Amidase Activity of Urokinase I. Hydrolysis of a-N-Acetyl-L-Lysine p-Nitroanilide

D Petkov; E Christova; M Karadjova

doi:10.1055/s-0038-1647770

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1973; 29(02): 276-285
DOI: 10.1055/s-0038-1647770

Original Article

Schattauer GmbH

Amidase Activity of Urokinase I. Hydrolysis of a-N-Acetyl-L-Lysine p-Nitroanilide

D Petkov

¹Laboratory of Protein Chemistry, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 13, Bulgaria

,

E Christova

¹Laboratory of Protein Chemistry, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 13, Bulgaria

,

M Karadjova

¹Laboratory of Protein Chemistry, Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia 13, Bulgaria

› Author Affiliations

Abstract

Summary

α-N-acetyl-L-lysine p-nitroanilide (ALNA) was synthesized as a low-molecular weight model of the natural urokinase substrate plasminogen. Urokinase was found to catalyse anilide bond cleavage. The extent of hydrolysis can be followed spectro- photometrically by using the strong absorption band of one of the products p-nitro- aniline. An accurate, continuous and convenient assay for urokinase is proposed. As determined by the kinetic study, V_max5 ^a measure for the sensitivity of an assay, is about 8 times higher than that obtained for the most sensitive urokinase ester substrates. This was demonstrated by its use for tracing the elution of urokinase during the purification of the enzyme by column chromatography.

The high apparant K_m value obtained is discussed in terms of the mechanism of serine proteinase action on esters and amides.

Full Text

References

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