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 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.

The epididymis contributes minimally to serum androgen-binding protein in the rat: a whole body kinetic study.

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)

Hormonal factors related to the incidence of testicular tumors in male pseudohermaphrodite rats.

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.

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.

Conservation of iron and hemoglobin during induced hemolytic stress in the amphibian Taricha granulosa.

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.

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.

Developmental pattern of androgen-binding protein secretion during the critical period of sexual differentiation.

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 hemoglobin-binding proteins identified in the plasma of the amphibian Taricha granulosa.

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.

Fructose-1,6-bisphosphate aldolase from Vibrio marinus, a psychrophilic marine bacterium.

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.

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 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.

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.

Comparison of the Structure of C- and N-Polyhedrins from Two Occluded Viruses Pathogenic for Orgyia pseudotsugata.

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.

Amino acid sequence of the hemoglobin of raccoon (Procyon lotor).

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.

Effects of testosterone and oestrogen on serum prolactin in the androgen-insensitive rat.

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.

Tryptic peptide analysis and NH2-terminal amino acid sequences of polyhedrins of two baculoviruses from Orgyia pseudotsugata.

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.

Tyrptic peptide compositions of hemoglobins from mink (Mustela vison) and hyena (Hyaenae hyaenae) and the alpha-chain of coatimundi (Nasua nasua).

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.