Amino Acid Composition Of Subunit Research Articles

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 μmol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, where those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotei and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex 1, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

NH2-terminal sequence of the isolated yeast ATP synthase subunit 6 reveals post-translational cleavage.

The three mitochondrially translated ATP synthase subunits of Saccharomyces cerevisiae were extracted from the enzyme and from whole mitochondria using an organic solvent mixture and then purified by reverse-phase HPLC. The amino acid composition of subunit 6 is close to the one predicted from the oli2 gene. The partial amino terminal sequence of subunit 6 reveals a post-translational cleavage site between the Thr-10 and Ser-11 residues of the precursor. Thus, mature subunit 6 contains 249 amino acid residues and displays a molecular mass of 27943 Da.

Comparison of Low and High Calcium Requiring Forms of the Calcium-Activated Neutral Protease (CANP) from Rabbit Skeletal Muscle1

Two distinct calcium-dependent neutral proteases (CANPs) with different sensitivities to calcium ions were purified concurrently by almost the same procedures from rabbit skeletal muscle and their enzymatic properties were compared (sensitivity to various divalent metal ions, the pH dependency and heat-stability of the activity, and the hydrolytic activity towards various substrates). They were further compared chemically in terms of the state of thiol groups, the amino acid compositions of subunits and the peptide fragments by digestion with S. aureus V8 protease. The low calcium requiring form of CANP (microCANP) was more sensitive to other divalent metal ions such as Sr2+ and Ba2+ than the high calcium requiring form of CANP (mCANP). The comparison of the pH dependency of these CANP activities showed that microCANP was active in a broader pH range than mCANP and the former was more heat-stable than the latter. Both CANPs had similar affinity to various substrates, but the hydrolytic velocity was several times higher with microCANP than with mCANP. Although they were inhibited by thiol protease inhibitors to the same extent, the states of thiol groups in them were quite different. The thiol group involved in the catalytic activity of the enzyme was exposed without adding Ca2+ in microCANP, whereas the group in mCANP became exposed only when sufficient Ca2+ was added. The large subunits of these two CANPs were different in their amino acid compositions and in the peptide fragment patterns produced by S. aureus V8 protease but the small subunits were indistinguishable from each other. These results led us to conclude that these two CANPs are quite different in nature and are not in a simple relationship, i.e., one of them is not derived from the other by autolysis or modification.

NADH-nitrate reductase in barley leaves: Identification and amino acid composition of subunit protein

NADH-nitrate reductase (NADH:nitrate oxidoreductase, EC 1.6.6.1) isolated from barley ( Hordeum vulgate L. cv. Steptoe) leaves was identified on a native slab gel after electrophoresis by staining for reduced methyl viologen nitrate reductase activity, NO 2 − production and diaphorase activity. The nitrate reductase protein, when subjected to a second-dimensional SDS-polyacrylamide gel electrophoresis, migrated as a single protein band of M r 110000. Thus, barley nitrate reductase (native molecular weight 221000) consists of two identical subunits. Preparative SDS-polyacrylamide gel electrophoresis was used to isolated highly homogeneous nitrate reductase subunit protein. Fluorograms of peptide maps indicated no homology among the nitrate reductase subunit and other polypeptides (60 kDa diaphorase, 41 kDa, 35 kDa and 25 kDa species) which are common contaminants of the affinity-purified nitrate reductase. Thus, the 60 kDa diaphorase and 41 kDa polypeptide observed under our experimental conditions are not breakdown products of the 110 kDa nitrate reductase subunit. The amino acid composition of the barley nitrate reductase subunit was similar to that reported for the nitrate reductase of Chlorella vulgaris and Ankistrodesmus braunii.

The structure of regular viruses in relation to their subunit amino acid composition

The surface area of that portion of the virus protein subunit exposed to nucleic acid and aqueous medium was estimated from the surface areas of charged and dipolar amino acids. This area and the protein subunit volume were determined for many viruses from their amino acid composition. The surface to volume ratios ( S V ) were from 0.05 A −1 to 0.06 A −1 regardless of the size or shape of the virus. The S V ratios of simple spherical and cylindrical shell models with virus dimensions were in agreement with the S V ratios predicted from amino acid composition for the icosahedral viruses. For the rod-shaped viruses, however, the surface of the embedded RNA had to be taken into account to obtain a better correlation between the S V value for the cylindrical model and that for the subunit.