1. A procedure for the purification of the cytoplasmic isoenzyme of aspartate aminotransferase from sheep liver is described. 2. The purified isoenzyme shows a single component in the ultracentrifuge at pH7.6 and forms a single protein band on agar-gel electrophoresis at pH6.3 or 8.6, as well as when stained for protein or activity after polyacrylamide-gel or cellulose acetate electrophoresis at pH8.8. 3. Immunoelectrophoresis on agar gel yields only one precipitin arc associated with the protein band, with rabbit antiserum to the purified isoenzyme. By immunodiffusion, cross-reaction was detected between the cytoplasmic isoenzymes from sheep liver and pig heart, but not between the cytoplasmic and mitochondrial sheep liver isoenzymes. 4. The s(20,w) of the enzyme is 5.69S and the molecular weight determined by sedimentation equilibrium is 88900; 19313 molecules of oxaloacetate were formed/min per molecule of enzyme at pH7.4 and 25 degrees C. 5. The amino acid composition of the isoenzyme is presented. It has about 790 residues per molecule. 6. The holoenzyme has a maximum of absorption at 362nm at pH7.6 and 25 degrees C. 7. A value of 2.1 was found for the coenzyme/enzyme molar ratio. 8. The purified enzyme revealed two bands of activity on polyacrylamide-gel electrophoresis at pH7.4 and an extra, faster, band in some circumstances. These bands occurred even when dithiothreitol was present throughout the isolation procedure. 9. Three main bands were obtained by electrofocusing on polyacrylamide plates with pI values 5.75, 5.56 and 5.35. 10. Structural similarities with cytoplasmic isoenzymes from other organs are discussed.

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.

A method for the purification of mitochondrial isoenzyme of sheep liver aspartate aminotransferase (EC 2.6.1.1) is described. The final preparation is homogeneous by ultracentrifuge analyses and polyacrylamide-gel electrophoresis and has a high specific activity (182 units/mg). The molecular weight determined by sedimentation equilibrium is 87,100 +/- 680. The amino acid composition is presented; it is similar to that of other mitochondrial isoenzymes, but with a higher content of tyrosine and threonine. Subforms have been detected. On isoelectric focusing a broad band was obtained, with pI 9.14. The properties of the mitochondrial aspartate aminotransferase are compared with those of the cytoplasmic isoenzyme. The Km for L-aspartate and 2-oxoglutarate for the cytoplasmic enzyme were 2.96 +/- 0.20 mM and 0.093 +/- 0.010 mM respectively; the corresponding values for the mitochondrial form were 0.40 +/- 0.12 mM and 0.98 +/- 0.14 mM. Cytoplasmic aspartate aminotransferase showed substrate inhibition by concentrations of 2-oxoglutarate above 0.25 mM in the presence of aspartate up to 2mM. The mitochondrial isoenzyme was not inhibited in this way. Pi at pH 7.4 inhibited cytoplasmic holoenzyme activity by up to about 60% and mitochondrial holoenzyme activity up to 40%. The apparent dissociation constants for pyridoxal 5'-phosphate were 0.23 micrometer (cytoplasmic) and 0.062 micrometer (mitochondrial) and for pyridoxamine 5'-phosphate they were 70 micrometer (cytoplasmic) and 40 micrometer (mitochondrial). Pi competitively inhibited coenzyme binding to the apoenzymes; the inhibition constants at 37 degree C were 32 micrometer for the cytoplasmic isoenzyme and 19.5 micrometer for the mitochondrial form.

Treatment of the purple membrane of Halobacterium halobium with tetranitromethane led to modification of tyrosine residues. Modification of more than 3-4 tyrosine residues per bacteriorhodopsin monomer caused a decrease in the light-induced proton-pumping ability of purple membrane in synthetic lipid vesicles, loss of the sharp X-ray-diffraction patterns characteristic of the crystal lattice, loss of the absorbance maximum at 560 nm, and change in the buoyant density of the membrane. No modification of lipid was detected. These changes were interpreted as a gradual denaturation of the protein component such that when 8-9 tyrosine residues are modified, no proton pumping is observed. Modification of less than 3-4 tyrosine residues with tetranitromethane caused an increse in light-induced proton pumping. It was possible to generate partly modified purple membrane which had completely lost the property of diffracting X-rays into the sharp pattern observed with native purple membrane, but which still retained the ability to pump protons in a vectorial manner. Retention of crystal lattice is not essential for proton pumping.