N- acetyl-/3-D-glucosamine respectively. The total neutral hexose content was 22811g/mg protein (23% neutral hexose) and 132 The present work was designed to investigate physicochemical properties of N-acetyl-I3-D-glucosaminidase purified from bovine kidney. The enzyme was purified by ammonium sulphate fractionation, ion exchange chromatography on DEAEceliulose and gel filtration chromatography on Sephadex G-200. The subunit (or apparent) molecular weight was estimated by SDS-PAGEto be 40,000 daltons (40kDa) and 18,000 daltons (18kDa) for the a and /3 subunits (two bands) respectively of NAGase A isoenzyme. NAGase B, on SDS-PAGEproduces a single band corresponding to a subunit molecular weight of 18,000 daltons (18kDa), suggesting that NAGaseA may be a heterodimer of a /3subunits while NAGaseB may be a homodimer of /3/3subunits. The Michaelis-Menten constant, Km, of the enzyme for p-nitrophenyl-Nacetyl-/ 3-D-glucosaminide (substrate) was 0.625mM while the maximum velocity, Vmax,was 0.913 nrnole/rnl/rnin for the NAGase A isoenzyme and 5.0nmole per ml/min for the NAGaseB isoenzyme. The activity of the enzyme was not significantly altered by the addition of 20mM NH4C1,(NH4)2S04,K2S04, ZnS04, Na2S04, MgS04 , NaCll, ZnCh, CaCh and MgCh while addition of 20mM CuCI2and CUS04significantly inhibited the enzyme activity. The inhibitor constant, Ki, of the enzyme was 0.09 and 1.22 mM for Nacetyl-/ 3-D galactosamine and N- acetyl-/3-D-glucosamine respectively. The total neutral hexose content was 22811g/mg protein (23% neutral hexose) and 132 The present work was designed to investigate physicochemical properties of N-acetyl-I3-D-glucosaminidase purified from bovine kidney. The enzyme was purified by ammonium sulphate fractionation, ion exchange chromatography on DEAEceliulose and gel filtration chromatography on Sephadex G-200. The subunit (or apparent) molecular weight was estimated by SDS-PAGEto be 40,000 daltons (40kDa) and 18,000 daltons (18kDa) for the a and /3 subunits (two bands) respectively of NAGase A isoenzyme. NAGase B, on SDS-PAGEproduces a single band corresponding to a subunit molecular weight of 18,000 daltons (18kDa), suggesting that NAGaseA may be a heterodimer of a /3subunits while NAGaseB may be a homodimer of /3/3subunits. The Michaelis-Menten constant, Km, of the enzyme for p-nitrophenyl-Nacetyl-/ 3-D-glucosaminide (substrate) was 0.625mM while the maximum velocity, Vmax,was 0.913 nrnole/rnl/rnin for the NAGase A isoenzyme and 5.0nmole per ml/min for the NAGaseB isoenzyme. The activity of the enzyme was not significantly altered by the addition of 20mM NH4C1,(NH4)2S04,K2S04, ZnS04, Na2S04, MgS04 , NaCll, ZnCh, CaCh and MgCh while addition of 20mM CuCI2and CUS04significantly inhibited the enzyme activity. The inhibitor constant, Ki, of the enzyme was 0.09 and 1.22 mM for Nacetyl-/ 3-D galactosamine and N- acetyl-/3-D-glucosamine respectively. The total neutral hexose content was 22811g/mg protein (23% neutral hexose) and 132 jJg!mg protein (13% neutral hexose) for the NAGaseA and NAGase'B isoenzvmes respectively. The free sialic acid content was 0.23 urnole/rnq protein and 0.16 urnole/rnq protein for NAGaseA and.B respectively. The specific activity of NAGase A enzyme was 62 nmole/rntn/mq protein from sephadex G-200 gel column while that of NAGaseB enzyme was estimated to be 114nmole/min/mg protein.