Abstract
Bovine hepatic gamma-glutamyl hydrolase (conjugase) has been purified to homogeneity. A feature of the purification procedure was the use of high affinity macromolecular polyanion enzyme inhibitors which formed tight complexes with the enzyme altering its solubility, gel filtration, and ion exchange properties. The enzyme, which cleaves the gamma-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with its lysosomal localization. Zinc is essential for enzyme stability. The presence of highly reactive sulfhydryl groups was evident from the extreme sensitivity to oxidizing agents and organomercurials. Very little thermal denaturation occurs below 65 degrees, but the enzyme is extremely sensitive to 0uffer anions, in keeping with the polyanionic nature of the substrate. In order to study the mechanism of action of the enzyme, a wide range of pteroylpolyglutamates, N-t-Boc polyglutamates and free polyglutamates were synthesized containing L-[U-14C]glutamic acid residues in different positions. Two pteroyltriglutamate derivatives were also synthesized in which an alpha bond replaced one of the two available gamma bonds. Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at gamma-glutamyl bonds and the presence of a COOH-terminal gamma bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should therefore be more appropriately classified as an acid hydrolase; (c) longer chain gamma-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of gamma bonds was independent of the NH2-terminal pteroyl moiety. Studies with polyanions such as the glycosaminoglycans and dextran sulfate supported the concept that the polyanion structure of the substrate was a major factor in substrate-active site interaction.
Highlights
Bovine hepatic y-glutamyl hydrolase has been purified to homogeneity
The enzyme, which cleaves the y-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with its lysosomal localization
Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at y-glutamyl bonds and the presence of a COOH-terminal y bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should be more appropriately classified as an acid hydrolase; (c) longer chain y-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of y bonds was independent of the NH,terminal pteroyl moiety
Summary
Pteroylglutamic acid was obtained from Sigma Chemical Co. N-t-Boc-r-glutamic acid-cu-benzyl ester, y-diglutamate (Glu-Glu), i y-triglutamate, a-diglutamate (c~-Glu-Glu), and a-triglutamate were purchased from the Fox Chemical Corp. Radioactive pteroyltriglutamate standard was a gift from Professor Charles Baugh while pure pteroic acid and pteroyldiglutamate standards were generously supplied by the American Cyanamid Corp. N-t-BOC-L- [U-“Clglutamic acid-a-benzyl ester was synthesized by New England Nuclear. 2000 were purchased from Pharmacia and Concanavalin A-fluorescein isothiocyanate from Miles-Yeda Ltd. Cibacron blue 3GA and Cibacron brilliant blue BRP were gifts from Ciba-Geigy (Australia). Polyethylene glycol 6000 was obtained from Osta Chemicals Ltd. Biochemical Corp. Chondroitin sulfates A, B, C, and D, heparin, and hyaluronic acid were obtained from Sigma. All other chemicals and materials were obtained through routine sources. Solvents and reagents required for solid phase peptide synthesis were routinely purified by fractional distillation prior to use
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
