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At least 595 records · Page 33Linked to original sources
PubMed1978

Characterization of DNA binding protein from rat liver chromatin which decreases during growth.

A nuclear nonhistone protein which decreases in chromatin during growth (Yeoman, L. C., et al. (1975) Cancer Res. 35, 1249) has been isolated in high purity from the chromatin of normal rat liver nuclei by gel electrophoresis and column chromatography. This protein, designated BA (Yeoman, L.C., et al. (1973) Biochem Biophys. Res. Commun. 53, 1067), has a molecular weight of 31 000, an acidic to basic amino acid composition ratio of 0.9, and contains one tryptophan residue per molecule. Hydrazinolysis indicated protein BA has a lysine carboxyl terminus; however, the amino terminal is blocked as no reaction occurred with dansyl chloride. Maps of tryptic peptides of protein BA contained 46 spots. Protein BA binding to various DNAs was examined by the nitrocellulose filter assay. Binding was slightly enhanced by 2mM Mn2+ion; Mg2+, however, decreased binding. Binding was optimal at neutral pH and an ionic strength of 0.2 M [NaCl]. Equilibrium competition binding studies indicated a binding preference of protein BA for dA-dT rich DNA.

Aging↗
PubMed1999

Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1967

An extracellular lipopolysaccharide-phospholipid-protein complex produced by Escherichia coli grown under lysine-limiting conditions.

Lysine limitation during growth of the lysine-requiring mutant of Escherichia coli 12408 resulted in the excretion of a complex containing 60% of lipopolysaccharide, 26% of extractable phospholipid and 11% of protein. The complex was obtained from the culture filtrate in yields of about 0.5g./l. by precipitation with chloroform or gel filtration; some purification steps are described. The greater part of the phospholipid consisted of phosphatidylethanolamine, which contained four main fatty acids; two monoenoic acids and a cyclopropane acid were esterified mainly in the beta-position, and a saturated acid was located mainly in the gamma-position. The protein component was relatively insoluble and contained an excess of acidic over basic amino acids and little cystine. The lipopolysaccharide resembled in composition the intracellular lipopolysaccharides from rough strains of E. coli. Both protein and lipopolysaccharide constituents of the complex were serologically active; the complex was less toxic than the purified lipopolysaccharide. In the electron microscope the complex showed a mixture of particles of various sizes and shapes. Rods and hollow spheroids (diameter 12-200mmu) were most common and resembled the particles previously found surrounding cells actively excreting the complex. The chloroform-precipitated material showed a tubular lamellar structure. Soluble lipopolysaccharide prepared from the complex also consisted of hollow spheres and rods.

Bacterial Proteins↗
PubMed1975

Studies on a wide-spectrum intestinal dipeptide uptake system in the monkey and in the human.

1. The intestinal transport of glycine and leucine residues of glycyl-L-leucine was studied in the monkey and in the human in vitro. Uptake of both [14C]glycyl-L-leuine and glycyl-L-[14C]leucine show similar Kt values, but there is a marked difference in the Vmax. values. Preliminary studies suggest that this anomalous difference in the Vmax. values may be due to the greater efflux rate of glycine from the tissue. 2. Arrhenius plots of both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake in the monkey intestine show a discontinuity at about 20 degrees C. The activation energies above and below the discontinuity are similar for both [14C]glycyl-L-leucine uptake and glycyl-L-[14C]leucine uptake. These similarities in uptake characteristics suggest that the dipeptide glycyl-L-leucine is transported as one unit. 3. In the monkey intestine, glycyl-L-leucine uptake is inhibited by a wide variety of dipeptides, including those containing acidic and basic amino acids. The inhibition was shown to be competitive by using four representative dipeptides namely: L-alanyl-L-alanine, L-alanyl-L-leucine, L-glutamyl-L-glutamic acid and L-lysyl-L-lysine. The results strongly suggest that in the monkey intestine there may be a dipeptide-uptake system with an extremely broad specificity. These results were also confirmed in the human in a limited way.

Alanine↗
PubMed1979

Studies on the high-mobility-group non-histone proteins from hen oviduct.

Nuclear high-mobility-group (HMG) proteins were isolated from hen oviduct. These were proteins HMG-1, -2, -3, -14 and -17, which are equivalent to the classification of calf thymus HMG proteins. Hen oviduct proteins HMG-1 and -2 were individually isolated by HCIO4.extraction and CM-Sephadex chromatographic separation. Their mol.wts. were determined as 28 000 and 27 000, respectively. The proteins have a high content of acidic and basic amino acids. The association of proteins HMG-1 and -2 with the genome of hen oviduct nuclei was probed by a limited digestion with nucleases. Hen oviduct nuclei were incubated with deoxyribonuclease I or micrococcal nuclease until 10% of the DNA was digested. The nuclear suspension was centrifuged and the contents of proteins HMG-1 and -2 in the supernatant and sediment fractions were analysed by polyacrylamide-gel electrophoresis. HMG proteins were found to be preferentially released by micrococcal-nuclease digestion rather than by deoxyribonuclease I.

Amino Acids↗
PubMed1979

Purification of a phosphoprotein from chromatin of rat liver.

A simple and effective method to purify a phosphoprotein (B2) (Mr 68,000, pI 6.2-8) from phenol-soluble non-histone chromatin proteins of rat liver is described. The purification involved only two steps, CM-cellulose chromatography and preparative SDS/polyacrylamide (10%)-gel electrophoresis. The purified phosphoprotein B2 was shown to be homogeneous by SDS/polyacrylamide-gel electrophoresis. The yield was 2% of total non-histone chromatin proteins. The acidic to basic amino acid ratio of phosphoprotein B2 was less than 1, with high contents of glutamic acid, aspartic acid, arginine, lysine, glycine and alanine. The phosphate content of this protein is 0.3%.

Amino Acids↗
PubMed1998

An alternatively spliced gene encoding a Y-box protein showing maternal expression and tissue-specific zygotic expression in the ascidian embryo.

An ascidian Y-box protein gene was cloned, designated as CiYB, which consists of a highly conserved cold shock domain and an auxiliary tail domain with alternating modules of acidic and basic amino acids. CiYB is a single copy gene in the ascidian genome. During oogenesis and early development, CiYB produces three different transcripts (CiYB1, CiYB2 and CiYB3) by alternate splicing. CiYB1 and CiYB2 were expressed during oogenesis, suggesting that they are recruited into maternal ribonucleoprotein particles. According to gel mobility shift assay, the CiYB1 protein has the ability to bind RNA. The sequence preference of RNA binding is similar to that of the Xenopus Y-box protein (FRGY2), which is a major component of the maternal messenger ribonucleoprotein particles (mRNP) in the oocyte. These results suggest that the ascidian Y-box protein may have an important role for masking and translational regulation of maternal mRNA. Furthermore, CiYB1, CiYB2 and CiYB3 were expressed zygotically in a tissue restricted manner. CiYB1 was expressed specifically in muscle precursor blastomeres and tail muscle cells suggesting its important role in muscle differentiation.

Age Factors↗
PubMed1997

Characterization of heparin and heparan sulfate domains binding to the long splice variant of platelet-derived growth factor A chain.

Platelet-derived growth factors (PDGFs) are homo- or heterodimers of two related polypeptides, known as A and B chains. The A chain exists as two splice variants due to the alternative usage of exons 6 (PDGF-AL, longer) and 7 (PDGF-AS, shorter). Exon 6 encodes an 18-amino acid sequence rich in basic amino acid residues, which has been implicated as a cell retention signal. Several lines of evidence indicate that the retention is due to binding of PDGF-AL to glycosaminoglycans, especially to heparan sulfate. We have analyzed the saccharide domains of smooth muscle cell-derived heparan sulfate involved in this interaction. Furthermore, we have employed selectively modified heparin oligosaccharides to elucidate the dependence of the binding on different sulfate groups and on fragment length. The shortest PDGF-AL binding domain consists of 6-8 monosaccharide units. Studies using selectively desulfated heparins and heparin fragments suggest that N-, 2-O-, and 6-O-sulfate groups all contribute to the interaction. Structural comparison of heparan sulfate oligosaccharides separated by affinity chromatography on immobilized PDGF-AL showed that the bound pool was enriched in -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- disaccharide units. Furthermore, analogous separation of a partially O-desulfated heparin decamer preparation, using a highly selective nitrocellulose filter-trapping system, yielded a PDGF-AL-bound fraction in which more than half of the disaccharide units had the structure -IdceA(2-OSO3)-GlcNSO3(6-OSO3)-. Our results suggest that the interaction between PDGF-AL and heparin/heparan sulfate is mediated via N-sulfated saccharide domains containing both 2-O- and 6-O-sulfate groups.

Animals↗
PubMed1970

Some properties of hyalin: the calcium-insoluble protein of the hyaline layer of the sea urchin egg.

The principal protein component of the hyaline layer of sea urchin eggs is the calcium-insoluble protein first described by Kane and Hersh. The protein hyalin is abnormally high in acidic amino acids, almost devoid of basic amino acids, and characteristically rich in valine and proline. Essentially all of the cysteine present is found in the disulfide form; no evidence points to intermolecular disulfide linkages. Hyalin from several species has a minimal subunit weight of about 100,000, though evidence exists for a particle three times this weight in urea or guanidine hydrochloride from one species. Optical rotatory dispersion measurements indicate no alpha-helix content, though the dispersion has unique characteristic features. Addition of small quantities of calcium causes hyalin to gel to a birefringent fibrous form. The fibrous, birefringent form of hyalin is rendered isotropic upon addition of EDTA, but the birefringence is restored with re-addition of divalent cation.

Animals↗
PubMed1977

Reversibly contractile nuclear matrix. Its isolation, structure, and composition.

From Tetrahymena macronuclei we have isolated a reversibly contractile nucleo-skeleton, i.e., an "expanded" nuclear matrix which reversibly contracts when the total concentration of the bivalent cations, Ca and Mg (3:2), is decreased to 5 mM or increased to 125 mM. During contraction the average diameter of the expanded matrix becomes reduced by about 24%; this corresponds to a volume contraction of about 55%. The reversible contraction of the nuclear matrix does not depend on ATP and cannot be inhibited by salygran. The expanded matrix is obtained by removing carefully from the macronuclei 89.7% of the phospholipid, 99.6% of the DNA, 98.5% of the RNA, and 74.8% of the protein by treatment with Triton X-100 and digestion with DNase and RNase followed by an extraction with 2 M NaCl. Electron microscopy reveals, within the expanded matrix, residual equivalents to the structures characteristic for macronuclei: (a) a residual nuclear envelope with nuclear pore complexes; (b) residual nucleoli at the periphery; (c) a fibrillar internal network. The expanded matrix is essentially composed of proteins (96.2%) and traces of DNA (0.8%), RNA (0.5%), phospholipid (1.6%), and carbohydrates (0.9%). The last, which have been determined by gas chromatography, contain glucose, mannose, and an unidentified sugar in the ratio 1:5.4:5.7. The ratio of acidic to basic amino acids of the expanded matrix is 1.55. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals a predominant protein with a mol wt of 18,000 which is apparently involved in the reversible contractile process. The mechanism of this reversible contraction of the expanded matrix remains to be elucidated, but it differs both from actin-myosin contraction systems and from the contractile spasmoneme system in vorticellids.

Calcium↗
PubMed2002

Characterization of signal that directs C-tail-anchored proteins to mammalian mitochondrial outer membrane.

We analyzed the signal that directs the outer membrane protein with the C-terminal transmembrane segment (TMS) to mammalian mitochondria by using yeast Tom5 as a model and green fluorescent protein as a reporter. Deletions or mutations were systematically introduced into the TMS or the flanking regions and their intracellular localization in COS-7 cells was examined using confocal microscopy and cell fractionation. 1) Three basic amino acid residues within the C-terminal five-residue segment (C-segment) contained the information required for mitochondrial-targeting. Reduction of the net positive charge in this segment decreased mitochondrial specificity, and the mutants were distributed throughout the intracellular membranes. 2) Elongation of the TMS interfered with the function of the C-segment and the mutants were delivered to the intracellular membranes. 3) Separation of the TMS and C-segment by linker insertion severely impaired mitochondrial targeting function, leading to mislocalization to the cytoplasm. 4) Mutations or small deletions in the region of the TMS flanking the C-segment also impaired the mitochondrial targeting. Therefore, the moderate length of the TMS, the positive charges in the C-segment, and the distance between or context of the TMS and C-segment are critical for the targeting signal. The structural characteristics of the signal thus defined were also confirmed with mammalian C-tail-anchored protein OMP25.

Amino Acid Sequence↗
PubMed2000

Convenient and efficient in vitro folding of disulfide-containing globular protein from crude bacterial inclusion bodies.

We investigated how the folding yield of disulfide-containing globular proteins having positive net charges from crude bacterial inclusion bodies was affected by additives in the folding buffer. In screening folding conditions for human ribonucleases and its derivative, we found that addition of salt (about 0.4 M) to a folding buffer increased the folding yield. This suggested that electrostatic interaction between polyanionic impurities such as nucleic acids and cationic unfolded protein led to the formation of aggregates under the low-salt conditions. Since inclusion bodies were found to contain nucleic acids regardless of the electrostatic nature of the expressed protein, the electrostatic interaction between phosphate moieties of nucleic acids and basic amino acid residues of a denatured protein may be large enough to cause aggregation, and therefore the addition of salt in a folding buffer may generally be useful for promotion of protein folding from crude inclusion bodies. We further systematically investigated additives such as glycerol, guanidium chloride, and urea that are known to act as chemical chaperons, and found that these additives, together with salt, synergistically improved folding yield. This study, suggesting that the addition of salt into the folding buffer is one of the crucial points to be considered, may pave the way for a systematic investigation of the folding conditions of disulfide-containing foreign proteins from crude bacterial inclusion bodies.

Bacterial Proteins↗
PubMed1975

Purification and some properties of cathepsin A of small molecular size from pig kidney.

Cathepsin A [EC 3.4.2.-] of small molecular size (cathepsin A, S) has been purified about 800-fold from pig kidney by procedures including chromatographies on DEAE-Sephadex, SP-Sephadex, and Sephadex G-150. 1. The homogeneity of the purified enzyme was proved by ultracentrifugation and polyacrylamide gel electrophoresis. The molecular weight (100,000) and isoelectric point (pI=5.0) were estimated. 2. The enzyme was remarkably stabilized by sucrose and KCl, and was most stable at pH 5-5.5 in the presence of both stabilizers. The enzyme had not only peptidase activity but also esterase and amidase activity; it was optimally active at pH 5.2 for peptide hydrolysis and at pH 8 for the hydrolysis of esters and amides. 3. Diisopropyl fluorophosphate and iodoacetamide completely inhibited these three activities. 4. The enzyme hydrolyzed various benzoyl- and benzyloxycarbonyl-dipeptides with neutral, acidic, and basic amino acids, and proline in the C-terminal position. The carboxypeptidase nature of the enzyme was proved by its action on an oligopeptide. 5. Several enzymatic properties of cathepsin A, S were almost the same as thoas of cathepsin A of large molecular size (cathepsin A, L) and the crude homogenate.

Animals↗
PubMed1990

Structural requirements of fibrinogen A alpha-(148-160) for the enhancement of the rate of plasminogen activation by tPA.

Fibrin, not fibrinogen, enhances the rate of tPA catalysed plasminogen activation. In earlier studies we have shown that a site involved in this rate enhancement is located in a tridecapeptide, i.e. fibrinogen A alpha-(148-160). This sequence comprises a special charge distribution in which a stretch with alternating neutral and acidic amino acids is embraced by basic amino acids. In this study we found that the disruption of charge distribution as caused by replacing valine 152 by other (charged and/or polar) amino acids leads to loss of rate-enhancing capacity. Also lysine at position A alpha-157 was replaced by lysine derivatives and other amino acids. We found that the side chain of the amino acid at position A alpha-157 must contain no (as in glycine) or one carbon atom without substitution (alanine). When the side chain contains two or more carbon atoms, there should also be a polar group in the side chain. We also synthesized a series of hexapeptides covering the sequence of A alpha-(148-160), and found that only A alpha-(154-159) is stimulatory, notwithstanding the fact that the peptides A alpha-(152-157), A alpha-(153-158) and A alpha-(155-160) also contain lysine A alpha-157. We conclude that the shortest peptide with stimulation activity is A alpha-(154-159); that the charge distribution in A alpha-(148-160) is important; that it is not lysine A alpha-157 per se that is crucial, but rather the properties and orientation of the side chain of A alpha-157.

Amino Acid Sequence↗
PubMed1994

A seven-gene operon essential for formate-dependent nitrite reduction to ammonia by enteric bacteria.

The DNA sequence of the regulatory region and the structural gene, nrfA, for cytochrome c552 of Escherichia coli K-12 have been reported. We have now established that nrfA is the first gene in a seven-gene operon, designated the nrf operon, at least five of which are essential for formate-dependent nitrite reduction to ammonia. This operon terminates just upstream of the previously sequenced gltP gene encoding a sodium-independent, glutamate and aspartate transporter. Expression of lac fused to nrfA, nrfE or nrfG is regulated by oxygen repression, FNR-dependent anaerobic induction, nitrite induction and nitrate repression during anaerobic growth, exactly as previously reported for the nrfA promoter. In contrast, expression of the gltP-lac fusion was FNR-independent. The open reading frame immediately downstream of nrfA encodes NrfB, a hydrophilic, penta-haem cytochrome c with an M(r) of 20,714. The structure of the N-terminal region is typical of a signal peptide for a periplasmic protein: cleavage at the putative signal peptide cleavage site, Ala-26, would result in a periplasmic cytochrome with a molecular mass of 18 kDa. The NrfC polypeptide, M(r) 24,567, contains 16 cysteine residues arranged in four clusters typical of the CooF super-family of non-haem iron-sulphur proteins. The NrfD sequence predicts a 318-residue hydrophobic protein with a distribution of acidic and basic amino acids which suggests that NrfD is an integral transmembrane protein with loops in both the periplasm and the cytoplasm. Proteins most similar to NrfD include the PsrC subunit of polysulphide reductase from Wolinella, but, as seven of the 10 most similar proteins are NADH-ubiquinone oxidoreductases, we propose that NrfD participates in the transfer of electrons from the quinone pool into the terminal components of the Nrf pathway. NrfE, M(r) 60,851, is predicted to be another hydrophobic, integral membrane protein homologous to the CdI1 protein of Rhodobacter capsulatus, which has been implicated in the assembly of periplasmic c-type cytochromes. The sequence of the 127 residue NrfF polypeptide, M(r) 14,522, is strikingly similar to the CcI2 protein of R. capsulatus, especially in the putative haem-binding motif, RCPQCQNQN.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1975

Preparation and some properties of strongly acidic proteins from calf-thymus nucleohistone.

The isolation of acidic proteins from calf-thymus nucleohistone (starting from purified nuclei) is reported. The method involved dissociation in 1 M KCl solution. Denaturating agents were not used at all. After electrophoresis on polyacrylamide gel, fractions containing a small number of components were obtained. The fractions display high ratios of acidic to basic amino acids, the ratios ranging from 4.0 to 1.5. In all fractions, the major components were of molecular weights in the ranges 12000-15000 and 24000-28000 as determined by gel-disc electrophoresis in dodecylsulphate and by equilibrium ultracentrifugation. Minor components of high molecular weights were also present. Amino-acid analyses are also reported. The tryptophan content was determined by a fluorometric method. Circular dichroism spectra depict a very low content of alpha-helicity that did not increase at higher ionic strength. A marked RNA-polymerase activity was found in one fraction.

Amino Acids↗
PubMed1977

The purification, characterization and partial sequence determination of a trout testis non-histone protein, HMG-T.

A specific non-histone chromatin-associated protein, having a high content of both acidic and basic amino acids has been isolated from the chromatin of rainbow trout (Salmo gairdnerii) testis cells. The protein has been prepared by the extraction of chromatin with 0.35 M sodium chloride and purified by chromatography on carboxymethyl-cellulose with a gradient of lithium chloride at pH 9.0. The amino acid sequence of the first 29 residues of the amino-terminal region has been determined using an automatic protein sequencer. The primary structure of this protein differs from that of any of the histones yet sequenced and, therefore, cannot be a degradation produce of any of them. Moreover, the N-terminal amino acid sequence shows considerable similarity to the HMG-1 and HMG-2 chromosomal proteins described by Goodwin et al. [Eur. J. Biochem. 38, 14-19 (1973)] whose-N-terminal sequences were also determined in this laboratory.

Amino Acid Sequence↗