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Non-competitive, sandwich enzyme immunoassay for both penicillin amidases from Escherichia coli is described. The assay involves the use of monospecific antibodies and their conjugates. The amidases inactivated by heating and by acid- or alkali-treatment cannot be assayed. Reproducible results for each amidase were achieved within 5-6 hours in the range of 3-500 ng/ml (0.25-40 mU/ml) and coefficient of variation was 13%.

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Immunoadsorbents were obtained by coupling antibodies to Sepharose 4 B activated with cyanogen bromide. Thus immobilized antibody directed against bovine kidney gamma-glutamyltransferase was used for immunoaffinity chromatography of the enzyme from bovine kidney and liver, from cow milk and from sheep kidney and liver. Immobilized anti-rat kidney gamma-glutamyltransferase antibody was used for purification of the enzyme from rat kidney, mouse kidney, hamster kidney and rat Morris hepatoma 5123D. Yields of the protease-solubilized gamma-glutamyltransferases isolated on immunoadsorbents columns were usually over 60%. The purified enzymes were almost homogenous in polyacrylamide gel electrophoresis. The enzymes showed different molecular weights and electrophoretic mobilities. The effect of antibodies on affinity of the enzymes to substrate and inhibition by synthetic anthglutin and its isomers were studied.

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Anti-BPTI-antibody inactivated the antitrypsin activity of basic pancreatic trypsin inhibitor. Esterification of BPTI with methanol did not affect its antitrypsin activity and precipitate formation with antibody. Acetylation, maleylation and hexa-S-carboxylation of BPTI completely inactivated the inhibitor reactivity and markedly diminished its precipitating ability. Performic acid oxidized BPTI and thermolysin digested BPTI lost its antitrypsin as well as antigenic activities. The both preparations as well as oxidized N-acetyl-L-cysteinyl-L-lysyl-L-alanylglycylglycyl-L-cysteine amide did not affect the complex formation between the inhibitor and antibody.

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From Escherichia coli PCM 271 cells two penicillin amidases were separated by affinity chromatography on immunoadsorbent column. In cells grown in organic medium the activities of the amidase 1 and 2 were 30 and 70% respectively, whereas the activity of the amidase 1 in the cells grown in inorganic medium increased up to 98%. The amidase 1 migrated faster in polyacrylamide gel electrophoresis and was retained on DEAE-cellulose in 10 mM phosphate buffer, pH 8. No catalytic differences were demonstrated between the amidases.

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By manifold immunizations of rabbits with virgin or modified trypsin inhibitor III from squash seeds and trypsin inhibitor II b from cucumber seeds, specific antibodies were produced. In double immunodiffusion the anti-squash inhibitor antibody also gave weak precipitate arcs with inhibitor I from squash, inhibitor II from summer squash and with inhibitor I from zucchini, but not with inhibitor II b from cucumber seeds. The genus of Cucurbita trypsin inhibitors, preincubated with the antibody, lost their antitrypsin activity. The antibody showed a significantly weaker effect on the activity of the inhibitor from cucumber sees. 1H-NMR and CD spectra also confirm structural differences between trypsin inhibitors from the genus of Cucurbita and the cucumber (genus of Cucumis) inhibitor.

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A simple and sensitive method of studies on interactions of antibodies with the light forms of gamma-glutamyltransferases was elaborated using polyacrylamide gel electrophoresis and the fluorescent technique for localization of the enzyme. Rabbit antibodies against bovine and rat kidney enzymes were used. gamma-Glutamyltransferases from various organs of the same animal were immunologically indistinguishable, but distinct immunological differences were observed between the enzymes from various animal species. The new method of studies on immunological reaction between antibody and the enzymes appeared more sensitive than the double immunodiffusion method.

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Using solubilization with bromelain, the light form of gamma-glutamyltransferase was purified from Morris hepatoma 5123D. Some properties of this enzyme were compared to those of the light form rat kidney GGT. Anthglutin and its isomer inhibit competitively the former enzyme but non-competitively the latter. On zymograms of rat control sera, five GGT fractions were noted, but in sera of rats with hepatoma 5123D also the light form and the increase of GGT activity ain region of fraction II were observed. Only these two enzyme fractions react with antibody anti heavy form of Morris hepatoma GGT.

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Light form of bovine kidney gamma-glutamyl transferase was isolated from heavy form of the enzyme after digestion with bromelain. Its apparent molecular weight was 95,000 and in SDS solution it dissociated into two non-identical subunits with molecular weights 26,000 and 69,000. No substantial differences between both forms in activation, kinetic parameters and inhibition with anthglutin and its isomers were noted. Using enzyme immunoassay it was possible to determine one enzyme form in the presence of the other. This was applied for studies of gamma-glutamyltransferase forms in cow serum and colostrum.

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Using 7-(gamma-L-glutamyl)-4-methyl-coumarylamide as the fluorogenic substrate a simple and sensitive method for the assay of transferase activity of gamma-glutamyl-transferase in human blood serum and some other biological fluids is described. The substrate was synthesized with a good yield by the King and Kidd method. Close correlation and good agreement was noted between activities measured by the fluorimetric method and by the old colorimetric one in which gamma-L-glutamyl-p-nitroanilide was used.

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