Pig gastric and small-intestinal mucus glycoproteins: proposed role in polymeric structure for protein joined by disulphide bridges.

Abstract

of the value before heating, but the rotation of the control enzyme decreased further, suggesting that cross-links were favouring the acquisition of native-like structure, whereas the unsubstituted protein was irreversibly denatured at 8OOC. Penicillinase activity was determined from the decrease in either A,,, (1 mM-benzylpeniciIlin) or (55pM-cephaloridine) in 1 mM-EDTA (disodium salt) and 39 rn~-KH,P0, /6 1 mMNa,HPO,, pH 7.0 at 2OOC. The benzylpenicillinase activity of the control enzyme was 201pmol/min per mg. The rate of cephaloridine hydrolysis after maximal deactivation was 0.053 pmol/min per mg. Treatment with dimethyl suberimidate for either 90, 150 or 1140min resulted in a decrease in benzylpenicillinase specific activity to 78% (s.D. = 2.0 (n = 3)l of that of the control enzyme. No deactivation by benzylpenicillin was observed with either the control or the treated enzyme. Both control and treated enzymes were deactivated by cephaloridine. However, the deactivated rate of cephaloridine hydrolysis with the treated enzyme was 45.5% [s.D. = 1.3 (n = 3)1 of the rate with the control enzyme. In summary, treatment with dimethyl suberimidate at pH 8.4 resulted in a product with almost normal activity with benzylpenicillin but with less than half of the normal deactivated activity with cephaloridine. The small effect on the activity with benzylpenicillin suggests that cross-linking did not directly involve the active site. We suggest that some of the (presumably heterogeneous) cross-links stabilize part of the protein conformation that is barely involved in any isomerization during the catalytic cycle with benzylpenicillin. The additional stability could contribute to lower activity in the deactivated state by an indirect effect on the active site. Alternatively, if the deactivated enzyme consists of an equilibrium mixture of active and inactive molecules, cross-linking could retard a conformational change that is necessary for the reactivation process but that is not necessary for deactivation. Our results contrast with the correlation between destruction of conformational 'flexibility' and protection against deactivation that has been reported for another penicillinase (Klemes 8c Citri, 1979). ,