Effect of cobra and viper venoms on alpha 2-macroglobulin activity in human, bovine, and goat sera.

Incubation of human serum with cobra or viper venoms (10 micrograms/0.1 ml serum) caused negligible decrease in total protease inhibitory activity whereas alpha 2-macroglobulin activity was reduced by 67.0-82.0% in 16 hr. The action of venoms on MG activity was time dependent. Human alpha 2-macroglobulin activity was reduced to a much greater extent than goat or bovine factors by the venoms. While 25 micrograms venoms/0.1 ml serum caused 60-100% inhibition of human alpha 2-macroglobulin activity, the bovine factor was not affected under similar conditions. Goat alpha 2-macroglobulin was affected to the extent of 0-20%. Evidence is provided to show that venom proteases generate endogenous proteases in situ in human plasma or serum which in turn bind to alpha 2-macroglobulin. The venom-mediated action was abolished by prior dialysis of the serum or its dilution. Ethylenediaminetetraacetate at 10(-3) M concentration also blocked the reaction. While phenylmethylsulfonyl fluoride had no effect, pepstatin in the concentration range 10(-2) to 10(-3) M caused partial inhibition of the venom-mediated inhibition of alpha 2-macroglobulin activity in human serum.

Isolation and characterization of a specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris).

A specific enterokinase inhibitor from kidney bean (Phaseolus vulgaris) was purified to homogeneity. It showed a single protein band on sodium dodecyl sulphate/polyacryl-amide-gel electrophoresis in the presence of mercaptoethanol, and the Mr was 31000. Aspartic acid was identified as the N-terminus of the inhibitor. The Mr by gel chromatography on Sephadex G-200 was found to be 60000, indicating the dimeric nature of the inhibitor. The inhibitor was found to be a glycoprotein. The monosaccharide moieties were glucose, mannose, glucuronic acid and glucosamine in the proportions 3.15%, 5.0%, 0.85% and 1.3% respectively. The inhibitor was most active on pig enterokinase, followed by bovine and human enterokinases. Maximal inhibitory activity was elicited by preincubation of the inhibitor with the enzyme for 15 min. Digestion with pepsin resulted in loss of inhibitory activity. The inhibitor was stable to exposure to a wide range of pH values (2-10), and exposure to pH above 10 resulted in loss of inhibitory activity. Modification of arginine residues by cyclohexane 1,2-dione and ninhydrin led to complete loss of enterokinase-inhibitory activity.

Variants of gamma-glutamyl transferase in human urine.

When dialyzed human urine was subjected to ultracentrifugation, gamma glutamyl transferase activity amounting to one-third or less of the total activity was found associated with the microsomal fraction and the remaining in the 100 000 X g supernatant. By ion-exchange chromatography on DEAE-cellulose at pH 7.5, the urinary enzyme was resolved into two fractions. The less anionic form (eluted with 0.15 mol/l NaCl) corresponded to the 100 000 X g supernatant fraction. The more anionic form (eluted with 0.5 mol/l NaCl) corresponded to the microsomal fraction. On chromatography on concanavalin A-sepharose, the urinary transferase activity separated into the unbound fraction and the bound fraction (eluted with alpha-methyl-D-glucoside). The bound fraction was retained more tightly on DEAE-cellulose (eluted with 0.5 mol/l NaCl). The unbound fraction had weaker interaction with the ionic-exchanger (eluted with 0.15 mol/l NaCl). Each of two fractions separated by affinity chromatography on concanavalin A-sepharose, resolved into two bands on cellulose acetate electrophoresis at pH 7.t. In addition, during gel chromatography on Sephadex G-200, they separated into a high molecular weight (greater than 200 000) fraction and a low molecular weight (55 000) fraction. The band that stayed at the origin during electrophoresis was high molecular weight in nature, whereas the fraction that moved towards the anode was found to be the smaller form of the enzyme. The study of the variants of urinary gamma glutamyl transferase was not found to be of clinical significance. The nature and origin of the variant forms of the enzyme in urine are discussed.