Guanidinobenzoatase on the surface of tumour cells

Abstract

Guanidinobenzoatase is a recently described serine enzyme (Steven & Al-Ahmad, 1983) that hydrolyses two active-site titrants for trypsin, namely 4-methylumbelliferyl p-guanidi nobenzoate (Coleman et al.. 1976) and 4-nitrophenyl pguanidinobenzoate (Chase & shaw, 1967). Unlike an active-site titration, the hydrolysis of these two substrates by guanidinobenzoatase is continuous over a 2h period. Each of these compounds acts as a competitive substrate for guanidinobenzoatase when the other compound is employed as the prime substrate in the enzyme assay. Guanidinobenzoatase is inhibited by a-N-benzoyl-DL-arginine ethyl ester and is unable to cleave this substrate for trypsin-like esterases and can therefore be distinguished from the esterase described by Walsh & Nadler (1980) that is assayed with tr-N-benzoyl-DL-arginine ethyl ester and also cleaves 4meth ylumbelliferyl-p-guanidinobenzoate. Guanidinobenzoatase is present in the extracellular fluid surrounding Ehrlich-ascites-tumour cells grown in mice (Steven & Al-Ahmad, 1982) and in the tumour fluid obtained from V,-carcinoma implanted in the leg muscles of rabbits (McCroskery et al., 1975). Normal rabbit muscle and rabbit serum lack guanidinobenzoatase, but an enzyme similar to guanidinobenzoatase is present in normal rabbit intestine and extracts of intestinal tissue. The enzyme can be easily purified from tumour fluids employing a two-step affinity Sepharose procedure. The tumour fluid ( 5 ml) is first mixed with loop1 of aprotinin (lo00 kallikrein-inhibitory units; Trasylol) in order to inhibit proteinases that may be present. The tumour fluid is then equilibrated with 2ml of damp Sepharose-2B in order to adsorb an enzyme which attaches to Sepharose and which rapidly degrades starch. Guanidinobenzoatase is not bound to Sepharose-2B but is bound to a second support made of agmatine-Sepharose-4B. The unbound protzins are eluted from the agmatine-Sepharose-4B with iso-osmotic NaCl and the guanidinobenzoatase is finally eluted with 0.2 M-Na,SO,, pH 6.0. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate demonstrated the presence of three polypeptide chains derived from guanidinobenzoatase. The guanidinobenzoatase eluted from agmatine-Sepharose-4B was successfully employed to raise antisera in rabbits against the mouse enzyme; these sera caused inhibition of guanidinobenzoatase assayed with 4meth ylumbelliferyl p-guanidinobenzoate. Agar gels (190) were prepared in 0.1 M-phosphate buffer,