Robert R. Becker

Specific indication of hemoproteins in polyacrylamide gels using a double-staining process

Hemoproteins were revealed in polyacrylamide gels in the presence of sodium dodecyl sulfate by staining with different benzidine derivatives. When the protein samples were treated with either beta-mercaptoethanol or dithiothreitol, a significant decrease in peroxidase activity of the proteins possessing noncovalently bound heme led to diminished staining. However, when Coomassie blue R-250 staining followed the hemespecific stain it was observed that the hemoprotein bands stained more intensely than duplicate sample bands that had been stained only with the Coomassie blue R-250. This staining property allows the indication of hemoproteins in gels even after the peroxidase yield has been significantly depleted by reducing agents.

Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria

The abilities of Staphylococcus aureus and Streptococcus pyogenes to remove iron from mouse 59Fe hemoglobin that was either in free form or complexed with human haptoglobin, were evaluated. 59Fe hemoglobin from the amphibian Taricha granulosa was also used in free form or complexed with the amphibians hemoglobin-binding proteins. Contrary to what was reported from a study using pathogenic Escherichia coli, haptoglobin failed to exhibit a bacteriostatic influence when complexed with hemoglobin. In our study, more 59Fe was removed by the bacteria from the haptoglobin-hemoglobin complex than from free mouse hemoglobin. The hemoglobin and hemoglobin-plasma protein complexes of Taricha were stripped of 59Fe at similar rates and extents by both bacterial species.

Purification and comparative properties of NADPH-cytochrome P-450 reductase from rat and rainbow trout: Differences in temperature optima between reconstituted and microsomal trout enzymes

NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of beta-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin-O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat.

The β-glucosidase system of the thermophilic fungus Chaetomium thermophile var. Coprophile n. var.

Abstract Chaetomium thermophile contains three distinct β-glucosidases. Two of the enzymes are cell bound while the third is extracellular. On the basis of relative substrate specificities toward p- nitrophenyl -β- d -glucoside and cellobiose, one of the cell-bound enzymes is classified as a cellobiase while the other two enzymes are classified as aryl-β- d -glucosidases. The enzymes were partially purified and characterized with respect to temperature stability and certain kinetic parameters. The enzymes exhibit greater temperature stability than analogous enzymes from mesophilic fungi. Cellobiose and cellolose are induceers of the cellobiase but not of the aryl-β-glucosidases. Inhibitors of protein synthesis (cycloheximide) and of metabolism (azide, dinitrophenol) prevent the induction of cellobiase. When the mycelia are grown on starch medium, all three β-glucosidase activities undergo large increases after the exponential growth phase.

The complete sequence of the acidic subunit from Mojave toxin determined by Edman degradation and mass spectrometry.

Mojave toxin, a heterodimeric, neurotoxic phospholipase complex from Crotalus scutulatus scutulatus, is one of a group of closely related rattlesnake toxins for which much structural information is still lacking. The complete amino-acid sequence of the acidic subunit from Mojave toxin was determined. The three individual peptide chains, derived from the acidic subunit by reductive alkylation, were separated by high-performance liquid chromatography. Fragmentations of the A and B chains were done using specific proteinases and the resulting peptide mixtures were fractionated by reverse-phase high-performance liquid chromatography. Sequence analyses on the intact chains and the fragments from digests were done by automated Edman degradation, carboxypeptidase Y degradation and triple-quadrupole and tandem-quadrupole Fourier-transform mass spectrometry. The sequence for each acidic subunit chain is very similar to the corresponding chain from the related neurotoxin complex, crotoxin, and overall the sequence is similar to the sequences of group I and II phospholipases A2. The N-terminus of the B chain is blocked by pyroglutamic acid. The existence of two distinct and closely related C chains was established. It is unlikely that the small sequence difference can account for the isoforms that are present in purified Mojave toxin and in unfractionated venom.

Amino acid sequences of myotoxins from Crotalus viridis concolor venom

Myotoxins I and II were isolated from the venom of Crotalus viridis concolor. Complete sequences were derived for each reduced, alkylated toxin with data obtained by a single run on a gas phase sequencer and from fragments derived by cyanogen bromide cleavage. The results demonstrate that microheterogeneity is present in myotoxin II. The newly established sequences were compared with 3447 protein sequences in the Protein Information Resource database. The only homologous proteins found were other known myotoxins from rattlesnake venoms, namely myotoxin a, crotamine and peptide C.

D-Ribulose-1,5-bisphosphate carboxylase/oxygenase from chemolithotrophically grown Rhizobium japonicum

Abstract Ribulose-1,5-bisphosphate carboxylase/oxygenase has been purified from chemolithotrophically grown Rhizobium japonicum SR and ribulose-5-phosphate kinase activity has also been detected in extracts of such cells. Electrophoretically homogeneous ribulosebisphosphate carboxylase/oxygenase purified in the presence of PMSF showed two types of large subunits of 55 000 and 53 000 daltons and small subunits of 14 200 daltons. The heterogeneity of large subunits was not observed when the enzyme was prepared in the presence of PMSF and DIFP. Ribulose-1,5-bisphosphate carboxylase from R. japonicum was inhibited by antibodies to this enzyme and a single precipitin band from the antibody-enzyme interaction was observed on double diffusion plates. Antibodies to R. japonicum enzyme did not cross-react on immunodiffusion plates with the ribulosebisphosphate carboxylase/oxygenases from wheat, spinach, soybean and tobacco.

Composition and synthesis during G1 and S phase of a high mobility group-E/G component from Chinese hamster ovary cells.

A perchloric acid soluble protein from the sedimented chromatin of blended Chinese hamster ovary (line CHO) cells has been isolated by guanidine hydrochloride gradient chromatography on Bio . Rex-70 ion exchange resin. The amino acid composition of the protein (designated as CHO HMG-E/G) is similar to that of mouse HMG-E, but it differs from that of bovine HMG-14 and HMG-17 or any possible mixture of the two. CHO HMG-E/G incorporates [32P]phosphate like HMG-14 and HMG-17 class proteins from other species, but all resolvable molecular species incorporate phosphate, and the more highly-phosphorylated band migrates faster, rather than slower, than the other in acid-urea gel systems. Incorporation of [3H]lysine into HMG-E/G following release from isoleucine deprivation G1 block indicates that the protein is extensively synthesized during both the G1 and S phases of the cell cycle.

THE EFFECT OF UNIVALENT CATIONS ON THE CIRCULAR DICHROISM OF PYRUVATE KINASE.

Abstract The circular dichroism spectrum of rabbit muscle pyruvate kinase in the near ultraviolet was altered by temperature changes and univalent cations. A large increase in ellipticity occurred in the 260–290-nm region when the temperature was lowered from 37 to 5°. At 25° 0.1 M K+, an activating univalent cation, and 0.1 M Li+, a non-activating univalent cation, caused significant and similar increases in ellipticity in the 250–290-nm region, both in the presence and absence of MgCl2 and phosphoenolpyruvate. Tetramethylammonium ion (0.1 M), a non-activating univalent cation, caused relatively small changes in the circular dichroism spectrum compared to the changes produced by K+ or Li+. Since pyruvate kinase is inactive in the presence of Li+, no correlation could be made between activity and enzyme conformation as determined by circular dichroism measurements.

Bisalbuminemia in an amphibian

Improved electrophoretic resolution revealed two albumin-like proteins in Taricha granulosa plasma (bisalbuminemia). The Taricha proteins were compared to mammalian, avian and reptilian serum albumins regarding molecular weight, amino acid composition, isoelectric character, solubility and the binding of hemin and dyes. The results indicate that although the two Taricha proteins have demonstrated hemoglobin-binding ability, they possess traits that characterize them to be true serum albumins.