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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.

A phylogenetic tree for occluded baculoviruses was constructed based on the N-terminal amino acid sequence of occlusion body proteins from six baculoviruses including three lepidopteran nuclear polyhedrosis viruses (NPVs), [two unicapsid (Bombyx mori and Orgyia pseudotsugata) and one multicapsid (Orgyia pseudotsugata)]; one granulosis virus (Pieris brassicae); and NPVs from a hymenopteran (Neodiprion sertifer) and a dipteran (Tipula paludosa). Amino acid sequence data for the B. mori NPV were from a report by Serebryani et al. (1977) and that for the O. pseudotsugata NPVs were reported previously by us (Rohrmann et al. 1979). The other N-terminal amino acid sequences are presented in this paper. The phylogenetic relationships determined based on the molecular evolution of polyhedrin were also investigated by antigenic comparisons of the proteins using a solid phase radioimmune assay. The results indicate that the lepidopteran NPVs are the most closely related of the above group of viruses and are related to these viruses in the following order: N. sertifer NPV, P. brassicae granulosis virus, and T. paludosa NPV. These data, in conjunction with Baculovirus distribution and evidence concerning insect phylogeny, suggest that the Baculovirus have an ancient association with insects and may havae evolved along with them.

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.

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.

Androgen-binding protein (ABP) distribution in the fluids and tissues of fetal and neonatal rats was determined by radioimmunoassay during the critical period of sexual differentiation from 12 days postconception (PC) to 6 days postpartum. No significant differences in plasma ABP concentrations were detected between litters of the same age. Fetal plasma ABP in both sexes was high at 18 days PC and decreased to birth. At birth, this level increased, then continued its decline during the neonatal period. After 4 days, the level in females was significantly lower than that of males; this was the only time a significant difference in ABP levels between the sexes was observed. Amniotic fluid ABP was low at 12 days and increased to a peak at 18 days PC. Thereafter, it decreased as in plasma. ABP concentrations in fetal livers and placentas were almost negligible and changed in parallel with fluid concentrations. No ABP was detected in maternal plasma. Fetal production of ABP begins at the approximate time of gonadal differentiation and reaches a peak at 18 days, the time of peak androgen production. ABP may function as a protection against excessive concentrations of free androgen in the developing fetus and neonate.

Two histone H1 fractions [H1(I) and H1(II)] and two histone H1(o) fractions (H1(o)a and H1(o)b) have been isolated from butyrate-treated Chinese hamster (line CHO) cells by guanidine hydrochloride gradient chromatography on Bio-Rex 70 ion-exchange resin. The fractions have been identified by electrophoresis and amino acid analyses. Electrophoretic analysis of cyanogen bromide treated H1(o) in long acid-urea-polyacrylamide gels suggests that H1(o)a and H1(o)b differ, at least, within the 20-30 residue fragment(s) removed by the cyanogen bromide cleavage. Shallow-gradient Bio-Rex 70 chromatography indicates that histones H1(o)a and H1(o)b are the same as the respective CHO histones, H1(III) and H1(IV), originally resolved by Gurley and co-workers [Gurley, L. R., Walters, R. A., & Tobey, R. A. (1975) J. Biol. Chem. 250, 3936]. This identification and the phosphate incorporation data of Gurley et al. (1975) reveal new features about H1(o) phosphorylation: (1) following release from G1 arrest, H1(o)a and H1(o)b become phosphorylated in late G1 prior to DNA synthesis; (2) H1(o)a and H1(o)b are phosphorylated at similar rates throughout the cell cycle. These and other data demonstrate that histone H1(o) is phosphorylated in a cell cycle dependent fashion which mimics that of histone H1.

An eight-iron, eight-sulfur ferredoxin from Rhizobium japonicum bacteroids of soybean root nodules has been purified to apparent homogeneity as judged by disc gel electrophoresis. The purification procedure included chromatography on DEAE-cellulose, Bio-Gel P-60, and hydroxylapatite. Specific activities of several purified preparations of bacteroid ferredoxin ranged from 1700 to 1900 nmol of C2H4 produced . min-1 . mg-1 in the reaction mediating electron transfer between illuminated chloroplasts and bacteroid nitrogenase. A molecular weight of 6740 for the protein was determined by low speed sedimentation equilibrium and a molecular weight of 6500 was estimated from the mobility of bacteroid ferredoxin relative to the mobility of standard proteins during sodium dodecyl sulfate disc gel electrophoresis. All of the common amino acids were present except arginine, methionine, and tryptophan. The absorbance spectrum of the oxidized protein exhibited maxima at 285 nm and 380 nm with a shoulder near 305 nm. The A380/A285 ratio was 0.76 and the extinction coefficient at 380 nm for the oxidized protein was found to be 30,800 M-1. Equilibration of bacteroid ferredoxin with methyl viologen at various potentials revealed a midpoint oxidation-reduction potential of -484 mV. Spectrophotometric examination of iron-sulfur clusters extruded from bacteroid ferredoxin with benzenethiol and the transfer of its iron-sulfur clusters to other ferredoxins established the presence of two [4Fe-4S] clusters in a molecule of bacteroid ferredoxin. The EPR spectrum of oxidized ferredoxin consisted of a small signal at g = 2.02 integrating to 0.19 spin/molecule. The EPR spectrum of ferredoxin reduced with 5-deazaflavin exhibited a signal with features at g values of 1.88, 1.94, 2.01, and 2.07, and integrated to 1.7 spins/molecule. The EPR properties of bacteroid ferredoxin are characteristic of a ferredoxin operating between the 1+ and 2+ oxidation levels. Bacteroid ferredoxin mediated electron transfer to clostridial hydrogenase, but was not reduced by the clostridial phosphoroclastic system in the presence of pyruvate. Bacteroid ferredoxin reduced by illuminated 5-deazariboflavin also supported a high rate of C2H2 reduction by bacteroid nitrogenase which was free of Na2S2O4. It was concluded, on this basis, that bacteroid ferredoxin has the capability of functioning as the electron donor for nitrogenase in R. japonicum.

The capability of Taricha granulosa to conserve hemoglobin upon in vivo hemolysis has been investigated. 59Fe incorporation into Taricha hemoglobin was similar in rate to mammals and birds. Phenylhydrazine-induced hemolysis resulted in comparatively low levels of 59Fe and no discernible amounts of hemoglobin excreted after 10 days. The addition of 59Fe Hb to Taricha circulation resulted in relatively low levels of 59Fe excretion and significant amounts of 59Fe incorporation into new hemoglobin within 10 days.

This study was designed to determine the utility of two newly developed self-report measures of group therapy effectiveness. Repeated administrations of these instruments indicated that group therapy was effective in inducing significant problem resolution and that these changes were stable on a 3-month follow-up. One of the measures also suggested that the nondirective style of therapy appeared to be more effective than the directive style. Results suggest that two self-report measures may be useful in studies designed to determine both the outcome as well as the process of group psychotherapy.

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.

The plasma of the amphibian newt Taricha granulosa has been shown to be devoid of haptoglobin. Upon hemolysis, Taricha albumin and another protein associate with hemoglobin. The acute-phase response to inflammation observed in birds and mammals appears to be absent in Taricha. Taricha hemoglobin failed to bind to human haptoglobin. Taricha hemoglobin not only failed to dissociate into alpha beta dimers as did human Hb, but formed alpha beta octamers.

The major portion of glutamine synthetase activity in root nodules of soya-bean plants is associated with the cytosol rather than with Rhizobium japonicum bacteroids. Glutamine synthetase accounts for about 2% of the total soluble protein in nodule cytosol. Glutamine synthetase from nodule cytosol has been purified by a procedure involving fractionation with protamine sulphate, ammonium sulphate and polypropylene glycol, chromatography on DEAE-Bio-Gel A and Bio-Gel A-5m and affinity chromatography on glutamate-agarose columns. The purified preparation appeared to be homogeneous in the analytical ultracentrifuge. From sedimentation-equilibrium experiments a mol. wt. of about 376000 was determined for the native enzyme and 47300 for the enzyme in guanidinium chloride. From these data and measurements of electron micrographs, we have concluded that glutamine synthetase from nodule cytosol consists of eight subunits arranged in two sets of planar tetramers which form a cubical configuration with dimensions of about 10 nm (100 A) across each side. Glutamine synthetase from nodule cytosol has a higher glycine and proline content and a lower content of phenylalanine than the glutamine synthetase that has been prepared from pea seed. The cytosol enzyme contains four half-cystine molecules per subunit, which is in contrast with two reported for the enzyme from pea seed. Enzyme activity is striking influenced by the relative proportion of Mg2+ and Mn2+ in the assay medium. Activity is inhibited by feedback inhibitors and is influenced by energy charge.

Trypsin digestion of the protamine-like protein from Spisula solidissima has revealed the existence of an internal resistant core. The peptide contains 75 amino acid residues, and its primary structure shows some conserved sequences that are common to those found in the core of the somatic histone H5 from chicken erythrocytes. The secondary structure of this core exhibits 33% antiparallel beta-sheet, 18% beta-turns, 37% random coil, and only 10% alpha-helix, in contrast to histone H5. Hydrodynamic measurements indicate a compact globular assembly for the tertiary structure of this peptide, when compared to the more extended shape observed for the whole protein. The possible relatedness of this protein to the histone H1 family is discussed.

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.

Pieces of testicular tumor from male pseudohermaphrodite (tfm) rats were transplanted between the skin and body wall musculature of 4-day-old female, male, and tfm hosts and to the anterior chambers of the eyes of gonadectomized and intact adult tfm, female, and male hosts. These transplants grew in all hosts except normal males. To determine whether these tumors were hormone-dependent, the levels of circulating gonadotropins were measured in all host types to ascertain which were low in the males and high in all of the others. Prolactin, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) levels were measured in intact and castrated normal and tfm rats. Normal males had low levels of prolactin, estrogen, LH, and FSH; however, transplants grew well in other hosts with low titers of these hormones. Except for normal males, all hosts had high serum levels of LH. Thus, testicular tumors in tfm rats appear to be LH-dependent.

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 amphibian's 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.

The synthesis and properties of a radiolabeled glycoside photoaffinity probe, [3H]-(3 beta,5 beta,14 beta, 20E)-24-azido-3-[(2,6-dideoxy-beta-D-ribo-hexopyranosyl) oxy]-14-hydroxy-21-norchol-20(22)-en-23-one, containing the photoactive group at the C-17 side chain of the steroid moiety are reported. The molecule binds to the sodium- and potassium-activated adenosinetriphosphatase from porcine kidney outer medulla under type II binding conditions [5 mM MgCl2, 3 mM phosphate, 2 mM ethylenediaminetetraacetic acid, 30 mM tris(hydroxymethyl)aminomethane, pH 7.2, 37 degrees C] in the dark with an equilibrium dissociation constant of (1.4 +/- 0.3) X 10(-7) M. Ultraviolet irradiation of a solution of enzyme plus 3H-labeled probe, followed by analysis of covalently incorporated radiolabel, shows ouabain-displaceable labeling exclusively of the alpha subunit of the sodium- and potassium-activated adenosinetriphosphatase. These data indicate that the binding site of the C-17 side group of cardiac glycosides is located on or near the alpha subunit of this enzyme.

Comparative analysis of the tryptic peptides and terminal amino acid sequence was made on polyhedrins from two genetically different baculoviruses that are naturally pathogenic for the same insect host. Comparison of the tryptic peptides of the nucleopolyhedrosis bundle virus and nucleopolyhedrosis single-rod virus of Orgyia pseudotsugata by means of cation-exchange resins indicated that the proteins have a closely related amino acid sequence. The NH(2)-terminal amino acid sequence of polyhedrins from the two viruses differed in only 4 out of 34 amino acids. The nucleopolyhedrosis bundle virus and the nucleopolyhedrosis single-rod virus also differed in 4 and 5 out of 34 terminal amino acids, respectively, from the sequence reported for polyhedrin of a baculovirus of Bombyx mori [Serebryani, S. B., Levitina, T. L., Kautsman, M. L., Radavski, Y. L., Gusak, N. M., Ovander, M. N., Sucharenko, N. V. & Kozlov, E. A. (1977) J. Invertebr. Pathol. 30, 442-443]. In addition, the nucleopolyhedrosis single-rod virus had two amino acids (Met-Tyr) on the NH(2) terminus that were not present on the terminus of nucleopolyhedrosis bundle virus or B. mori baculovirus polyhedrin. Approximately half (six) of the total tyrosine residues are clustered in the terminal 20 amino acids of the polyhedrins. Secondary structures predicted from the primary sequence suggest that the tyrosines are clustered in two areas. This nonrandom distribution and the pK(a) of about 10 for tyrosine may be related to the alkali solubility of the polyhedrin.

The amino acid composition of the tryptic peptides from hyena hemoglobin, two hemoglobins of the mink and the alpha-chain of coatimundi have been determined, allowing comparison with data previously obtained from other carnivores. The two hemoglobins of mink differ at only one amino acid residue, alpha 15, which is glycyl in one hemoblobin and aspartyl in the other.

Fructose-1,6-bisphosphate aldolase (Fru-P2A) from a psychrophilic marine bacterium was found to be Class II aldolase based on activation by K+, activation by divalent cations, inactivation by EDTA, low molecular weight, and similar values for Km, Vmax, and Arrhenius activation energy. This enzyme was not markedly different in amino acid composition from the enzymes from mesophilic and thermophilic organisms, yet it has unusual thermal properties.

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.

The half-times, MCRs, and secretion rates of androgen-binding protein (rABP) were determined in male rats under a variety of conditions. After orchiectomy, the disappearance of endogenous immunoassayable rABP from serum was described by a single exponential term with half-lives of 21 +/- 0.2 and 20 +/- 0.8 h at 25 and 90 days, respectively. The MCR (milliliters per g/day) was not affected by age or hormonal status of the animals. The secretion rate of rABP into the blood was higher in the immature animals than in adults. The decrease in serum rABP concentrations after 20-25 days of age was due to a decrease in the rate of secretion into blood rather than an increase in MCR, a finding consistent with the observation that after formation of the blood-testis barrier, most of the rABP is secreted into the seminiferous tubular lumen. The disappearance curve after injection of purified epididymal rABP was best described by two exponential terms. The first component disappeared very rapidly and the second more slowly, with a half-time corresponding to that of endogenous rABP. The MCR calculated from the latter component was the same as that for endogenous rABP. Having established the kinetic parameters for rABP in serum, a series of experiments was conducted to determine whether it was possible for the epididymis to release this protein into the blood. The apparent half-time of rABP measured in rats in which the testes had been removed and the epididymides left intact was found to be 65 +/- 3 to 70 +/- 5 h in three separate experiments. This increase over the actual half-life of rABP (20 h) was due to the release of rABP from the epididymis into the blood. A similar experiment was performed in an identical group of animals (testes removed, epididymides intact) that had been treated with testosterone via Silastic implants. In these animals the apparent half-time (24 +/- 4 to 28 +/- 2 h; three experiments) closely approximated the actual half-life (20 h). These findings indicate that androgens retarded degeneration of the epididymides, thus minimizing their release of rABP into blood. Our experimental findings suggest the following conclusions. The dramatic rise and subsequent decline of serum rABP concentrations that occur before puberty are due to changes in the secretion rate rather than in the MCR, which is unaffected by age or hormonal states.(ABSTRACT TRUNCATED AT 400 WORDS)

C-and N-polyhedrins from a cytoplasmic polyhedrosis virus (a double-stranded RNA virus) and a nuclear polyhedrosis virus (a DNA virus), respectively, of Orgyia pseudotsugata were compared. Although both polyhedrins appear to stabilize their respective virions and have similar molecular weights, they differed in amino acid composition, tryptic peptide elution profiles from a cation-exchange resin, and N-terminal amino acid sequence and showed no antigenic relatedness. This suggests that these two proteins originated independently of one another.

Serum prolactin levels in intact, castrated, and castrated adrenalectomized normal and male pseudohermaphrodite (Tfm) rats were compared before and after administering testosterone oenanthate and oestradiol benzoate. Without steroids, castration alone and castration plus adrenalectomy effected a drop in prolactin titres which was greater in the castrated-adrenalectomized rats. Although testosterone did not raise the prolactin titres in any of the animals studied, oestrogen raised them far above normal in all. The fall in prolactin following castration and castration + adrenalectomy could be the result of removal of organs that were producing oestrogen.

A fraction enriched in the butyrate-enhanced protein (BEP) has been isolated from Chinese hamster (line CHO) cells by perchloric acid extraction and Bio-Rex 70 chromatography. Amino acid analyses indicate that the composition of BEP resembles that of CHO H1; however, BEP contains 11% less alanine than H1, and, in contrast to H1, BEP contains methionine. Treatment of BEP with cyanogen bromide results in the cleavage of a small fragment of approximately 20 amino acids so that the large fragment seen in sodium dodecyl sulfate--acrylamide gels has a molecular weight of approximately 20 000. Radiolabeling and electrophoresis indicate that BEP is phosphorylated in a cell cycle dependent fashion. In G1-arrested cells, little or no phosphate is incorporated into BEP. As cells progress through interphase, BEP becomes phosphorylated so that 12--35% of the BEP molecules are phosphorylated at one to two sites by late interphase. During mitosis, all BEP molecules become phosphorylated at approximately four sites per molecule (BEPM). Electrophoresis and the analysis of cell populations by electron microscopy indicate that the appearance of BEPM is temporally correlated with the mitotic phosphorylation of histone H1 (H1M) and with chromosomal condensation during prophase, metaphase, and anaphase. During exit from mitosis, BEPM undergoes dephosphorylation. The dephosphorylation of BEPM is temporally correlated with dephosphorylation of H1M and with the unraveling of fully condensed chromosomes near the anaphase--telophase transition. These data suggest that (1) BEP is a specialized histone of the H1 class and (2) BEP is the species equivalent of calf lung histone H1(0) [Panyim, S., & Chalkley, R. (1969) Biochem. Biophys. Res. Commun. 37, 1042], rat H1(0) [Medvedev, Zh. A., Medvedeva, M. N., & Huschtscha, L. I. (1977) Gerontology (Basel) 23, 334], and IP25, a protein enhanced in differentiated Friend erythroleukemia cells [Keppel, F., Allet, B., & Eisen, H. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 653]. The data also indicate that putative HMG1 and HMG2 proteins do not undergo the extensive cell cycle dependent phosphorylations measured for histone H1 and BEP.

The amino acid sequences of the hemoglobin alpha- and beta-chains of raccoon have been determined by a combination of manual and automatic sequencing procedures. The raccoon beta-chain shows 16 amino acid differences from that of dog. The alpha chain shows 10 differences. These values are identical with those predicted by analogy from their tryptic peptide compositions.