Lack of effect of carbohydrate depletion on some properties of human mast cell chymase

Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This e...

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Bibliographic Details
Published in:Biochimica et biophysica acta 1999-03, Vol.1427 (1), p.74-81
Main Authors: Takao, Kazumasa, Takai, Shinji, Shiota, Naotaka, Song, Keifu, Nishimura, Kazuo, Ishihara, Takafumi, Miyazaki, Mizuo
Format: Article
Language:English
Subjects:
Angiotensin
Carbohydrates - analysis
Chymase
Chymases
Deglycosylation
Enzyme Stability
glycoproteins
Glycosylation
Hot Temperature
Human
Humans
Hydrogen-Ion Concentration
Kinetics
Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase - chemistry
Mast Cells - enzymology
n-glycosylation
Polysaccharides - analysis
Serine Endopeptidases - chemistry
Serine Endopeptidases - isolation & purification
serine proteinases
Substrate Specificity
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Summary:Human chymase from vascular tissues was purified to homogeneity by heparin affinity and gel filtration chromatography. Treatment of human chymase with endoglycosidase F resulted in cleavage of the carbohydrate moiety yielding a deglycosylation product that did not lose its catalytic activity. This enzymatic deglycosylation product was enough to explore possibilities that N-glycan might modify some properties of human chymase. Substrate specificity, optimum pH and the elution profile from the heparin affinity gel were not affected by the deglycosylation. Only a slight but significant difference was observed in the K m value for conversion of angiotensin I to angiotensin II. Other kinetic constants such as k cat were not influenced. The kinetics of conversion of big endothelin-1 to endothelin-1(1–31) were not significantly affected. The deglycosylated human chymase was more susceptible to deactivation under alkaline pH and thermal stress. Even at physiological temperature and pH, the activity of glycosylated human chymase was more stable. From these results, it appears that the N-glycan of human chymase contributes to the stability of this enzyme but not to its functional properties.
ISSN:0304-4165
0006-3002
1872-8006
1878-2434
DOI:10.1016/S0304-4165(99)00002-1