Thomas J. Andary

A sensitive polyacrylamide disc gel method for detection of proteinases

Abstract To enable direct detection of proteinase activities subsequent to electrophoresis, a technique utilizing the incorporation or diffusion of protein substrates into polyacrylamide disc gels was developed. Denatured insoluble substrates, casein or hemoglobin, were added to acrylamide solutions prior to polymerization of the gel mixture. Alternatively, soluble protein substrates were diffused into gels after electrophoresis. In either case, an incubation period ensued at the pH optimum of the proteinases to allow for their detection. Classification of resolved proteinases was accomplished subsequent to electrophoresis by incubation of gels in media containing either synthetic substrates, as the naphthylamide derivatives, or specific inhibitors of the enzymes. Separation of purified trypsin from chymotrypsin, and proteinases in preparations of seminal plasma and mouse blastocysts homogenates demonstrated the efficacy of the method at the submicrogram enzyme level.

Effects of prothrombin fragments on thrombin, on thrombin formation, and separation from Ac-globulin (factor V)

Abstract Bovine prothrombin fragment 2 (profragment 2) and prethrombin 1 were found in normal oxalated bovine plasma after extensive adsorption with barium carbonate. This implies continuous physiological utilization of prothrombin. Ac-globulin-profragment 2 complex was isolated from plasma by the method of Dombrose and Seegers. This purified complex dissociated at pH 7.2 in a buffer consisting of 0.15 M NaCl in 0.15 M phosphate buffer. The two proteins were separated by ultrafiltration. The profragment 2 passed through the filter while the Ac-globulin (Factor V) was retained in pure form. Purified bovine profragment 2 enhanced esterase activity of thrombin and retarded the clotting function, thus producing a large esterase/coagulation ratio in prethrombin 1 activation mixtures. Inhibition of the thrombin-fibrinogen reaction by profragment 2 must be taken into account in some assays for prothrombin or prethrombin 1. If profragment 2 is not bound in the activation mixture of a prothrombin assay, a low thrombin titer may be recorded. Contrary to a previous conclusion reached in this laboratory, there is no enzyme with only esterase activity in the following degradation sequence: Prothrombin → Prethrombin 1 → Prethrombin 2 → Thrombin. The esterase enhancement of thrombin and its coagulation inhibition by profragment 2 were neutralized by additions of purified Ac-globulin or by specific antibodies to profragment 2. The procoagulant or anticoagulant properties of purified bovine prothrombin fragment 1 (profragment 1) were studied. For that purpose, purified bovine profragment 1 was incubated with purified Ac-globulin, purified autoprothrombin C (Factor Xa), crude “cephalin,” and calcium ions. Due to profragment 1, the procoagulant function of the mixture diminished when tested against purified prothrombin as substrate, but the procoagulant function increased when tested against purified prethrombin 1 as substrate. Thus, depending upon conditions, profragment 1 can function as a procoagulant or as an anticoagulant.

Tryptic- and chymotryptic-like proteinases in early and late preimplantation mouse blastocysts.

Neutral tryptic- and chymotryptic-like enzyme activities have been identified in extracts of early and late mouse blastocyts. The enzyme activities are distinguishable my means of: (a) reactivity with specific naphthylamide derivatives; (b) reactivity with tosyl-L-lysine-chloromethyl ketone or L-tosylamido-2-phenylethyl-chloromethyl ketone, respectively; (c) electrophoretic mobilities; and (d) specific activity profiles of late vs. early blastocysts.

Blood coagulation as a cybernetic system: Control of autoprothrombin C (factor Xa) formation☆

Abstract The procoagulants of the intrinsic system are very powerful, but under physiological conditions seem to be weak because of their presence in functionally low concentrations. Autoprothrombin C (Factor Xa) activity generated rapidly from purified autoprothrombin III (Factor X) in a reaction mixture that contained optimum amounts of purified Factor VIII, a platelet factor 3 preparation (PF-3), a Factor IXa preparation, and 0.01 M calcium ions at pH 7.2. The initial generation of Auto-C was very rapid. Lowering the concentration of any one of the procoagulants below the optimum reduced the yield of Auto-C. In this system, Auto-C generation is governed by apparent stoichiometric reactions. At the completion of the reaction, very little Factor VIII activity remained and was apparently destroyed by Auto-C. Factor IXa generated some Auto-C without either PF-3 or Factor VIII. To meet the phospholipoprotein requirement, it was possible to substitute phosphatidylserine for PF-3, but not phosphatidylcholine or phosphatidylethanolamine. This enlarges on reports in the literature, where it is recorded that the latter two function with the protein of thromboplastin in Auto-C generation, and phosphatidylserine functions in thrombin generation. For the acceleration or initiation of blood coagulation, plasmatic factors as well as platelet materials are needed.

Bovine antithrombin-III: Miscellaneous observations

Abstract Bovine antithrombin-III was purified by two procedures. With the ultracentrifuge we found S 20,w = 4.18 − 0.015X. It was found to have an NH 2 -terminal histidine residue, neutral sugars, sialic acid and disulfide bonds. It formed a complex with purified bovine thrombin in a 1:1 molar proportion with residual quantities of either component remaining. It formed a 1:1 molar complex with purified Factor Xa. In the ultracentrifuge this complex sedimented as a single component with S 20,w = 4.65 − 0.034X. Antibodies were used to deplete plasma of antithrombin-III. The depleted plasma progressively inactivated purified thrombin.