Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation最新文献

UDPG pyrophosphorylase (UTP:α-d-glucose-1-phosphate uridyltransferase EC 2.7.7.9) has been purified from bovine mammary gland acetone powder. The enzyme is specific for UTP and glucose 1-phosphate. The enzyme is inhibited by anions and galactose 1-phosphate is a competitive inhibitor, $\text{K}{}_{\mathrm{i}}\text{= 8·10}{}^{\mathrm{-3}}\text{M}$. The enzyme requires Mg²⁺ for activity. The $\text{K}{}_{\mathrm{m}}$ for all the substrates have been determined and they are a function of the Mg²⁺ concentration. The pH optimum is between 8 and 9.

Suitable assay conditions are described for the NADPH-dependent oxidative desaturation of stearyl-CoA by rat-liver microsomes.

NADH is an even more effective electron donor than NADPH. Ascorbate at high concentrations also acts as a donor, but with low efficiency. Unlike the NADPH-dependent drug hydroxylations, the desaturation reaction does not seem to involve the microsomal hemoprotein P-450. Instead, a hitherto unknown cyanide-sensitive factor appears to be involved in the desaturation mechanism, regardless of the electron donors employed. The microsomal NADPH-specific flavoprotein with a cytochrome $\text{c}$ reductase activity seems to participate, not only in the NADPH-dependent drug hydroxylations, but also in the NADPH-supported desaturation. Microsomal oxidation of methanol, which requires NADPH and is sensitive to cyanide, appears to be catalyzed by a mechanism which differs from that involved in desaturation.

On the basis of these findings the electron-transfer mechanisms associated with these microsomal reactions are discussed.

• 1.

  1. From Saccharomyces cerevisiae, incubated on a glucose-free medium with acetate as the only carbon source, two different malate dehydrogenases (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) have been isolated by DEAE-cellulose ion-exchange chromatography. One of these enzymes was only found in the mitochondria and is called enzyme A or m-malate dehydrogenase; the other enzyme was found in the extramitochondrial c-space and is called enzyme B or c-malate dehydrogenase. At present it cannot be decided whether m-malate dehydrogenase also exists in the c-space or leaks when the mitochondria are injured.

• 2.

  2. The reaction velocity plotted against the concentration of oxaloacetic acid showed a characteristic substrate inhibition in the case of m-malate dehydrogenase In contrast, c-malate dehydrogenase showed no substrate inhibition. This difference corresponds to the behaviour of m-malate dehydrogenase and c-malate dehydrogenase from liver.

• 3.

  3. In yeast grown on glucose only m-malate dehydrogenase could be found, but after incubating the cells on acetate as the sole carbon source, both m-malate dehydrogenase and c-malate dehydrogenase were found. In reference to earlier experiments concerning the regulation of malate dehydrogenase activity in yeast, it is concluded that a repression of c-malate dehydrogenase synthesis by glucose occurs. This regulating mechanism is useful for the cell, because in the glycoxylate cycle c-malate dehydrogenase participates in the gluconeogenesis from acetate or ethanol. This enzyme is not necessary when glucose is in the medium.

An enzyme has been found in rat-kidney homogenates with the ability to release bradykinin or related peptides from plasma globulin. The system is normally inactive but can easily be activated in hypotonic media at pH 5.0.

Centrifugation studies have shown that most of the enzymatic activity is concentrated in particles with the sedimentation characteristics of lysosomes or droplets.

The sediment is inactive and can be activated by procedures used to release phosphate from lysosomes.

The kidney enzyme has many properties of the urinary kallikrein found in rat urine.

The conformation of the extracellular acid proteinase of Aspergillus saitoi (aspergillopeptidase A, EC 3.4.4.17) has been investigated in aqueous solution. The optical rotation, $\text{[α]}{}_{\mathrm{<mtext>D</mtext>}}$, was −35°. The optical rotatory dispersion constant, $\text{λ}{}_{\mathrm{<mtext>c</mtext>}}$, was 207 mμ, and the Moffitt-Yang parameter, $\text{b}{}_0$, was zero. The $\text{−a}{}_0$ value in the Moffitt-Yang parameter or levorotation of the aspergillopeptidase. A molecule increased markedly in the presence of urea, while the value of $\text{b}{}_0$ remained unchanged. This finding indicates the absence of a helical conformation.

The infrared result indicates that the deuterium-exchanged aspergillopeptidase A exists in the antiparallel β structure. The location of an amide I band at 1632 cm⁻¹ has been observed. The spectrum has shown the presence of a weak band around 1685 cm⁻¹.

The location of the single tryptophan residue in aspergillopeptidase A is discussed.

• 1.

  1. The inactivation of citrate lyase (citrate-oxaloacetate lyase, EC 4.1.3.6) by oxaloacetate has been investigated.

• 2.

  2. Studies of the pH profiles of inactivation at pH 7–9 with varying total oxaloacetate and magnesium concentrations show that the magnesium complexes of enolic oxaloacetate are involved.

• 3.

  3. The inhibitory effects of the following structural analogues of the keto and enol forms of oxaloacetate were examined: l-malate, α,α-dimethyloxaloacetate, tartronate, α,α-difluorooxaloacetate, pyruvate, ketomalonate, and isomalate. These results, and the effect of other divalent metal cations, indicate that the site of the inactivation is identical with the active site for citrate cleavage.

• 4.

  4. The inactivation is irreversible and is time and concentration dependent. Free oxaloacetate does not inactivate the enzyme.

• 5.

  5. The inactivation phenomenon has high structural specificity, requiring a straight-chain, four-carbon dicarboxylic acid with an ionisable α-hydroxy group, and the presence of a divalent metal cation.