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alpha-Lactalbumin was purified to homogeneity from rat milk. Rat alpha-lactalbumin, in contrast to other alpha-lactalbumins, is a glycoprotein and exhibits an abnormally high molecular weight when obtained by gel filtration or electrophoresis in sodium dodecyl sulfate. The molecular weight by sedimentation equilibrium is 15 400 +/- 5% and of the reduced and alkylated protein is 16 000 when determined by thin-layer chromatography in 6 M guanidine hydrochloride. At least, three major charge forms, all containing carbohydrate and active in the lactose synthetase reaction were demonstrated. The amino acid composition reveals a high proline content which is reflected in a low alpha-helical content.
Intraperitoneal injection of tritiated folic acid (PteGlu) into rats has revealed the presence of three separate protein fractions in the cytosol fraction of the liver and one in the mitochondria which bind folate derivatives. The proteins in the cytosol (cytosol I, II and III) have approximate molecular weights of 350,000, 150,000, and 25,000 and the protein in the mitochondria has an approximate molecular weight of 90,000 as estimated by gel filtration. The bound folate derivatives are primarily polyglutamate forms while cytosol II contains primarily bound 5-methyltetrahydrofolate polyglutamate derivatives. Little binding of radioactively labeled folic acid or 5-methyltetrahydrofolate to these fractions was observed when binding was carried out in vitro. Significant binding in vitro was observed, however, when a mixture of biosynthetically labeled natural folate derivatives was used. These proteins have not been purified, but cytosol III partially consists of the enzyme, tetrahydrofolate dehydrogenase (EC 184.108.40.206). Studies on the time course of folic acid incorporation into the liver showed that soon after injection nonmetabolized folic acid was bound to the plasma membrane fraction of the liver cell. It is suggested that at least one of the binding proteins in the cytosol may be involved in storage of the vitamin while the binding of nonmetabolized folic acid to the plasma membrane may reflect the existence of a carrier for folic acid transport into the cell.
The structure of rabbit transferrin was investigated with regard to number, size, and composition of the heteropolysaccharide units and their relative location on the polypeptide chain. The composition and molecular weight of the Pronase glycopeptides revealed that rabbit transferrin contains two heteropolysaccharide units, each composed of 2 sialic acid residues, 2 galactose residues, 3 mannose residues, and 4-N-acetylglucosamine residues. The composition and molecular weight of the tryptic glycopeptides further substantiated the existence of two identical heteropolysaccharide units and revealed that both units have identical amino acid residues in the immediate vicinity of the carbohydrate attachment sites to the polypeptide chain, suggesting a sequence homology surrounding the two glycosylation sites. Characterization of the cyanogen bromide fragments from rabbit transferrin indicated that both heteropolysaccharide units are located within a single polypeptide fragment representing approximately one-third of the molecule.
The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.
Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.
Renal tubules from rabbit kidneys were isolated from thin shavings of the kidney surface. Basement membrane was then prepared following sonication of the isolated tubules. To insure preservation of the integrity of the basement membrane polypeptides, the protease inhibitors, diisopropyl fluorophosphate, ethylenediaminetetraacetic acid, N-ethylmaleimide, and epsilon-amino-caproic acid were used at all stages of the preparations. The optimal conditions of sonication and centrifugation were established and the chemical composition of basement membrane prepared under these conditions was examined in detail. Glycine, hydroxyproline, and hydroxylysine were found in concentrations of 206, 65, and 18 residues per thousand, respectively, in basement membrane from young kidneys. About 38% of the basement membrane was found to be soluble in sodium dodecyl sulfate upon incubation at 90 degrees C, and to possess relatively low amounts of the amino acids characteristic of collagen. Electrophoretic analysis of this fraction revealed that the major subunits ranged in approximate molecular weight from 18,500 to greater than 10(6). When analyzed with disulfide bonds reduced, a molecular weight range from 31,000 to 275,000 was observed for this fraction. The sodium dodecyl sulfate-insoluble fraction could be dissolved upon reduction and alkylation and its composition was enriched in the amino acids characteristic of collagen. Polypeptides from this fraction were analyzed by electrophoresis in agarose and in agarose-acrylamide gels. The approximate molecular weight of the smallest component was 164,000. Additional polypeptides were observed whose molecular weights occurred in multimers of this component, up to 1.1 x 10(6), possibly indicating covalent cross-linked multimers of a basic collagen-like polypeptide(s).
Cell free extracts of Pseudomonas MS previously have been shown to carry out the synthesis of a novel amino acid, N-methylalanine (Kung, H.F., and Wagner, C. (1970) Biochim. Biophys. Acta 201, 513-516). An enzyme has been isolated from this organism which is responsible for the synthesis of N-methylalanine. The stoichiometry of the reaction catalyzed by this enzyme leads to the following formulation: Methylamine + pyruvate + NADPH + H-+ yields N-methylalanine + NADP-+ + H2O. This enzyme has been physically separated from alanine dehydrogenase, which is also present in these extracts. This new enzyme has been named N-methylalanine dehydrogenase. It has been purified to near homogeneity as judged by disc gel electrophoresis. Gel filtration chromatography showed that N-methylalanine dehydrogenase has an apparent molecular weight of 77,000, while electrophoresis in sodium dodecyl sulfate gave rise to a single band with a molecular weight of approximately 36,500. The enzyme is optimally active in the pH range between 8.2 and 8.6. The apparent K-m values for pyruvate, NADPH, and methylamine, respectively, are 1-5 times 10 minus 2 M, 3-5 times 10 minus 5 M, and 7.5 times 10 minus 2 M.