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1. 1. In beef heart AMP-deaminase (EC 3.5.4.6.), 7 SH-groups out of 26 half-cysteine residues in the protein molecule have been shown to be accessible to alkylation by DTNB in the absence of ATP. The addition of ATP showed that only 6 SH-groups were accessible. 2. 2. DTNB-modified enzyme showed about 30% of the native catalytic activity but no sensitivity to the ATP-activating effect. 3. 3. Almost full reactivation of the modified enzyme and the restoration of the activatory effect of ATP could be achieved by exhaustive dialysis against mercaptoethanol.

1. 1. Kinetic properties of the inhibitory effect of inorganic phosphate and fluoride and of the activatory effect of potassium ion on human skeletal muscle AMP-deaminase (E.C. 3.5.4.6) have been investigated 2. 2. It has been shown that phosphate is a competitive inhibitor ( K 1 , ≈0.8 × 10 −3 M) and fluoride a noncompetitive inhibitor ( K 1 ≈3.2 × 10 −3 M) of human muscle AMP-deaminase. 3. 3. The changes of potassium ion concentration between 20 and 200 mM did not influence the Michaelis constant which was about 0.9 x 10 −3 M at 30°C. Also the change of substrate concentration in the range 40–300 μM did not influence the activation constant for potassium ( K a ≈0.4 × 10 −1 M). 4. 4. Higher concentraion of potassium (200mM) was found to diminish the “temperature sensitivity” of the enzyme activity. 5. 5. The energy of activation (E) in the presence of 150 mM KC1 calculated from Arrhenius plot was about 4600 cal/mole of substrate. The heat of the enzyme-substrate complex formation obtained from the plot of log K m vs T −1 was shown to have positive value (+2200 cal/mole) at the temperatures lower than 23°C and negative value (—4100 cal/mole) at the temperatures higher than 23°C.

1. 1. High activity of malic enzyme ( l -malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) has been shown in brown adipose tissue isolated from cold-adapted rats. The enzyme was located mainly within extramitochondrial compartment. 2. 2. The extramitochondrial malic enzyme has been partially purified by ammonium sulphate fractionation, chromatography on DEAE-cellulose and Ultrogel AcA-34. Specific activity of the purified enzyme was 40 μmol · min−1 per mg of protein which corresponds to about 175-fold purification. 3. 3. The enzyme was shown to carboxylate pyruvate in the presence of high concentration of KHCO3 and pyruvate at about 80% of the rate of forward reaction. The Km values determined at pH 7.2 for malate, NADP and Mn2+ were 0.18 mM, 8.6 μM and 14.5 μM respectively. The Km values for pyruvate, NADPH and KHCO3 were 3.1 mM, 6.3 μM and 24.3 mM respectively. 4. 4. In isolated mitochondria, pyruvate, bicarbonate and NADPH (the substrates for malic enzyme) and extramitochondrial malic enzyme were able to replace added malate in supplying oxaloacetate, a condensing partner for acetyl-CoA formed during β-oxidation of fatty acids. 5. 5. Possible metabolic role of extramitochondrial malic enzyme in brown adipose tissue is discussed.

1. 1. Lactate dehydrogenase has been purified from skeletal muscle of 14-day chick embryo, of chick i day after hatching and of adult hen. 2. 2. The reaction rate measured at low substrate concentrations (IO−4 M pyruvate) did not depend on temperature between 10° and 50° C when catalyzed by the enzyme from adult muscle. The rate showed a sharp temperature optimum at 35° and 40° C when the reaction was catalyzed by the enzyme from embryonic and I-day chick muscle enzyme respectively. 3. 3. The dependence of Km on temperature for embryonic and adult enzyme was also different; the value of Km for adult enzyme increased steadily with temperature whereas the value of Km for embryonic enzyme increased sharply with the rise of temperature from 35° to 50° C, showing little change in the temperature range 10°–35°C. 4. 4. These results are discussed and compared with experiments on AMP-aminohydrolase, another enzyme the kinetics of which have been investigated previously in developing chick muscle.

1. 1. AMP-aminohydrolase has been purified from skeletal muscle of chick embryo, I-day-old chick, and adult hen. 2. 2. The reaction rates measured at low substrate concentrations (5.5 × 10 −5 M ) at temperatures between 10 and 50° C. showed that ‘ adult ’ enzyme has a temperature optimum of 35° C., whereas ‘ embryonic ’ enzyme has no temperature optimum in this temperature range, the rate increasing steadily with the rise of temperature. 3. 3. The dependence of K m on temperature was different for ‘ adult’ and for ‘ embryonic ’ enzyme, indicating that the differences mentioned above consist of different enzymesubstrate affinity rather than of different molecular activity of the enzyme.

1. 1. Alanine, a significant substance that takes part in skeletal muscle metabolism, has been found to inhibit human skeletal muscle AMP-aminohydrolase activity. 2. 2. The type and the degree of this inhibition depends on the KCl concentration in the incubation medium. At a low KCl concentration (7 m m ) the inhibition by alanine occurred at all substrate concentrations tested (from 0.04 to 1.0 m m ), whereas at a higher KCl concentration ∗ (150 m m ) the inhibition was found only at lower concentrations of AMP. 3. 3. A possible metabolic significance of the inhibitory effect of alanine on AMP-aminohydrolase activity is briefly discussed.