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Bovine kidney monoamine oxidase (amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4) has been purified to one band on disc electrophoresis, and is shown to be localized in the intra- and extramitochondrial membrane. Kinetic models have been used to determine the effect of different substances on the enzyme activity. This enzyme shows a very high substrate specificity. It is suggested that phenol ring and one hydrogen atom each on the methylene and amine groups are responsible for the enzyme activity. N-methylbenzylamine exhibits a homotropic negative cooperative effect which is also supported by the n and Rs values. Benzylhydrazine is apparently a good substrate unlike phenylhydrazine, semicarbazide, harmaline and alpha- and beta-naphthol which show an inhibitory effect on the enzyme activity. Methylamine has no effect. It is suggested that the enzyme may have different sites or different conformations for different substrates. The results of this communication demonstrate bovine kidney monoamine oxidase to be different from monoamine oxidase from other sources.

Pigeon liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP forming), EC 6.4.1.1) shows allosteric properties similar to those of chicken or rat liver enzyme. Kinetic methods have been used to determine the effect of Ca2+ on this enzyme. The Ca2+ activation effect is absolutely dependent on the Mg2+ concentration; in the absence of Mg2+, pyruvate carboxylase has no catalytic activity. Furthermore, Ca2+ cannot replace Mg2+ and also shows a paradoxical effect on the liver enzyme activity. It is an activator at low pyruvate or Mg2+ concentrations; at increased pyruvate concentrations, however, it becomes an inhibitor. At low levels of ATP a pronounced activation of pigeon liver pyruvate carboxylase by Ca2+ has been demonstrated. The results of this communication demonstrate pigeon liver pyruvate carboxylase to be different from pyruvate carboxylase from other sources.

Kinetic methods have been used to determine the interrelationship between HCO-3, pyruvate and acetyl-CoA and their effect on pigeon kidney pyruvate carboxylase (pyruvate: CO2 ligase [ADP], EC 6.4.1.1). HCO-3 shows a negative co-operative effect (biphasic kinetics with two different Km values). Pyruvate influences the attachment of HCO-3 to this enzyme. The same has been shown for acetyl-CoA. Contrary to the results of other investigators no co-operative effect was seen with pyruvate even at different concentrations of acetyl-CoA. HCO-3 itself shows hardly any effect on the homotropic positive co-operativity (sigmoidal kinetics) of acetyl-CoA. The negative co-operative effect of HCO-3 could not be removed even at saturating concentrations of pyruvate and/or acetyl-CoA, which is also supported by the n and Rs values. The results of this communication bring out differences between pigeon kidney pyruvate carboxylase and pyruvate carboxylase from other sources. It is also suggested that there may be different allosteric and regulatory sites for acetyl-CoA, HCO-3 and pyruvate.

Kinetic methods have been used to determine whether Mg2+ and MgATP2- play an important role in regulating pigeon liver pyruvate carboxylase [pyruvate: CO2 ligase (ADP), EC 6.4.1.1]. Mg2+ not only forms a complex with ATP4- (MgATP2-) but is also required for the enzyme activation (and probably for the binding of MgATP2- to this enzyme). Contrary to the results of other investigators, the MgATP2- complex was not found to activate pigeon liver pyruvate carboxylase. We could not demonstrate homotropic cooperativity with MgATP2-. Excess Mg2+ induced allosteric stimulation of the enzymatic activity at different concentrations of MgATP2-. With different Mg2+ concentrations, changes also occurred in the apparent Km, Vmax and Rs values. Without excess of Mg2+ (heterotropic effector) only about 2% of the total enzymic activity available could be demonstrated in the presence of MgATP2-. It is concluded that Mg2+ exhibits a homotropic cooperative effect and is required for the activation of this enzyme. Mg2+ may bind either to a specific effector site, at the active site, or at the binding site for MgATP2- which is capable of functioning as an effector site and in this way facilitates the carboxylation of pyruvate.

The effect of varous compounds on 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was studied. N-Acetylcysteine inhibited the nezyme non-competitively (Ki 3.2 mM), whereas 3-hydroxybutanone inhibited competitively (Ki 4.1 mM). Methionine, isoleucine and cystathionine apparently enhanced the enzyme activity. The enzyme had a mol. wt. of 63000 as determined by gel filtration. The present studies differentiate between the aspartylglucosylaminase from human liver and that obtained from various other sources.

The activity of 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was measured in normal and diseased human liver, brain and kidney. Organs from patients with aspartylglucosaminuria show very little activity. Crude homogenates of human organs show a reaction catalysed by a complex enzyme system. With homogenate, the formation of product was linear with time up to about 6 h. Reaction times longer than 6-7h resulted in a decrease in the total concentration of product. This phenomenon was not found with the partially purified enzyme fraction. Linearity of the enzyme activity with different protein concentrations was found, independent of the incubation time. Longer incubation of the crude homogenate resulted in the utilization of the product, N-acetylglucosamine. This phenomenon was not observed with the partially purified enzyme fraction. This amidase from human organs differs from that obtained from other sources and apparently represents a rather complex enzyme system.

Human liver 1-aspartamido-beta-N-acetylglucosamine amidohydrolase (aspartylglucosylaminase, EC 3.5.1.26) was purified 17 500-fold to apparent homogeneity as judged from polyacrylamide-gel disc electrophoresis. A pH optimum of 7.7-9.0 was found. The Km value was pH- and temperature-dependent. At 37 degrees C and pH 7.7, Km was 0.16 mM and it increased to 0.29 at pH 6.0 and 0.23 at pH 9.0. At 25 degrees C and pH 7.7, a Km value of 0.99 mM was obtained. When the substrate concentration was varied, apparent Michaelis-Menten kinetics were obtained. p-Hydroxymercuribenzoate, glutathione or cysteine had no effect on the enzyme activity; 5 mM-N-acetylcysteine inhibited about 47% of the total enzyme activity. Apart from Cu2+, other bivalent ions were virtually ineffective at 1 mM. The kinetic study differentiates this enzyme from aspartylglucosylaminase from other sources.