Phosphopeptides in highly purified calf thymus DNA

Highly purified DNA obtained from calf thymus nuclei was found to cleave after reaction with a chelating agent and subsequent dialysis against 0.01 M phosphate. During the cleavage release of proteineous material into the dialysate was observed. By means of anion exchange resin column chromatography, this material was separated into 9 main fractions. Two of these fractions P1 and P5) were found to contain the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by Chelex 100 treatment. The proportion of phosphoserine was much greater in P5 than in P1. P1 and P5 contained essentially no nucleotide material. All other fractions (P2, P3, P3a, P4, P5a, P6, P7, P8, P6a, P9) were found to contain ribonucleotides and deoxynucleotides. The deoxynucleotide content was about 10% of total nucleotide content. After a deionizing treatment with Chelex, the amounts of nucleotides were extensively reduced to a level corresponding to about 1 nucleotide of 10 amino acids. In separate experiments, commercial DNA (S-DNA) was ultrasonicated, and digested with pancreatic DNAase, exonuclease III, and S1 nuclease. From DEAE Sephacel chromatography of this material the fraction obtained having the highest proportion of protein aceous material was hydrolyzed with Pronase and again chromatographed on DEAE Sephacel. From this fractionation a single fraction containing deoxynucleotides and amino acids was found. The mixture obtained by hydrolysis of this fraction with snake venom diesterase and was again rechromatographed, which revealed two peaks, one corresponding to deoxynucleotide material and a second one to a mixture of 4 amino acids, phosphoserine, asp, glu, and gly. From this it was concluded that the fraction used for diesterase digestion consisted of deoxynucleotide-amino acids, with covalent diester bonds between their deoxynucleotide and amino acid portions. The results indicate that in purified S-DNA phosphopeptides are linked through covalent bonds to the terminal deoxynucleotide residues.     

Characterization of phosphopeptides released during dialysis of EDTA-reacted highly purified DNA prepared from calf thymus nuclei

The treatment of highly purified DNA obtained from calf thymus nuclei (N-DNA) with a chelating agent and subsequent repeated dialyses led to release of phosphopeptides (PPs) into the dialysates. By means of anion exchange column chromatography, the PPs were separated into 9 main fractions. Two of them (P1 and P5) contained the amino acids phosphoserine, asp, thr, ser, glu, gly, ala, val, ile, leu, and arg, as well as metal ion complexes of phosphoserine. The complexes were dissociated by deionization with nitrilotriacetate + Chelex. The proportion of phosphoserine was about twice as great in P5 as in P1. Whereas P1 and P5 contained essentially no nucleotide material, the other fractions contained ribonucleotides and deoxynucleotides. The deoxynucleotide content was less than 10% of that of total nucleotides. After a deionizing treatment, the amounts of nucleotides in these fractions were reduced to a level corresponding to 1 nucleotide per peptide of 5-15 amino acids.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Amino acid analysis of proteins and tightly bound phosphopeptides in calf thymus DNA: indications of a novel regulatory mechanism

Purification of DNA prepared from calf thymus nuclei, which were isolated at temperatures of -10 degrees to -15 degrees C, termed N-DNA, reduced the protein content from 17% to 0.7%. Amino acid analysis of the crude N-DNA revealed that the protein material removed in this process contained lys and phe, whereas in purified N-DNA neither of these amino acids was present. The crude N-DNA had a lower total Pser molar ratio (5.9) than that of the purified N-DNA (22.6). The purification also effected a large decrease of molecular weight, from 170 to 15 million daltons, roughly corresponding to the size of replicons. The amino acid composition of the N-DNA was compared with those of the two PP fractions, a) the PPs released into the dialysates, and b) the core material bound in the DNA, probably covalently, after reaction of N-DNA with EDTA and subsequent dialysis. N-DNA had an intermediate total Pser molar ratio between that of a) (35.3) and b) (11.9). Analysis of a standard preparation of DNA (S-DNA), which involved initial rupture of nuclei in the presence of cytoplasmic components, gave elevated values of asp and glu, a total Pser average value of 8.7 before, and 9.0, after the purification, and again phe and lys in the crude DNA.     

Relationship between bound and free amino acids in the brain of growing rats]

Total pool of glutamate, glutamine and GABA in the hemispheres increases during postnatal life of rats, the increase being due to that in free and bound forms of amino acids. In the cerebellum of 1-day rats, the content of free and bound glu, gln asp, GABA, bound ala and free gly is lower, whereas the level of free glu and ala, bound gly is higher than in mature animals. To the end of the 1st week, total amino acid content decreases, except GABA, which is increased. Aminon acid content begins to increase at the 21th and 28th days of postnatal life.     

Purification and characterization of the myoglobins of Paramecium tetraurelia.

1. The molecular variants of the myoglobins of Paramecium tetraurelia have been purified as five separate components and designated Mb 1 to Mb 5. 2. The five molecular forms are homogeneous on polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IF). 3. The myoglobin species appear to have identical molecular weights and amino acid compositions and differ only in their isoelectric points and relative concentrations in vivo. 4. The myoglobin species have an apparent molecular weight of 15,000 +/- 500 and possess a single heme group per mole which appears to be protoporphyrin IX. 5. The amino acid composition of the five species is: lys12, his3, arg4, asp17, thr9, ser6, glu16, pro4, gly11, ala15, cys0, val8, met2, ile7, leu10, tyr5, phe7. 6. The spectra of several ferrous and ferric derivatives of the mixture Mb 1-Mb 5 are presented.     

THE IN VITRO FORMATION OF ACETYL-ASPARTYL-PEPTIDO-SEROTONIN COMPLEXES BY PIG HYPOTHALAMIC TISSUE EXTRACTS

Peptido-serotonin complexes are formed on incubation of pig hypothalamic tissue extracts with acetyl-aspartate, the amino acids glu. ser, gly and ala. an ATP-regenerating system and serotonin. The purification and partial characterisation of two acetyl-aspartyl-peptido-serotonin complexes is described.     

Behavioural responses of glass eels (Anguilla anguilla) towards amino acids

The behavioural responses of glass eels of Anguilla anguilla towards amino acids were investigated by binary choice experiments testing different concentrations of 14 L-amino acids in fresh water and salt water. Glass eels responded to solutions of individual amino acids down to a concentration of 10⁻⁸M, 10⁻⁹m. Media of different salinities influenced the responses. In freshwater, gln and thr were strongly attractive; asn, ala, met, glu and ile induced significant avoidance; gly elicited multimodal reactions depending on concentrations; his, lys, phe, val, leu and asp hardly influenced behaviour. In salt water, only gly, asn and lys significantly influenced the behaviour of the glass eels.     

Variations circadiennes des acides aminés libres de l'hemolymphe de Penaeus japonicus

The non-essential free amino acids of Peneaus japonicus hemolymph (asp, ser, glu, pro, gly, tyr, arg, ala, cys, tau) are more common than the essential ones: 750 pmol μl^?1 of hemolymph vs 330 pmol. Under a light-dark (L-D); 12-12 photoperiod, tricircadian variations of males or tetracircadian variations of females are more pronounced for non-essential amino acids then for essential ones. In the first case, free amino acid concentrations of hemolymph can be multiplied by a factor of three, and in the second case by a factor of six; but circadian variations of females are greater than those of males. Differences between the maximum and minimum of essential free amino acid concentrations of male and female hemolymph are more frequent than for non-essential amino acids. A differences of 2 h between the minimum of essential and non-essential amino acid concentrations in males only appeared during the afternoon. The more concentrated free amino acids in P. japnicus hemolymph are glycine, proline, histidine, and alanine; the less concentrated are lysine, cysteine and glutamic acid while others, like leucine, isoleucine, phenylalanine and tyrosine can only be estimated at 10.00 and 24.00 h.     

Preliminary characterization of inhibitors of Neisseria gonorrhoeae produced in vitro by Eubacterium limosum

Among anaerobic bacteria normally found in the urogenital flora, Eubacterium limosum was found to inhibit the in vitro growth of Neisseria gonorrhoeae. The antigonococcal activity produced by E. limosum was soluble in methanol and in a chloroform--methanol mixture (30:70). The fraction soluble in chloroform--methanol (30:70) yielded eight absorbance peaks when chromatographed on Bio-Gel P-2 and the inhibitory activity was found in the first two peaks. This activity was not absorbed on DEAE Sephacel and was eluted with distilled water in a peak considered as peak 1, on which preliminary characterization was done. The inhibitory activity of peak 1 was found to be heat and pH resistant and not susceptible to proteases, lipase, or amylases. When peak 1 was chromatographed on cellulose paper using a butanol--acetic acid (4:1) solvent system, eight different spots were detected upon spraying the paper with ninhydrin. No spot was detected with anthrone, bromothymol, nor Sudan black reagents used for the detection of carbohydrates and lipids. Based on sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis and gel chromatography on Sephadex G-25, peak 1 appeared either as a diffuse band and as a single peak, respectively. The molecular weight of the inhibitory complex was estimated to be 2400. All these results suggest that the antigonococcal activity produced by E. limosum is composed of more than one low molecular weight amino compound.     

A monoclonal antibody (OT145) specific for the T cell antigen receptor V beta 6.7a allele detects an epitope related to a proposed superantigen-binding site.

A mAb, OT145, recognizes a TCR allotype encoded by one of two alleles of the V beta 6.7 gene. The peptide products of the two V beta 6.7 alleles differ because of nonconservative amino acid substitutions at positions 38 and 72. V beta 6.7a encodes ser38 and gly72, whereas V beta 6.7b encodes arg38 and glu72. We show here that the binding of mAb OT145 ot the beta-chain of TCR is lost when residue 72 of V beta 6.7a is mutated from gly to glu. The binding of OT145 is not affected by mutation of residue 38 from ser to arg. Thus, OT145 recognizes an epitope related to position 72. Residue 72 of the beta-chain of the TCR is located at a putative superantigen-binding site.     

A unifying concept for the amino acid code

The structure of the genetic code is related to a Gray code, which is a plausible theoretical model for an amino acid code. The proposed model implies that the most important factor in shaping the code was the effects of mistakes in translation, not effects of mutations. Another possible implication is that the preservation of stiffness and flexibility at appropriate places in a protein chain is as important in protein structure as the appropriate placement of hydrophilic (external) and hydrophobic (internal) residues. Other results are a simple conceptualization of the relationships among the 20 amino acids and their relations to their codons. The detailed relationships are summarized in the following ‘similarity alphabet’: ala, thr, gly, pro, ser; asp, asn, glu, gln, lys; his, arg, trp, tyr, phe; leu, met, ile, val, cys; (ATGPS DNEQK HRWYF LMIVC in the one-letter code). This alphabet falls into four groups of amino acids: small, external, large, internal. The approximate relation of the groups to their codons is expressed as: the first base of a codon controls size—a purine means a small amino acid, a pyrimidine means large; the middle base controls cloisterednes—purine means external, pyrimidine means internal. These relationships express the minimum change principle upon which the code appears to be founded.     

兔阑尾中一种新的21kD的钙结合蛋白的纯化与鉴定

纯化与鉴定了Ｂ淋巴细胞中一种新的分子量为２１ｋＤ的钙结合蛋白（ＣａＢＰ２１）。兔阑尾淋巴细胞匀浆经热变性,Ｐｈｅｎｙｌ－Ｓｅｐｈａｒｏｓｅ与ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ柱层析,自每１ｋｇ细胞沉积物中获得ＳＤＳ－ＰＡＧＥ均一的ＣａＢＰ２１５．３ｍｇ。ＨＣｌ水解后的酸性氨基酸（Ａｓｐ＋Ｇｌｕ）含量为２６％。如同大多数钙结合蛋白一样,Ｎ末端封闭阻止其进行Ｅｄｍａｎ降解。ＣａＢＰ２１中疏水性氨基酸（计Ｇｌｙ,不计Ｔｒｐ）约占４６％,碱性氨基酸１０％,酸性氨基酸与极性氨基酸约４４％。ＣａＢＰ２１有较高的Ｓｅｒ、Ｔｙｒ含量。肽谱分析等确证ＣａＢＰ２１为２个相同或相似亚基二聚体。以ＡｒｓｅｎａｚｏⅢ作Ｃａ２＋结合分析表明每分子ＣａＢＰ２１可结合４分子Ｃａ２＋,对Ｃａ２＋的结合常数约为１０－５ｍｏｌ／Ｌ。各种性质表明ＣａＢＰ２１是一种不同于其他已知钙结合蛋白的新钙结合蛋白。     

De novo origin of periodic proteins

Summary Silk fibroin, collagen, freezing point depressing glycoproteins, keratin and protamines have periodic amino acid sequences which are unlikely to have arisen by amino acid replacements and internal duplications of non-periodic DNA. Evidence here discussed suggests that such proteins arise by a single evolutionary event, an iterativede novo synthesis of DNA.Abbreviations used a ala - r arg - n asn - d asp - c cys - q gln - e glu - h his - i ile - l leu - k lys - m met - f phe - p pro - s ser - t thr - w trp - y tyr - v val - z gln or glu (Dayhoff, 1969) Where appropriate, the now accepted one letter notation will be used for amino acidsWhen attention is drawn to some residue it is capitalized.^* signifies the amino or carboxy terminus of a sequence.When attention is drawn to some residue it is capitalized.^* signifies the amino or carboxy terminus of a sequence.     

Solubilization and partial purification of squalene synthase from daffodil microsomal membranes

A squalene synthase was solubilized from daffodil (Narcissus pseudonarcissus L.) microsomes with CHAPS, a zwitterionic non-denaturating detergent. By successive chromatography on DEAE Sephacel and APP Sepharose a fraction enriched in this enzyme (21-fold) was prepared.     

MDCK microcarrier cultures: Seeding density effects and amino acid utilization

Summary The growth of Madin Darby canine kidney cells on microcarriers was studied at different cell seeding densities. Maximum growth was attained when a cell-to-bead ratio of 7∶1 was used. Under these conditions an initial concentration of above 3×10⁵ cells/ml resulted in a yield of over 2×10⁶ cells/ml in 2 d. The amino acid utilization of cells from Dulbecco's modified Eagle medium was studied under the above conditions. Eight amino acids (arg, cys, gln, ile, leu, met, ser, and val) showed rapid and near complete depletion from the medium. Five amino acids (his, lys, phe, thr, and tyr) showed limited depletion. Two amino acids (ala and gly) were released into the medium by the cells.     

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.     

The Synthesis of Neuroactive Amino Acids from Radioactive Glucose and Glutamine in the Rat Retina: Effects of Light Stimulation

The effect of light stimulation in vitro on the labelling of neuroactive amino acids derived from [14C]glucose or [14C]glutamine in the rat retina has been studied. [14C]Glutamine, at 700 microM, provided about 50% of the tissue pools of glutamate, aspartate, and GABA; and the labelling of these decreased on light stimulation, both in the photoreceptor cells (glu and asp) and in the inner retina (glu, asp, and GABA). In contrast, there were no significant changes in the entry of label derived from [14C]glucose, although similar trends were apparent in the data obtained for the photoreceptor cell layer. The pools may, therefore, be separate. Other results support the contention that glucose is the principal energy source for the retina, its entry into non-amino acid derivates being decreased on light stimulation.     

Interactions of gold with cytosolic selenium-containing proteins in rat kidney and liver

Rats injected with aurothioglucose (ATG) for 5 days were subsequently injected with [75Se]selenious acid and killed after 3 days. Kidney and liver cytosols were chromatographed on Sephadex G-150. 75Se in kidney was associated with high molecular weight (HMW), 85,000 Mr, 26,000 Mr, and 10,000 Mr proteins and with a nonprotein fraction. The elution profile of liver cytosol was similar to that of kidney, but without a 26,000 Mr protein. ATG injection increased the association of 75Se with all fractions of kidney cytosol except the 85,000 Mr fractions, which contained Se-glutathione peroxidase (SeGSHPx) activity; 75Se in liver was increased only in HMW fractions. Unfractionated kidney cytosolic SeGSHPx activity was decreased 14% by ATG injection, but liver enzyme activity was not changed. However, Sephadex G-150 chromatography showed that total and specific activities, respectively, were decreased 28 and 23% in kidney and 25 and 16% in liver. Au coeluted with HMW and 10,000 Mr 73Se-containing kidney proteins; the latter contained 50% of the Au eluted from the column. DEAE Sephacel chromatography of the 10,000 Mr kidney protein showed that both Au and 75Se were tightly associated with metallothionein-like proteins. This study demonstrates the interaction of Au with rat liver and kidney 75Se-containing proteins.