Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects最新文献

• 1.

  1. Bacillus subtilis alanine dehydrogenase (L-alanine: NAD oxidoreductase (deaminating), EC_1.4.1.1. has been purified by column chromatography using calcium phosphate gel, DEAE-Sephadex and Ecteola-cellulose; it has been obtained in crystalline form.

• 2.

  2. The sedimentation pattern indicated a homogenous preparation. The sedimentation constant at infinite dilution was 8.8 S. The molecular weight estimated by the sedimentation equilibrium method was 228 000.

• 3.

  3. Amino acid analysis gave the following ratios of amino acid residues: Asp, ₄₃; Thr, ₃₄; Ser, ₂₀; Glu, ₅₁; Pro, ₂₅; Gly, ₅₁; Ala, ₆₅; CySH, ₂; Val, ₄₉; Met, ₁₁; iLeu, ₂₈; Leu, ₄₂; Tyr, ₁₅; Phe, ₇; Try, ₁; Lys, ₂₈; His, ₉; Arg, ₁₄.

A method is described for the purification of ribonmuclease (EC 2.7.7.16) from rabbit reticulocytes by heart treatment at pH 2.5 followed by chromatography on a carboxymethylcellulose column. The enzymei was purified 5000-fold, and was free from both acid and alkaline phosphatases (EC 3.I.3.2 and EC 3.I.3.I, respectively), non-specific phosphodiesterase (EC 3.I.4.I) and deoxyribonuclease (EC 3.I.4.5).

The enzyme had a pH optimum at 5.8. It was heat and acid stable and could be stored at —20° for I year without loss of activity. The products of hydrolysis of yeast ribonucleic acid by the enzyme were found to contain both 2′- and 3′-purine and pyrimide nucleotides,t he ratio of ratio of adenylic to cytidylic acid being approximately I:I. An incomplete digest of yeast ribonucleic acid by the enzyme was separated on a diethylaminoethyl-cellulose column and both mono- and oligonucleotide fractions were identified. Nucleoside 2′- and 3′-cyclic phosphates were hydrolyzed to their corresponding non-cyclic mononucleotides by the enzyme.

A possible physiological role of the enzyme is discussed on the basis of the following experiments: (I) degradation of rabbit reticulocyte ribosome by the enzyme; (2) chronological difference in its activity in rabbit reticulocytes; and (3) intracellular localization of the enzyme.

l-Rhamnose isomearse (l-rhamnose ketol-isomerase) has been purified about 50-fold from extracts of Escherichia coli. THe enzyme appears to be an SH enzyme, and catalyzes the reversible reaction l-rhamnose ⇆ l-rhamnulose. The enzyme was activated by the addition of manganous ions. At equilibrium about 60% of the total methylpentose was present as l-rhamnulose.

The enzyme was specific for l-rhamnose and l-rhamnulose; no other sugars were found to react.

Extracts of Escherichia coli, prepared from cells grown on l-rhamnose, contained a l-rhamnulose kinase (ATP:l-rhamnulose i-phosphotransferase, EC 2.7.1.5) which has been purified about 13-fold. The enzyme appears to be an SH enzyme, which catalyzes the phosphorylation of l-rhamnulose in the presence of ATP and some divalent metal ions but seems not to reactive with other sugars. Analytical studies on the phosphorulated sugar suggest that the reaction product is l-rhamnulose i-phosphate.

Evidence is present which indicates that the first step in the fermentation of l-rhamnose is the conversion of the methylpentose to l-rhamnulose followed by a phosphorylation of the ketose.

The mechanism of catabolite repression has been tested in mutants of Escherichia coli and a mutant of Aerobacter aerogenes, JF-4, using the technique of nutrient limitation. Two distinct types of enzyme control have been identified: (1) a control which is antagonized by inducer and is called “inducer-specific repression”; and (2) a control which is independent of the concentration of inducer and is called “inducer-independent represseion”. A metho of analysis is employed which allows each of the two types of repression to be measured quantitavely. The restriction of catabolic activities leads to derepression while the restriction of anabolic activities leads to repression; however, the relative change in inducer-specific and inducer-independent repression are not the same. Carbon limitation leads to a preferential decrease in inducer-specific repression while nitrogen and sulfur limitation lead to a preferential increase in inducer-independent repression.

• 1.

  1. The irreversible inhibition of ficin (EC 3.4.4.12) by chloroacetamide or iodoacetamide is due to the alkylation of the thiol group of one particular cysteine residue in the enzyme.

• 2.

  2. The variation of the rate of alkylation of ficin with pH shows that the reactive thiol group of ficin behaves essenitally as a freely ionising roup, pK_a′ = 8.55 (25 °; I, 0.1). Hence it is concluded that this group is unlikely to exist in a hydrogen-bonded form in the free enzyme.

• 3.

  3. The rates of reaction of ficin with chloroacetamide or iodacetamide were accelerated in the presence of substrates or products of catalytic activity without the stoichiometry of the overall reaction being affected. The rate of addition of the essential thiol of ficin to N-ethylmaleimide was unaffected by substrate or substrate analogues.

• 4.

  4. The nucleophilicity of the essential thiol group of ficin in S_N² reactions was found to be greater than that of simple thiol compounds whereas in the addition reaction to N-ethylmaleimide the converse was found.

• 5.

  5. A possible reason for the high nucleophilicyt of ficin in S_N² reactions and the acceleration caused by the presence of substrate or substrate analogues is given.

• 1.

  1. Human erythrocyte membrane contains a Mg²⁺-dependent adenosinetriphosphatase activity. The enhancement of this activity requires the simultaneous presence of Na⁺ and K⁺. When the membranes were broken by ultrasonic vibrations, the adenosinetriphosphatase activity was enhanced with either Na⁺ and K⁺, and in this case enhancement was no longer affected by ouabain.

• 2.

  2. The effects of Mg²⁺, Ca²⁺, pH, and detergents on the various manifestations of the adenosinetriphosphatase activity, namely: (a) the Na⁺-plus-K⁺-dependent form, (b) the single-ion-dependent form, and (c) the ion-independent form, were studied. The results did not suggest the presence of more than one enzyme.

• 3.

  3. Since the single-ion-dependent adenosinetriphosphatase activity was revealed merely by a physical change in the method of preparation (sonication), it is reasonable to suspect that it and the Na⁺-plus-K⁺-dependent activity are different manifestations of a single enzymic activity.