Purification and characterization of Mr 43,000 protein similar to Mr 25,000 protein, a substrate for protein Ser/Thr kinases, identified as a part of Xenopus laevis vitellogenin B1

Mr 25,000 protein (pp25), a substrate for protein Ser/Thr kinases, was recently shown to consist of a portion of the Xenopus laevis vitellogenin B1 protein. By Western blot analyses using antibodies against pp25, a minor protein band with Mr 43,000 (pp43) was detected in purified preparations of pp25. In this study, pp43 was highly purified through several column chromatography steps and its protein structure was analyzed. The amino acid sequence of the amino-terminal region of pp43 was the same as that of pp25. pp43 contained about two times more phosphates than pp25. These phosphates in pp43 were more resistant to acid phosphatase attack than those of pp25. pp43 was able to bind to pNiXa, a binding protein of pp25. Alpha-chymotryptic digestion generated a common fragment with molecular mass of 23,000 from both pp43 and pp25. These results suggest that pp43 may be a precursor of pp25 generated during processing of vitellogenin B1.     

Degradation of M r 25,000 Protein by Cathepsin L-like Protease in Xenopus laevis Oocytes

A phosphorylated protein with a molecular mass of 25,000 (pp25) is involved in Xenopus laevis vitellogenin B1 and partially overlaps with phosvitin and lipovitellin 2. The protease responsible for pp25 degradation was studied in vitro since this occurs during embryogenesis. Initially, a protease thought to be a contaminant of the purified pp25 preparation was analyzed and an antipain-sensitive protease presumed to be involved. When commercially available proteases were examined, pp25 was not degraded by calpain I or 20S proteasome, but it was degraded by cathepsin L in vitro. A survey of the protease responsible for pp25 degradation in the cytoplasm of Xenopus oocytes found partially purified pp25 was degraded in partly antipain-sensitive manner. These results suggest that an antipain-sensitive protease or cathepsin L (or a related protease) is a candidate for pp25 degradation.     

Modulation of Protein Phosphorylation by M r 25,000 Protein Partially Overlapping Phosvitin and Lipovitellin 2 in Xenopus laevis Vitellogenin B1 Protein

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35–45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.     

Regulation of Protein Synthesis in Plant Embryo by Protein Phosphorylation : I. Purification and Characterization of a cAMP-Independent Protein Kinase and Its Endogenous Substrate

A cyclic AMP-independent protein kinase, which strongly inhibits in vitro protein synthesis, was purified to homogeneity from barley embryo by affinity and ion exchange chromatography. The M_r of the purified enzyme is 95,000 with two nonidentical subunits of M_r 58,000 and 39,000. The enzyme activity is not stimulated by cAMP, cGMP, or calmodulin. The endogenous phosphate acceptor of this kinase is a protein of M_r 52,000, was isolated by purified protein kinase immobilized Sepharose column. Using antibodies raised against this protein kinase, the levels of the enzyme during embryogenesis and germination are determined. An inverse relationship has been observed between protein kinase level and rate of protein synthesis.     

Mr 25 000 protein,a substrate for protein serine/threonine kinases,is identified as a part of Xenopus laevis vitellogenin B1

A phosphorylated protein with a molecular mass of 25 000 (pp25) previously purified from the cytosolic fraction of Xenopus laevis oocytes is an effective phosphate acceptor for casein kinases and protein kinase C. In this study, based on the partial amino acid sequence of pp25, a cDNA was isolated that encodes a new yolk precursor protein, Xenopus vitellogenin B1, which contained the sequence encoding pp25. Both mRNA and protein of vitellogenin B1 were expressed in all of the female organs examined. In agreement with a previous report, the amount of vitellogenin B1 protein in the liver increased after stimulation with estrogen. These results suggest that pp25 is a cytosolic non-crystallized yolk protein nutrient source, but it might also play a role in rapid development.     

Biosynthesis,purification, and characterization of Aedes aegypti vitellin and vitellogenin

Vitellin, the major egg yolk protein, and vitellogenin, the hemolymph precursor of egg yolk protein, have been purified to apparent homogeneity from the mosquito Aedes aegypti. The purification procedure included chromatography on ion exchange, hydrophobic, and gel filtration columns. Vitellin and vitellogenin have a similar molecular weight (M_r 300,000) on gel filtration columns. However, the molecular weights of vitellin and vitellogenin, as determined from SDS electrophoresis, were 393,000 and 337,000, respectively. Vitellin in sodium dodecyl sulfate released six subunits of molecular weight 116,000, 83,000, 75,000, 54,000, 36,000, and 29,000, whereas vitellogenin released only three subunits (155,000, 120,000, and 62,000). The average molecular weights of vitellin and vitellogenin after gel filtration and SDS electrophoresis were 346,000 and 318,000, respectively. Vitellin has a high content of aspartic acid and glutamic acid, and a low content of histidine, methionine, cysteine, and tryptophan. Vitellin also contains 0.9% mol of glucosamine and no galactosamine. The isoelectric points of vitellin and vitellogenin are at pH 6.4 and 6.3, respectively. Aedes aegypti fat bodies incubated for short intervals in tissue culture medium in the presence of [³H]valine showed incorporation by radio-immunoprecipitation and SDS electrophoresis into three primary vitellogenin polypeptides of molecular weights (± SEM) 156,000 ± 4,000, 114,000 ± 5,000, and 62,000 ± 400 inside the fat body and 162,000 ± 3,000, 118,200 ± 2,000, and 63,000 ± 300 in the medium. These results suggest that the molecular weight of vitellogenin synthesized inside the fat body (M_r 332,000) remains unchanged when secreted into the hemolymph (M_r 343,000). The three vitellogenin subunits are processed by the ovary into six subunits which are then deposited in the yolk granules as vitellin.     

Herpes Simplex Virus DNA Cleavage and Packaging: Association of Multiple Forms of UL15-Encoded Proteins with B Capsids Requires at Least the UL6, UL17, and UL28 Genes

The U_L15 gene of herpes simplex virus (HSV) is one of several genes required for the packaging of viral DNA into intranuclear B capsids to produce C capsids that become enveloped at the inner nuclear membrane. A rabbit antiserum directed against U_L15-encoded protein recognized three proteins with apparent M_rs of 79,000, 80,000, and 83,000 in highly purified B capsids. The 83,000-M_r protein was detected in type C capsids and comigrated with the product of a U_L15 cDNA transcribed and translated in vitro. The 83,000- and 80,000-M_r proteins were readily detected in purified virions. Inasmuch as (i) none of these proteins were detectable in capsids purified from cells infected with HSV-1(ΔU_L15), a virus lacking an intact U_L15 gene, and (ii) corresponding proteins in capsids purified from cells infected with a recombinant virus [HSV-1(R7244), containing a 20-codon tag at the 3′ end of U_L15] were decreased in electrophoretic mobility relative to the wild-type proteins, we conclude that the proteins with apparent M_rs of 83,000, 80,000, and 79,000 are products of U_L15 with identical C termini. The 79,000-, 80,000-, and 83,000-M_r proteins remained associated with B capsids in the presence of 0.5 M guanidine HCl and remained detectable in capsids treated with 2.0 M guanidine HCl and lacking proteins associated with the capsid core. These data, therefore, indicate that U_L15-encoded proteins are integral components of B capsids. Only the 83,000-M_r protein was detected in B capsids purified from cells infected with viruses lacking the U_L6, U_L17, or U_L28 genes, which are required for DNA cleavage and packaging, suggesting that capsid association of the 80,000- and 79,000-M_r proteins requires intact cleavage and packaging machinery. These data, therefore, indicate that capsid association of the 80,000- and 79,000-M_r U_L15-encoded proteins reflects a previously unrecognized step in the DNA cleavage and packaging reaction.     

Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca²⁺, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K_M of 450 micromolar for ATP and a V_max of 25 picomoles of ³²P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K_i 150 micromolar).     

The purification and partial amino acid sequence of a polypeptide from the glutelin fraction of rice grains; homology to pea legumin

The glutelin fraction was extracted from grain meals of rice (Oryzea sativa) with 50 mM Tris-HCl buffer (pH 8.8) containing 6 M urea and 10 mM 2-mercaptoethanol. Polypeptides of glutelin were separated and purified by ion-exchange chromatography under denaturing conditions. Analysis by two-dimensional gel electrophoresis showed that 2 major polypeptides of the rice glutelin fraction, M_r 36 000 and 22 000, were linked in disulphide bonded pairs containing one M_r 36 000 and one M_r 22 000 subunit. A partial amino acid sequence of the purified M_r 22 000 glutelin subunit showed it to be homologous to the β-subunit of pea legumin, a storage protein which also contains disulphide-linked subunit pairs (M_r 38 000 and M_r 22 000). It is therefore proposed that the major component of rice glutelin is a legumin-like protein.     

Identification of a novel calcium binding protein from bovine brain

A novel Ca²⁺ binding protein, named caligulin, was extracted from the heat-treated 100 000 × g supernatant of bovine brain and purified to electrophoretic homogeneity. The apparent M_r of caligulin determined on sodium dodecyl sulfate polyacrylamide gels was 24 000. Analysis by gel filtration chromatography indicated an apparent M_r of 33 000, suggesting a monomeric protein. Amino acid composition data demonstrated the presence of 25% acidic residues, 12% basic residues and 10% leucine. In the presence of 1 mM MgCl₂ and 0.15 M KCl, caligulin bound 1 mol Ca²⁺/mol protein with half-maximal binding at about 0.2 μM Ca²⁺.     

Protein synthesis by Beggiatoa alba B18LD in the presence and absence of sulfide

The interaction of sulfide oxidation and protein synthesis by Beggiatoa alba B18LD was investigated using the incorporation of radiolabeled leucine to estimate protein synthesis. Leucine was assimilated into whole cells in the presence of 6.1 mM acetate at a rate of 0.6 nmol · min^-1 · mg protein^-1, 43% of which was incorporated into the protein fraction. Protein synthesis by B. alba was unaffected by 1 mM sulfide, whether or not the cells had been preincubated with sulfide. B. alba oxidized radioactive sulfide to sulfur within 30 s of addition of the label, whether or not the organism was preinduced by sulfide. Furthermore, chloramphenicol, which inhibited protein synthesis, did not significantly inhibit sulfide oxidation by sulfide-induced or uninduced B. alba. This indicates that sulfide oxidation is a constitutive process. Enrichments of sulfur inclusions from B. alba B18LD that were analyzed by polyacrylamide gel electrophoresis demonstrated two enriched peptides with M_r values of 13,000 and 15,000. The 13,000 and 15,000 M_r peptide bands were more evident in cells grown in a medium containing sulfide than in cells from a medium lacking sulfide. Although sulfide did not increase the rate of overall protein synthesis, the synthesis of a few peptides was increased by the addition of sulfide to the growth medium. Among those, the 15,000 M_r peptide was one of the most distinctive.Non-standard abbreviations SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis - PPO 2,5-diphenyloxazole - POPOP 1,4-bis [5-phenyl-2-oxazolyl]-benzene - BSS basal salts solution - BH Beggiatoa heterotrophic (medium) - BSO Beggiatoa sulfide oxidation (medium) - CM chloramphenicol - TCA trichloroacetic acid - M_r molecular mass     

Immunological characterization of rat cardiac gap junctions: Presence of common antigenic determinants in heart of other vertebrate species and in various organs

Summary Antibodies to the following synthetic peptide, SALGKLLDKVQAY, were purified by affinity chromatography and characterized by ELISA and immunoblotting. These antibodies, shown to be specific to the major protein constitutent of isolated rat heart junctions: connexin 43, cross-reacted with a homologous protein in immunoreplicas of whole heart fractions of trout, frog, chicken, guinea pig, mouse and rat, suggesting a phylogenic conservation of connexin 43 in vertebrates. By immunoblotting of whole organ fractions it was also demonstrated that these antibodies cross-reacted with major proteins ofM _r 32 and 22 kD in rat and mouse liver, ofM _r 41 kD in rat cerebellum, ofM _r 43 kD in uterus, stomach and kidney of rat, ofM _r 46 and 70 kD in rat lens, suggesting that these proteins share common or related epitopes with the synthetic peptide and connexin 43.     

Identification of an Arylphorin-type Storage Protein in the Sweet Potato Hornworm, Agrius convolvuli

A major hemolymph protein (M_r 480,000) in the larvae of the sweet potato hornworm, Agrius convolvuli, was purified and characterized. This protein was isolated with a high yield from the hemolymph of day 3 fifth final instar larvae by ammonium sulfate precipitation and Phenyl-Sepharose and Q-Sepharose column chromatographies. The protein has two subunits, an M_r 84,000 subunit (α) and an M_r 80,000 subunit (β), and the native protein was composed of a heterohexamer (α₃β₃). The two subunits have similar amino acid compositions, with high contents of aromatic amino acids (about 15% phenylalanine plus tyrosine) and low levels of methionine. The N-terminal amino acid sequences of both subunits showed high homologies with insect arylphorin-type storage proteins. The protein concentration in the hemolymph increased steeply from day 3 final instar larva and reached a maximum level of 42 mg/ml in females and 41 mg/ml in males among wandering larvae. The concentration in the hemolymph declined once during the larval–pupal transformation but remained high during the early–mid pupal period and almost disappeared after adult emergence. These quantitative changes were the same for males and females. Based on these characteristics, we identified the hemolymph protein as an arylphorin-type storage protein.     

Characterization of alpha-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris

The common bean, Phaseolus vulgaris, contains a glycoprotein that inhibits the activity of mammalian and insect α-amylases, but not of plant α-amylases. It is therefore classified as an antifeedant or seed defense protein. In P. vulgaris cv Greensleeves, α-amylase inhibitor (αAl) is present in embryonic axes and cotyledons, but not in other organs of the plant. The protein is synthesized during the same time period that phaseolin and phytohemagglutinin are made and also accumulates in the protein storage vacuoles (protein bodies). Purified αAl can be resolved by SDS-PAGE into five bands (M_r 15,000-19,000), four of which have covalently attached glycans. These bands represent glycoforms of two different polypeptides. All the glycoforms have complex glycans that are resistant to removal by endoglycosidase H, indicating transport of the protein through the Golgi apparatus. The two different polypeptides correspond to the N-terminal and C-terminal halves of a lectin-like protein encoded by an already identified gene or a gene closely related to it (LM Hoffman [1984] J Mol Appl Genet 2: 447-453; J Moreno, MJ Chrispeels [1989] Proc Natl Acad Sci USA 86:7885-7889). The primary translation product of αAl is a polypeptide of M_r 28,000. Immunologically cross-reacting glycopolypeptides of M_r 30,000 to 35,000 are present in the endoplasmic reticulum, while the smaller polypeptides (M_r 15,000-19,000) accumulate in protein storage vacuoles (protein bodies). Together these data indicate that αAl is a typical bean lectin-type protein that is synthesized on the rough endoplasmlc reticulum, modified in the Golgi, and transported to the protein storage vacuoles.     

Identification and characterization of an immunologically conserved adenovirus early region 11,000 Mr protein and its association with the nuclear matrix

Antisera from some hamsters bearing adenovirus-induced tumors contain antibodies to an 11,000 M_r adenovirus-induced protein. In adenovirus-infected HeLa cells, this early viral protein was specifically associated with the nuclear matrix fraction. After two-dimensional gel electrophoresis, two forms of the 11,000 M_r protein at pI 5.6 and pI 5.4 were found. Only the pI 5.4 form of this protein was associated with the nuclear matrix fraction. Adenoviruses from groups A, B, C, D and E all produced an early viral protein (10,000 to 12,000 M_r) that reacted with group C antibody to the 11,000 M_r protein. To date, this is the only known early viral protein that is immunologically conserved in all of the human adenovirus groups.The positions of two methionine and seven leucine residues were determined by sequencing the first 35 amino acids from the N terminus of the adenovirus serotype 2 group C 11,000 M_r protein. The positions of these amino acid residues were compared to the adenovirus serotype 2 nucleotide sequence, which uniquely localized the structural gene of the 11,000 M_r protein to region E4, subregion 3 in type 2 adenovirus. A frameshift mutant, which contained a deletion of one base-pair in the structural gene of the 11,000 M_r protein, was isolated and mapped by marker rescue and nucleotide sequence analysis. This mutant failed to produce immunologically detectable 11,000 M_r protein. The mutant had a viable phenotype, producing normal levels of infectious virus in both HeLa cells and WI38 cells in culture. These experiments identify the first adenovirus early region 4 protein detected in virus-infected cells.     

Complex formation between metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes leads to a shift of the photoproduct equilibrium

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M_r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low-M_r immunoreactive peptides (M_r 43000 and M_r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M_r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.     

Purification and characterization of a multifunctional calmodulin-dependent protein kinase from canine myocardial cytosol

A calmodulin-dependent protein kinase from canine myocardial cytosol was purified 1150-fold to apparent homogeneity with a 1.5% yield. The purified enzyme had a M_r of 550,000 with a sedimentation coefficient of 16.6 S, and showed a single protein band with a M_r of 55,000 (55K protein), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 1.6 μmol/mg protein/min, and K_a values of 67 nM and 1.1 μM for calmodulin and Ca²⁺, respectively, using chicken gizzard myosin light chain as substrate. Calmodulin bound to the 55K protein. The purified enzyme had a broad substrate specificity. Endogenous proteins including glycogen synthase, phospholamban, and troponin I from the canine heart were phosphorylated by the enzyme. These results suggest that the purified enzyme works as a multifunctional protein kinase in the Ca²⁺, calmodulin-dependent cellular functions of the canine myocardium, and that the enzyme resembles enzymes detected in the brain, liver, and skeletal muscle.     

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.     

Nadph: Biochanin a oxidoreductase from the fungus Fusarium javanicum

A soluble enzyme which catalyses the NADPH-dependent reduction of the heterocyclic double bond of the isoflavone biochanin A (5,7-dihydroxy-4′-methoxy-isoflavone) yielding the corresponding isoflavanone was isolated from the fungus Fusarium javanicum. The NADPH: biochanin A oxidoreductase was constitutively present in the mycelium with an extractable average activity of 4 pkat/g fresh weight. The enzyme was purified ca 4500 fold to apparent homogeneity. The native enzyme had M_r, of ca 87 000 and consisted of two identical subunits of M_r, 43 000. The enzyme reaction showed a pH-optimum at pH 7.5 and a temperature optimum between 30 and 35°. The apparent K_m values were 43 μM for biochanin A and 190 μM for NADPH with a maximum velocity of 4 mkat/kg protein. The enzyme exhibited a remarkable substrate specificity for biochanin A.     

Mass-spectrometric Identification of Binding Proteins of <Emphasis Type="BoldItalic">M</Emphasis><Subscript>r</Subscript> 25,000 Protein,a Part of Vitellogenin B1, Detected in Particulate Fraction of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Oocytes

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. Our previous report showed the existence of several binding proteins of pp25 in the particulate fraction of Xenopus oocytes. In an attempt to elucidate the function of pp25, two of these binding proteins were purified, analyzed by mass-spectrometry, and identified as ribosomal proteins S13 and S14. Other binding proteins in the particulate fraction mostly corresponded to those derived from purified 40S and 60S ribosomal subunits, as shown by the overlay assay method. However, pp25 did not show any effect on protein synthesis in the rabbit reticulocyte lysate system. A model in which pp25 connects a type of serpin (serine protease inhibitor), the only pp25-binding protein detected in the cytoplasm, to the endoplasmic reticulum through two serine clusters is proposed to explain a possible function of this protein.