Basic Protein Fraction Research Articles

Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins.

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.

D-Aspartic acid in purified myelin and myelin basic protein

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.

Estimation of hydrophobicity of the 12 S globulin and the basic low molecular protein fraction from rapeseed (Brassica napus L.) by partition in an aqueous biphasic polymeric system

Estimation of hydrophobicity of the 12 S globulin and the basic low molecular protein fraction from rapeseed (Brassica napus L.) by partition in an aqueous biphasic polymeric system

The major basic proteins of bull seminal vesicle secretion.

We have employed HPLC on reversed phase columns to analyse the major basic proteins from bull seminal vesicle secretion. The identification of proteins was achieved by comparison with authentic protein samples from bull seminal plasma as well as immunological characterisation using antisera directed against the latter proteins. The major basic proteins from bull seminal plasma: bull seminal proteinase inhibitor II (BUSI II), the seminal ribonuclease BS1, the protein P6 as well as the antimicrobial protein were also identified as the main constituents of the fraction of basic proteins derived from seminal vesicle secretion. FPLC using Mono S HR columns was also found to resolve the mixture of basic proteins and proved to be especially useful with respect to the isolation of the antimicrobial protein from basic proteins of seminal vesicle secretion. The identity of the antimicrobial protein from bull seminal plasma with the respective protein from seminal vesicle secretion was confirmed by amino-acid analysis and comparison of tryptic peptide patterns by HPLC. The antimicrobial protein was isolated from seminal vesicle secretion with a yield of 3 mg/ml of secretion.

Identification and partial purification of fibroblast growth factor from the brains of developing rats and leucodystrophic mutant mice

High titres of fibroblast growth factor activity (assessed by mitogenicity for Balb/c 3T3 fibroblast cells in vitro) have been extracted from the brains of foetal and neonatal rats long before myelinogenesis commences, from the brains of hypomyelinated, leucodystrophic, murine mutants and from normal adult rats. Partial purification, by ion-exchange chromatography and gel filtration, indicates that the brain fibroblast growth factor activity from these sources is associated with very similar basic protein fractions. These results, together with the observation that none of the samples of partially purified basic fibroblast growth factor elicits the experimental allergic encephalomyelitis response attributed to myelin basic protein in the rat, suggest that basic fibroblast growth factor is not a degradation product of myelin basic protein.

Characterization of basic proteins of bull seminal plasma.

We have employed high-performance liquid chromatography on reversed phase columns to analyse the major basic proteins from bull seminal plasma. The proteins were separated preparatively and characterized with respect to molecular mass, amino-acid composition as well as by means of immunodiffusion against specific antisera. The following proteins could be identified: bull seminal proteinase inhibitor II (BUSI II), two seminal RNAases, the seminal antimicrobial protein and proteolytic fragments, derived from it, and a hitherto unknown protein P6 of molecular mass 20 000 Da. Another unknown protein, P5, found to be formed during preparation of the basic protein fraction turned out to be a proteolytic fragment of protein P6 with a molecular mass of 8 750 Da for the polypeptide chain. Antisera against the isolated proteins were raised in rabbits and their specificity established. Single radial immunodiffusion was used to determine the concentration of the above basic proteins in bull seminal plasma: BUSI II (0.25 mg/ml), seminal RNAases (6.5 mg/ml) and protein P6 (2.9 mg/ml).

Ethionine inhibits in vivo methylation of nuclear proteins.

Nuclear protein methylation was studied in regenerating rat liver by giving [methyl-3H]methionine 45 h after partial hepatectomy. Ethionine, a liver carcinogen, has been shown to alter the methylation patterns in a basic protein (histone) fraction, as well as an acidic protein (non-histone) fraction present in a 0.25 N HCl nuclear extraction. The proteins present in the 0.25 N HCl extraction were separated by chromatography using a Bio-Rex 70 cation exchange column. Polyacrylamide gel electrophoresis and total amino acid analysis showed the first protein fraction contained acidic large molecular weight non-histone proteins, while the second fraction contained basic small molecular weight histone proteins. Both fractions were then hydrolyzed, and the amino acids chromatographed on an Aminex A-5 cation exchange column. The histones were found to contain epsilon-N-mono, di and trimethyllysine derivatives; whereas the non-histone fraction contained these lysine derivatives and additional basic amino acid identified as NG,NG-dimethylarginine. Ethionine (0.5 mg/g body weight) was found to inhibit in vivo methylation of lysine to form epsilon-N-mono, di and trimethyllysine, 46, 52 and 68%, respectively. The formation of NG,NG-dimethylarginine was inhibited by 85%. Ethylation of these proteins was also studied by giving [ethyl-3H]ethionine. After hydrolysis, the non-histones were found to contain a labeled lysine and arginine derivative, but in the histone fraction only labeled lysine was found.

Isolation and characterization of the main toxic fraction from the venom of the false horned viper ( Pseudocerastes fieldi)

R. Batzri-Izraeli and A. Bdolah. Isolation and characterization of the main toxic fraction from the venom of the false horned viper ( Pseudocerastes fieldi). Toxicon 20, 867–875, 1982—The venom of Pseudocerastes fieldi was subjected to gel filtration on Sephadex G-75. Most of the protein and lethality of the venom were eluted in a major symmetrical peak (C). The lethality of this peak is confined to a basic protein fraction, Cb (pI > 9.5) separable by DEAE-cellulose chromatography. Two proteins with molecular sizes close to 16,000 daltons were isolated from this fraction by preparative acidic gel electrophoresis in the presence of Triton X-100. One of the proteins (CbII) is lethal to mice ( ld 50 = 1 mg/kg ) and shows phospholipase A activity as well as direct hemolytic activity. The other protein (CbI) does not reveal any known biological activity. However, upon recombination of the two a synergistic lethal activity is evident ( the ld 50 of the mixture = 0.25 mg/kg ). It is suggested that CbI may be a specifier which potentiates the toxicity of the phospholipase A at the target site.

Microelectrophoretic analysis of basic protein changes during spermiogenesis in the dogfish Scylliorhinus caniculus (L.)

Spermatogenesis in the dogfish is characterized by the synchronous development of germinal cells inside follicles. This particularity has permitted studies on precise stages of cell differentiation, especially on the evolution of chromatin structure. A microelectrophoretic method has been devised for the determination of the basic nuclear protein content of accurately identified homogeneous stages of spermatid differentiation. No significant difference was observed during the first stages of spermiogenesis, i.e., in round spermatids, where a typical histone complement was present. At the beginning of nuclear elongation, two new basic protein fractions appeared and coexisted for some time with typical histones; they replaced somatic histones progressively. Later, during elongation, four proteins of high electrophoretic mobility appeared and gradually replaced the intermediary basic proteins. In elongated spermatids, DNA was found tightly packed by these four proteins: three are arginine- and cysteine-rich (Z1, Z2 and S4), the fourth is arginine-rich (Z3). At first, these fractions are all soluble in 0.25 M HCl but during sperm maturation only one (Z3) remains acid-soluble, the others being extractable only after reducing and alkylating treatments. This modification of solubility of Z1, Z2 and S4 corresponded to the oxidation of cysteine residues to form SS crosslinks in chromatin of mature sperm cells. Thus spermiogenesis of the dogfish shows two basic nuclear protein transitions which both occur during nuclear elongation.

Isolation and characterization of multiple components of basic gonadotropin from bullfrog (Rana catesbeiana) pituitary gland.

Four gonadotropin components were purified from the basic protein fraction of bullfrog pituitary glands. All the components had high potencies not only in the ovulation assay using Xenopus laevis ovaries but also in the competitive binding assay for rat follicle-stimulating hormone (FSH) using Xenopus laevis testis. The isoelectric points of the four components determined by isoelectric focusing were at pH 8.8, 9.0, 9.1, and 9.3, respectively. No appreciable difference in the physical and chemical properties other than isoelectric points was found among the four components. Their Stokes radius estimated by gel filtration was 2.36 nm. This value was appreciably lower than that reported for mammalian luteinizing hormone (LH). On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, reduced preparations of the four components each showed two bands, indicating a subunit structure similar to that of mammalian gonadotropins. Their mobilities were greater than those of mammalian LH. The molecular weights of subunits estimated by high-speed gel filtration in 6 M guanidine hydrochloride were lower than those of mammalian LH. The amino acid composition of the four basic components of bullfrog gonadotropin was appreciably different from that of mammalian LH.

Turnover of basic chromosomal proteins in fertilized eggs: a cytoimmunochemical study of events in vivo.

The chromosomal complements of mouse oocytes, ova, and fertilizing sperm have been studied by immunofluorescence with specific antisera to the basic protein fraction of sperm nuclei and to histones H2b and H4, and by staining with ethidium bromide. These studies support the hypothesis, previously proposed (Rodman and Barth, 1979, Dev. Biol. 68:82-95), that the chromosomes of the oocyte in maturation incorporate unique basic protein(s) similar to those incorporated during spermiogenesis. That similarity is characterized, here, by immunologic cross-reactivity. The basic proteins of the fertilizing sperm nucleus and the cross-reactive moiety of the two haploid complements of the ovum are displaced simultaneously, shortly after sperm entry. However, because the unique basic proteins incorporated into the oocyte chromosomes do not, as in the spermatogenic sequence, entirely replace the histones, the maternal chromosomes display histones H2b and H4 at all postfertilization stages examined, whereas the decondensing paternal complement, for an interval before maturation of the pronuclei, contains neither sperm basic chromosomal proteins nor histones. Sequential staining of the same specimens with ethidium bromide revealed well-organized nuclear morphology of the residual DNA complex. Those observations suggest that, for an as yet undefined period in the transformation from spermatozoal to embryonic genome, the chromatin is devoid of a complement of basic proteins.

Age-changes in basic protein contents of liver and muscle of the fresh water teleost, Channa punctatus

The basic protein fractions and the total protein contents of liver and muscle of the murrel ( Channa punctatus) were estimated to evaluate the effect of age. In the muscle of males and in both muscle and liver of females the basic protein contents showed a rising trend with advancing age. In males, the total protein content of liver showed a peak during maturity and declined thereafter but in muscle a continuous decline occurred with increasing age. Neither in the basic protein content of liver of males nor in the total protein contents of liver and muscles of females were appreciable age-changes observed.

Subunit structure of Trypanosoma cruzi chromatin.

Chromatin of Trypanosoma cruzi epimastigotes was found to be arranged in a subunit structure by the demonstration of (i) nu-bodies, (ii) monomeric and oligomeric DNA fragments, and (iii) four basic nuclear proteins which presumably correspond to the intrinsic protein component of nucleosomes. A discrete core fragment of 140 base pairs and a basic nuclear protein fraction clearly similar to most histones H1 were not detected. In contrast with vertebrate material, the nuclear preparations were physically and enzymatically fragile, the basic proteins showed different mobilities in electrophoresis, but the DNA repeat length was similar. The data are discussed in relation to histone regional charge, histone content, and chromatin condensation.

Life history of mouse sperm protein. Intratesticular stages.

A basic protein fraction, migrating as a single band in acetic acid-urea gel, distinct from histones, was isolated from mouse sperm collected from vasa deferentia and caudae epididymides and was used to immunize female rabbits. The presence of antibodies to the mouse sperm protein (MSP) in the rabbit antisera was demonstrated by a cytoimmunofluorescence procedure using the cells of origin of the antigenic protein, the mature mouse sperm. The specificity of the antisera was verified by fluid and gel precipitation tests and by crossed immunoelectrophoresis. The latter procedure demonstrated the presence of two antigen-antibody systems, consonant with earlier reports that the basic chromosomal protein of mouse sperm is heterogeneous. MSP antigen in situ was recognized by the specific antibodies of the rabbit antisera only after the smear of mature sperm was treated with either of two reducing agents: 2-mercaptoethanol or dithiothreitol. However, when the immunofluorescence procedure was applied to untreated smears of mouse testicular cells, spermatids of all stages from 1 to 14-15 were positive, while spermatocytes, stage 16 spermatids and spermatozoa were negative. After treatment of testes smears with reducing agent, only spermatocytes remained negative. Those observations indicate the following: (a) MSP is immunogenic in a heterologous species; (b) its antigenic sites are detectable in spermatozoa and spermatids of all stages, but not in primary spermatocytes; (c) those antigenic sites become masked at about stage 15 of spermiogenesis and may be unmasked by treatment with a reducing agent. The interpretation is made, therefore, that one or more components of MSP are assembled at the beginning of spermiogenesis and undergo an alteration in the final intratesticular stage of spermatid maturation. That alteration may be presumed to be the formation of disulfide linkages between the cysteine residues.

Fertility of boar spermatozoa after freezing in the absence of seminal vesicular proteins

Because exposure to seminal plasma enhances the detrimental effects of cold shock on boar spermatozoa (Lasley & Bogart, 1944; Pursel, Johnson & Rampacek, 1972), many procedures for preserving semen by deep freezing employ sperm-rich ejaculate fractions (Pursel & Johnson, 1975; Larrson & Einarsson, 1975), thereby excluding much of the seminal plasma. Our previous studies showed that a basic protein fraction of seminal vesicle origin binds irreversibly to boar spermatozoa at ejaculation (Moore & Hibbitt, 1976) and during cooling promotes sperm membrane disruption (Moore, Hall & Hibbitt, 1976). We therefore tested the possibility that boar spermatozoa could be frozen more effectively in the absence of seminal vesicular fluid.

Adaptation of the Three Stage Extraction Process to Rapeseed Meal for Preparation of Colourless Protein Extracts

The three stage extraction method reported for preparation of four different protein fractions from detoxified rapeseed flour (Kodagoda et al., 1973) was unsuitable for application to commercial oil-extracted meals since the separation of the basic protein fraction from the acidic and neutral fractions was incomplete. Adaptation of the original method to commercial rapeseed meals became feasible by decreasing the number of water extractions in the first stage from three to two. Further repetition of the water extraction caused contamination of the acid-neutral protein fraction with the basic proteins. The oxalic acid treatment used in the original procedure to decrease the ash content in the basic proteins was no longer required. The basic proteins separated in the second stage contained higher lysine, histidine and cystine than the acid-neutral proteins extracted in the first stage and also exhibited excellent emulsifying and foaming properties.Of the four different oxidizing agents, hydrogen peroxide was the most effective in decolourizing the dark brownish or greenish protein extracts. However, a significant loss in the sulfur containing amino acids occurred by this treatment. A sodium sulfite or sulfur dioxide treatment was found effective in conjunction with protein precipitation from ethanol solutions; after drying, the powders were nearly colourless.

Iodination-deiodination: A radiochemical method for detection of structure and changes in structure in RNA

Bound iodine is released from radioiodinated nucleotides in polymers exposed to sodium bisulfite. The rate of bisulfite-catalyzed deiodination of pyrimidines can be controlled both by change of temperature or pH and is also dependent on the molecular association of the nucleotide. The rate of release of iodine from iodocytidine in polycytidylate is greater than the rate of elimination from RNA. Experiments testing the influence of base-pairing of the iodopyrimidines in synthetic polynucleotides showed that pairing of the substituted nucleotide protected the iodine bond. The rates of bisulfite-catalyzed deiodination of several radioiodinated RNAs were measured. The action of bisulfite on all single stranded RNAs tested was multiphasic consisting of a rapid early deiodination reaction supplanted by a slower phase which was followed by reacceleration of release. The release of iodine from double stranded RNA and DNA-RNA duplexes was retarded in comparison with the release from ribosomal and messenger RNA fractions. The deiodination profiles of single and double stranded RNA suggested that the intermediate stage iodine release is governed by melting of paired zones of low stability. Late release may result from destabilization of the molecule through the addition of bisulfite to the pyrimidine ring or deamination. The effect of several substances expected to complex with polynucleotides was tested. Acridine orange and ethidium bromide increased loss of iodine from ribosomal RNA but slightly decreased elimination from double stranded viral RNA. A basic protein fraction isolated from ribosomal particles accelerated the deiodination of ribosomal RNA. While the destabilization caused by this protein fraction was greater than that caused by an equal amount of albumin, as tested the effect was non-specific. The results show that a change in sensitivity to chemical deiodination may follow the interaction of small amounts of protein with polynucleotides.

A new rat liver phospholipid exchange protein

The soluble fraction from several mammalian tissue homogenates is known to stimulate phospholipid exchange between cell membrane fractions or artificial vesicles. All phospholipid exchange proteins purified to data exhibit an acidic isoelectric point. Using an assay that measures the transfer of [ 32P]phosphatidylcholine from liposomes to beef heart mitochondria, we report the presence of a new phospholipid exchange protein with a basic isoelectric point (8.4) in rat liver cytosol. A purification procedure, consisting of pH adjustment to 5.1, gel filtrations on Sephadex G-75 and DE 52 cellulose, isoelectric focusing between a pH of 5 and 10, and gel filtration on Sephadex G-50, yielded a fraction with high phosphatidylcholine exchange activity per mg of protein. This fraction exhibits a major band and two minor bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the major band (18 700) is close to that for basic exchange protein fraction obtained by gel filtration (17 000). The distribution of basic and acidic exchange proteins differs markedly in various tissues and animal species. About 50 and 35% of phosphatidylcholine exchange activity from rat liver and rat intestine respectively are due to basic phospholipid exchange proteins. In contrast, no basic exchange protein was found in beef heart and only a small amount in beef liver. In the latter organ, less than 10% of phosphatidylcholine exchange activity was due to a basic phospholipid exchange protein fraction.

Nuclear components responsible for the retention of steroid–receptor complexes, especially from the standpoint of the specifcity of hormonal responses

1. By covalently linking nuclear components from hormone-sensitive cells to Sepharose 2B, it is possible to investigate the interaction between nuclear components and cytoplasmic receptor-steroid complexes by affinity chromatography. 2. Many factors are implicated in the specifity of nuclear-cytoplasmic interactions, including the nature of the nuclear components, the presence of the cytoplasmic receptor protein and the provision of the appropriate steroid ligand. 3. Two distinct sets of binding sites are present in nuclear extracts immobilized to Sepharose 2B, namely a small number of specific high-affinity sites and a larger number of non-specific low affinity-sites. 4. Considerable evidence supports the importance of the high-affinity binding sites in the manifestation of hormonal specificity in different tissues. Although the study has centred largely on androgenresponsive systems, the findings are germane to cytoplasmic-nuclear interactions in general. 5. The high-affinity or acceptor sites in nuclear extracts reside in the basic but non-histone protein fraction. 6. Hormonal specificity is seemingly maintained by both the cytoplasmic and nuclear components, and the results are discussed in the context of the mechanism of action of steroid hormones.

Interaction of the estradiol receptor from calf uterus with its nuclear acceptor sites

The specific interaction between 17 beta-estradiol-receptor complex and nuclear acceptors was analyzed by immobilizing various nuclear proteins to CNBr-activated agarose. The specific, high affinity sites identified in a fraction of basic proteins that can be solubilized from purified nuclei of calf uterus (Puca, G.A., Sica, V., and Nola. E (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 979-983) were chromatographed on Sephadex G-100 columns. Elution of the acceptor activity depends on the pH and ionic strength of the buffer used. With 5 mM HCl, however, a peak of acceptor activity with a molecular weight of about 70,000 was partially dissociated from the other basic nuclear proteins. The high affinity binding of the receptor to the acceptor proteins was estradiol-, but not progesterone-, cortisone-, or testosterone-dependent; it was very sensitive to ionic strength and showed a physiological pH optimum. Low affinity binding, such as that seen between receptor and histone, showed no estradiol dependence and little ionic strength and pH sensitivity. Native or heat-denatured DNA strongly modified the receptor-acceptor interaction, reducing the number of binding sites of acceptor for the receptor without changing the high affinity of the interaction. Heating of the acceptor protein before its covalent linkage to agarose considerably increased the affinity of the resulting agarose derivative. Free sulfhydryl groups of the receptor but not of the acceptor molecule play an important role in the acceptor-receptor interaction. When receptor and acceptor preparations were incubated in solution, the resulting complex was included on a Sephadex G-100 column and it eluted from DEAE-cellulose columns at lower ionic strength than the receptor alone. Even though not absolutely specific, these two properties allowed determination of the molecular weight (85,000) of the acceptor protein at neutral pH and more nearly physiological ionic strength. The apparent KD of the acceptor-receptor interaction was determined to be 2 x 10(-10) M at O degrees. Apparently similar, high affinity binding sites for estradiol receptors are also present in nuclei of other tissues.