A two-receptor model for salmon gonadotropins (GTH I and GTH II).

The possible existence of distinct receptors for salmon gonadotropins (GTH I and GTH II) and the distribution of the receptor(s) were studied through examination of the binding of coho salmon (Oncorhynchus kistuch) GTH I and GTH II to membranes from thecal layers and granulosa cells of salmon ovaries. Purified coho salmon gonadotropins were iodinated by the lactoperoxidase method. Crude membrane preparations were obtained from thecal layers, granulosa cells, and whole ovaries of coho salmon in the postvitellogenic/preovulatory phase. Binding of 125I-GTH I to membranes from thecal layers, granulosa cells, and whole ovaries, and binding of 125I-GTH II to thecal layer cell membranes could be inhibited by both GTHs, but GTH I was more potent than GTH II. In contrast, GTH II was more potent than GTH I in inhibiting 125I-GTH II binding to membranes from granulosa cells and whole ovaries, but the inhibition curves were not parallel. Scatchard plot analysis suggested that there was a single type of receptor in the thecal layers for both GTHs, whereas in the granulosa cells there was more than one type of receptor for both GTHs. Based on these results, a two-receptor model for the postvitellogenic/preovulatory salmon ovary is proposed with the following features: 1) there are two types of gonadotropin receptors in the salmon ovary, type I and type II; 2) the type I receptor binds both GTHs, but with higher affinity for GTH I, whereas the type II receptor is highly specific for GTH II and may have only limited interaction with GTH I; and 3) the type I receptor is present in both thecal cells and granulosa cells, whereas the type II receptor is present in granulosa cells.     

Isolation and characterization of chum salmon growth hormone

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by iso-electric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 microgram/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.     

Occurrence of a new corticotropin-like intermediate lobe peptide in salmon pituitary glands

A new corticotropin-like intermediate lobe peptide (CLIP) has been identified in the pituitary of chum salmon, Oncorhynchus keta. The newly isolated peptide is a tetracosa peptide, which is two residues longer than the predominant form, CLIP I, with the following amino acid sequence, H-ArgProIleLysValTyrAlaSerSerLeuGlu GlyGlyAspSerSerGluGlyThrPheProLeuGlnAlaOH. This peptide, named CLIP II is the fourth line of evidence in the teleost that the pituitary gland secretes two different forms of processed hormones, for which precursor molecules are coded on two separate genes. Together with the structures of α-melanotropin I and II, two putative ACTH molecules are proposed.     

Isolation and characterization of calcitonin from pericardium and esophagus of eel.

Calcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs. The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on the Sephadex G-50, chromatography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.     

Seawater adaptability of underyearling coho,chinook and chum salmons in the estuary of Avacha River (Kamchatka)

Synopsis The 96 h LC50 tests were conducted with chum, coho and chinook salmon alevins caught in the Avacha River (Kamchatka) estuary. Coho and chinook alevins exhibited very low seawater a adaptability when compared with chum. It is likely that coho and chinook young were pulled downstream by high velocity flow during the summer freshets.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

The subunit structure and active site sequence of porcine spleen deoxyribonuclease

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Fish gonadotropin(s). II. Isolation of gonadotropin(s) from chum salmon pituitary glands using affinity chromatography.

A highly purified gonadotropic hormone preparation has been obtained from chum salmon (Oncorhynchus keta) pituitaries by extraction with ethanolic or aqueous buffer, affinity chromatography on Con A Sepharose and gel filtration on Sephadex G-75 superfine. A purified fraction from Sephadex G-75 averaged 448 mug NIH-LH-S18/mg glycoprotein as measured by the uptake of radiophosphate into chick testes. A total of 1.1 g of salmon gonadotropin (s) (SG)/kg fresh tissue was recovered when the isolation began with an aqueous extraction. Analytical polyacrylamide gel electrophoresis (P.A.G.E.) of the purified fraction from Sephadex G-75 displayed a single broad zone in non-dissociating conditions and two bands in 8 M urea. Polyacrylamide gel electrofocusing yielded six sharp bands with an isoelectric point range of 4.38 to 5.05, and four bands with an isoelectric point range of 4.31 to 4.95 in 8 M urea. A molecular weight of 41,000 was determined by gel filtration. A subunit molecular weight of 17,800 +/- 10% was found by P.A.G.E. in 0.1% sodium dodecyl sulphate (SDS), suggesting that native SG consists of two subunits. Purified preparations were highly stable in Tris-Cl buffers and retained their activity for several months when stored at -73 degrees C.     

猪骨胶原蛋白酶解物中血管紧张素转换酶抑制剂的提纯

将猪骨胶原蛋白粗提物用胰蛋白酶水解,经阳离子交换树脂层析,ＳｅｐｈａｄｅｘＧ－２５柱凝胶过滤,以及数次反相高效液相层析,最终获得一具有抑制血管紧张素转换酶（ＡＣＥ）活性的单一峰值的多肽。其氨基酸组成为Ｉｌｅ,Ｈｉｓ,Ｓｅｒ,Ｇｌｙ,Ａｌａ,Ｐｒｏ,Ｔｙｒ,Ｌｅｕ,Ａｓｐ．以Ｈｉｐ－Ｇｌｙ－Ｇｌｙ为底物,在ｐＨ为７．１的条件下,此肽对猪肺ＡＣＥ的Ｉ＿（５０）值为２６μｍｏｌ／Ｌ。     

Chemical identification of catfish growth hormone and prolactin.

Isolation and primary structure of growth hormone (GH) and prolactin (PRL) from the pituitary gland of catfish (Ictalurus punctatus) are described. Alkaline extract of the pituitary glands was fractionated by gel filtration on Sephadex G-75, ion-exchange chromatography on DEAE-cellulose, and reversed-phase high-performance liquid chromatography on Octadecyl silica ODS. Catfish GH and PRL were identified by Western blotting with antisera against chum salmon GH and PRL. The catfish GH consists of 178 residues and is the most similar to carp GH, with sequence identity of 77%, although there is an uninterrupted deletion of 10 amino acid residues that corresponds to carp GH (90-99). The PRL is composed of 187 residues, which also exhibits the highest identity (79%) with carp PRL. Sequence identity between catfish GH and PRL is only 27%.     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

Partial purification of pepsins from adult and juvenile salmon fish Oncorhynchus keta. Effect of NaCl on proteolytic activities

1. Two pepsins, designated Pepsin I and Pepsin II, were isolated and partially characterized from the stomach of the adult stage salmon Oncorhynchus keta. This stage is developed in a marine environment. 2. One pepsin, designated Pepsin II, was isolated from the stomach of the juvenile stage salmon Oncorhynchus keta. This stage is developed in an estuarine environment. 3. The enzymes were partially purified by ammonium sulfate precipitation, ion exchange chromatography and gel filtration. 4. Pepsins I and II from adults and Pepsin II from juvenile showed proteolytic activity on acid-denatured hemoglobin with a pH optimum of 3. 5. The mol. wt determined by gel filtration on Sephadex G-100 of Pepsin I from juvenile species was found to be 32,000 whereas a value of 27,000 was determined for Pepsin II from juvenile and adult fish. 6. In contrast with Pepsin II, Pepsin I was activated by NaCl. It is suggested that the appearance of NaCl-activated pepsin would represent and adaptive response of the organism to the change from a low to a high salinity environment.     

Studies on epsilon-prototoxin of Clostridium perfringens type D. Physicochemical and chemical properties of epsilon-prototoxin.

epsilon-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asp52 Thr31 Ser25 Glu28 Pro12 Gly17 Ala14 Val28 Met5 Ile15 Leu18 Tyr17 Phe8 Lys31 His2 Arg5 Tyr2. It has no free cysteine but contains one blocked cysteine. The N-terminal as well as the C-terminal residue is lysine. The ultracentrifuge pattern gave one single boundary having S020,w = 2.15 S and Do20,w = 5.56-10(-7) cm2/s. Calculation of the molecular weight from D020,w and S020,w gave a value of 34 250. The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 +/- 1000. On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500. This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of epsilon-prototoxin by Sephadex.     

Fish gonadotropin(s). III. Evidence for more than one gonadotropin in chum salmon pituitary glands.

Two proteins with gonadotropin activity have been isolated from a highly purified chum salmon (Oncorhynchus keta) gonadotropin preparation (G-75 Fraction II) by chromatography on DEAE Bio Gel A. These gonadotropins exhibited distinct behaviour in polyacrylamide gel electrophoresis, chromatography on Sephadex G-75 superfine, and ratios of cAMP stimulation in immature rainbow trout ovaries and testes. Rechromatography of G-75 Fraction II on Sephadex G-75 superfine gave a symmetrical protein peak with a coincident cAMP activity profile. Repeated freezing and thawing elicited a shift in the cAMP activity profile toward the trailing edge of the protein peak. Data are discussed in terms of two gonadotropin molecules which respond differently to phase changes. Charge polymorphism was exhibited by isoelectric focusing in polyacrylamide gels of one of the DEAE fractions. Five UV absorbing bands were observed which stimulated cAMP production in immature rainbow trout gonads. Three of these bands increased adenyl cyclase activity in trout ovaries and testes. One of the bands stimulated cAMP production primarily in trout testes and the other stimulated trout ovaries, providing evidence for two gonadotropins, each of which is sex specific.     

Primary structures of carp gonadotropin subunits deduced from cDNA nucleotide sequences

The alpha and beta subunits of carp gonadotropin (cGTH) were isolated by high performance liquid chromatography. They were identified to be the subunits of cGTH by bioassay and by partial N-terminal amino acid sequence analysis. To elucidate the complete primary structures of the alpha and beta subunits of cGTH, cDNA cloning technique was employed. The alpha and beta subunits consist of 95 and 115 amino acid residues, respectively. Homology of the alpha subunit of cGTH to those of mammalian GTH is around 70%. In comparison, the extent of homology of the beta subunit between carp and salmon GTH (75%) is higher than that between fish and mammalian GTH (39-47%). Such comparative data suggest that the alpha subunit is highly conserved while the beta subunit is diversified during the molecular evolution of vertebrate GTH.     

Isolation and properties of a ferredoxin from leaves of Sambucus racemosa L.

Ferredoxin was isolated in good yield from leaves of Sambucus racemosa L. by the following procedure: (1) homogenization in buffered acetone-water (1:1v/v); (2) ion-exchange chromatography on several columns of DEAE-cellulose; and (3) purification by gel filtration with Sephadex G-75. The ultraviolet and visible spectrum showed maxima at 277, 331, 423, and 466 nm. The electron paramagnetic resonance spectrum was centered around g = 1.957. The protein sustained an initial photoreduction rate of 86 mumol NADP per milligram chlorophyll per hour. The amino acid composition was found to be Lys 5, His 2, Arg1, Asx11, Thr5, Ser7, Glx17, Pro6, Gly7, Ala6-7, Cys4, Val8, Ile5, Leu7, Tyr3, Phe2, and Trp1. The molecule had a molecular weight of 10 700 and contained two atoms of iron. The amino-terminal residue was alanine. These properties are highly similar to those of other angiosperm ferredoxins. Sambucus ferredoxin was found to be most closely related to that of Leucaena.     

Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.     

Differential production and regulation of gonadotropins (GTH I and GTH II) in the pituitary gland of rainbow trout,Oncorhynchus mykiss,during ovarian development

Summary Biosynthesis of salmon gonadotropins, GTH I and GTH II, during ovarian development, were examined by means of in situ hybridization histochemistry and indirect immunocytochemistry. In rainbow trout pituitary glands, expression of GTH I- and II-subunit genes appeared separately in distinct cells (GTH I- and GTH II-cells), whereas the GTH -subunit gene was expressed in both cell-types. In the GTH I-cells, coordinated increases in GTh, and I messenger ribonucleic acids (mRNAs) occurred coincident with the onset of vitellogenesis, indicating active synthesis of GTH I during vitellogenesis. In contrast, in the GTH II-cells, both GTH -and II-mRNA signals markedly increased from a later stage of vitellogenesis and persisted throughout oocyte maturation and ovulation, supporting the idea that GTH II is actively synthesized as a maturational GTH. GTH -mRNA levels in the GTH I-cells selectively decreased prior to final oocyte maturation, although I-mRNA levels remained elevated, thus suggesting a decline of biosynthesis of GTH I after vitellogenesis. These findings clarify how the synthesis of GTH I and GTH II are coordinated in the piscine pituitary, and indicate that the expression of GTH subunit genes during gametogenesis is regulated differentially in a cell-specific manner, both temporally and spatially.     

Suppressor cell activity in tumor-bearing mice. III. Co-purification of a factor inhibiting cellular DNA synthesis and DNA polymerase activity

The factor(s) derived from fibrosarcoma-induced suppressor T cells was sensitive to pronase and neuraminidase, but not to trypsin, beta-galactosidase, DNase, or RNase. Protein and RNA, but not DNA, synthesis were required to mediate suppression. Suppressor T cell-derived factor(s) could be precipitated by a 50% saturated ammonium sulfate (SAS) solution. The 50% SAS fraction inhibited both in vitro and in vivo spleen cell blastogenesis, whereas the 80% and unprecipitated fractions had no inhibitory activity. Using Sephadex G-200 chromatography, the 2nd protein fraction (fraction II) contained an inhibitor of both DNA polymerases (IDP) and DNA synthesis (IDS) activity, which possessed no cytotoxic activity. In vitro DNA polymerase alpha activity was suppressed by fraction II, whereas DNA polymerase beta and gamma activities remained unchanged. Molecular weight of IDP/IDS, as determined by Sephadex G-200 gel filtration chromatography, was approximately 14,500. Attempts to separate IDP/IDS activities found in fraction II by anion-exchange chromatography and slab gel electrophoresis were not successful, which suggested that the 2 activities were the same or very similar molecules.