Isolation,purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun.60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ?-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A₁ and A₂ peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.     

Chromatographic Purification and Determination of the Carboxy-Terminal Sequences of Photosystem II Reaction Center Proteins, Dl and D2

The Dl and D2 subunits of the reaction center of photosystemII are intrinsic proteins, each with a molecular mass of about30 kDa. They exhibit considerable homology to each other interms of primary structure. A procedure was developed for theseparation and purification of these two proteins on a largescale from the photosystem II reaction center complex of spinachby high-performance liquid chromatography on a gel-permeationcolumn in the presence of sodium dodecyl sulfate. The purificationwas achieved by a combination of two gel-permeation chromatographicsteps performed with different concentrations of phosphate buffer,200 mM and 50 mM, as the mobile phase. The purified Dl and D2proteins were subjected to determination of their carboxy-terminalsequences by digestion of the proteins with carboxypeptidaseY. Comparison of the sequences deduced from the enzymatic analysiswith the sequences deduced from the psb A and psb D genes ofspinach indicates that the Dl protein ends at Ala-344 and theD2 protein at Leu-353. Thus, it appears that the Dl proteinloses 9 amino acid residues from the carboxy-terminus, fromAla-345 to Gly-353, during maturation, while the D2 proteindoes not lose any amino acid residues from the carboxy-terminus. (Received July 27, 1989; Accepted December 28, 1989)     

Improved polyacrylamide gel electrophoresis with different amino acids as the trailing constituent

Using a polyacrylamide disc gel electrophoretic system similar to that described by J. T. Clarke (1964, Ann. N. Y. Acad. Sci.121, 428–436), we have achieved an improved separation of hemoglobins from Rana catesbeiana tadpoles by substituting one of several amino acids in the place of glycine in the electrode chamber buffer. The relative migrations (R_f) and degree of separation of these similar hemoglobins are proportional to the pK′ of the α-amino group of the amino acid used in the buffer. Specifically, for these proteins, log (R_f × 100) was found to be directly proportional to the pK′₂ of the amino acid divided by the volume conductivity (specific conductance) of the electrode chamber buffer. For example, improved separation of these hemoglobins in short electrophoretic times can be achieved, at low cost, by using dl-alanine instead of glycine in the buffer. Improved separation of other proteins which migrate at basic pH might be achieved by a similar approach.     

Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins

The ras genes, which were first identified by their presence in RNA tumor viruses and which belong to a highly conserved gene family in vertebrates, have two close homologs in yeast, detectable by Southern blotting. We have cloned both genes (RAS1 and RAS2) from plasmid libraries and determined the complete nucleotide sequence of their coding regions. They encode proteins with nearly 90% homology to the first 80 positions of the mammalian ras proteins, and nearly 50% homology to the next 80 amino acids. Yeast RAS1 and RAS2 proteins are more homologous to each other, with about 90% homology for the first 180 positions. After this, at nearly the same position that the mammalian ras proteins begin to diverge from each other, the two yeast ras proteins diverge radically. The yeast ras proteins, like the proteins encoded by the mammalian genes, terminate with the sequence cysAAX, where A is an aliphatic amino acid. Thus the yeast ras proteins have the same overall structure and interrelationship as the family of mammalian ras proteins. The domains of divergence may correspond to functional domains of the ras proteins. Monoclonal antibody directed against mammalian ras proteins immunoprecipitates protein in yeast cells containing high copy numbers of the yeast RAS2 gene.     

A program to find regions of similarity between homologous protein sequences using dotmatrix analysis

MATRIX is a program designed primarily to enable the user to visualize all regions of similarity between two proteins at a glance. The program helps the user to see where they are similar—at what relative positions in the amino acid sequences of the two proteins in question does the similarity exist; how they are similar—what functional characteristics the two similar sequences have in common; and to what extent they are similar—is the similarity significant, if so how significant relative to other similar sequences in the protein. This is achieved by constructing a diagram in which quantitative parameters of amino acids are used to compare every amino acid residue of the first protein with every amino acid residue of the second.Another function of the program is, given two sets of atomic coordinates—either of different proteins or for the same protein (for self-comparison)—to demonstrate which residues of the two proteins, when the two proteins are superimposed upon each other, appear in the same space (or are close to each other).     

Reactions of the amino groups in ribonuclease A: II. Identification of reactive amino groups in ribonuclease

Ribonuclease A has been trinitrophenylated to varying degrees by reaction with trinitrobenzenesulfonic acid. The reactive amino groups were identified by use of the peptides obtained from the oxidized TNP-RNase by tryptic and chymotryptic hydrolysis. From a quantitative study of the TNP-peptides it was possible to associate each amino group with values of pK_ma. It was shown that the lys-41 amino group had a pK_a of 9.03 in TEA buffer. The pK_a values of all of the other amino groups were dependent on the nature of the buffer (triethanolamine and phosphate) and on the pH.     

天花粉同工凝集素－1双链的拆分和鉴定

天花粉同工凝集素－１经巯基乙醇还原,碘代乙酰胺保护,其链间二硫键被打开,但仍非共价结合在一起。我们利用尿素变性的Ｑ－Ｓｅｐｈａｒｏｓｅ离子交换层忻分离了此凝集素的两条链。氨基酸组成测定与其他３种肽链作一比较,它们都含有较多的酸性和羟基氨基酸。蛋白质印迹显示ＴＫＬ的抗血清不仅能与ＴＫＬ－１的两条链分别反应,也能与天花粉毒蛋白及蓖麻毒蛋白的Ａ链起作用。溴化氰裂解的ＳＤＳ－ＰＡＧＥＲ肽谱表明天花粉凝集素的两条链与天花粉毒蛋白含有类似的裂解片段,在分子量１６ｋｄ左右有相同的电泳条带。ＴＫＬ－１两亚基的Ｎ末端序列已经测定,同源性比较发现其３３ｋｄ亚基的Ｎ末端序列与天花粉毒蛋白、蓖麻毒蛋白的一些肽段类似。迄今已有的证据表明ＴＫＬ与ＴＣＳ等是一些非常相关的蛋白质。     

Biochemistry of Fern Spore Germination: Globulin Storage Proteins in Matteuccia struthiopteris L

Two globulin storage proteins have been identified in spores of the ostrich fern, Matteuccia struthiopteris (L.) Todaro. The two proteins comprise a significant amount of the total spore protein, are predominantly salt-soluble, and can be extracted by other solvents to a limited extent. The large 11.3 Svedberg unit (S) globulin is composed of five polypeptides with molecular weights of 21,000, 22,000, 24,000, 28,000 and 30,000. Each polypeptide has several isoelectric point (pI) variants between pH 5 and 7. The small 2.2S storage protein has a pI > 10.5 and is composed of at least two major polypeptides of 6,000 and 14,000 M_r. The amino acid composition of both storage proteins reveals that the 11.3S protein is particularly rich in aspartic and glutamic acid, while the 2.2S protein has few acidic amino acids. During imbibition and germination the globulin fraction declines rapidly, with a corresponding degradation of individual polypeptides of each protein. Polyclonal antibodies against each of the two proteins were produced and used for immunolocalization to determine the site of storage protein deposition within the quiescent spore. The proteins were sequestered in protein bodies of 2 to 10 micrometers, that are morphologically similar to those found in the seeds of flowering plants. The results suggest that spore globulins are biochemically similar to seed globulins, especially those found in some cruciferous seeds.     

Isolation and partial characterization of human parotid basic proteins

Methods are presented for the isolation of basic proteins (Pb proteins) from human parotid saliva collected from humans possessing different alleles at the Pb locus. The proteins were found to be extremely basic, with an isoelectric point above 9.5. They contain approximately 45% of the basic amino acids histidine, lysine, and arginine, and are devoid of cysteine, proline, threonine, valine, methionine, and tryptophan. They are free of carbohydrate. A comparison of the amino acid sequence data of Pb protein to all available amino acid sequences revealed that no sequence similarities exist between the Pb proteins and any other proteins reported, although proteins of similar amino acid compositions have been reported by others. A model is presented with accounts for the several forms of allelic proteins based on observed amino acid sequence differences.     

Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.     

The isolation of carcinogen-binding protein from livers of rats given 4-dimethylaminoazobenzene

1. Three azo-dye-binding proteins were identified in the soluble cell supernatant fraction from livers of rats that had received 4-dimethylaminoazobenzene by intraperitoneal injection. 2. One is basic and was highly purified. It has an isoelectric point of pH8.4 in barbital-sodium chloride buffer, I0.1, an S(20,w) value of 3.5s and a molecular weight determined by Sephadex chromatography of 45000. 3. It does not have N-terminal amino acids with free alpha-amino groups. 4. Digestion with Pronase gives rise to a single azo-dye-bound peptide, which on hydrolysis is shown to contain glycine, alanine, serine, threonine, glutamic acid and aspartic acid. The amino acid that binds the azo-dye was not identified. 5. On starch-gel electrophoresis the basic protein separates into a double band, indicating microheterogeneity. 6. The other two proteins were partially purified and occur in a fraction together. They have isoelectric points near neutrality and a molecular weight as determined by Sephadex chromatography of 13800. 7. The absorption spectra in formic acid of both the basic and the low-molecular-weight proteins are similar. The azonium ion has an absorption maximum at 518mmu and another adsorbed chromogen is present with an absorption maximum at 395mmu.     

Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana

Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Höfte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. α-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; γ-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins.     

State of oncomarker protein B23/nucleophosmin in HeLa cells

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.     

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds

Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC₅₀ ≤ 1 μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3′-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.     

Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans

The histones of Caenorhabditis elegans (Nematoda) have been identified by correlating criteria of electrophoresis and amino acid composition with the five main histones from calf thymus. C. elegans H1(1) consists of at least two subtypes with approximate molecular weights of 20,000 and 18,500 daltons as resolved by SDS polyacrylamide gel electrophoresis. They are some 10% smaller than the two subtypes of calf histone H1. The differences are also corrobated by the amino acid composition of the nematode and calf H1 complements. Nematode H2A resembles calf H2A in chromatographic and electrophoretic properties and in the amino acid composition, although it lacks histidine, which seems to be replaced by lysine. Like calf H2A, it is dimorphic as shown by Triton/acid/urea polyacrylamide gel electrophoresis. The H2B complement from C. elegans consists of two proteins with a molecular weight of approximately 12,500. They can be separated by ion-exchange chromatography, but they are very analogous to each other and to calf H2B in amino acid composition. Each form is also resolved into two more subtypes by Triton/acid/urea polyacrylamide gel electrophoresis. Nematode H3 resembles calf thymus H3 in its electrophoretic behaviour; three subfractions can be distinguished in Triton/acid/urea gels. C. elegans H4 is very similar to calf H4 in its chromatographic, electrophoretic and solubility properties, but differs significantly in composition. The meaning of this difference is discussed with regard to the generally observed stringent conservation of H4 sequences between distantly related species.     

Genetic variability and improvement of seed proteins in wheat

Summary Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins.     

Amino acid uptake by isolated renal brush border membrane vesicles in various buffers

The uptake of amino acids by isolated rat renal brush border membrane vesicles in a modified Krebs-Ringer bicarbonate buffer and a phosphate buffer was compared to the uptake in the standard membrane vesicle buffer, Tris-Hepes-mannitol. The uptake in the modified Krebs-Ringer bicarbonate buffer was similar to that in the Tris-Hepes-mannitol buffer. Removal of the ionic constituents other than NaCl and NaHCO3 in the modified Krebs-Ringer bicarbonate buffer (KCl, CaCl2, KH2PO4 and MgSO4) did not affect the amino acid uptake by the isolated membrane vesicles. The timed uptake of proline under sodium gradient conditions in a phosphate buffer had a markedly dampened overshoot. Kinetic analysis of the initial rate of proline uptake in a phosphate buffer compared to a Tris-Herpes-mannitol buffer showed two entry systems for proline in each buffer with similar Km values, but the maximal rate of transport (V) for each system in the phosphate buffer was much lower than that in the Tris-Hepes-mannitol buffer. From these data, phosphate buffer does not appear to be a suitable medium for the study of amino acid uptake by isolated brush border membrane vesicles.     

Molecular cloning of two novel stearoyl-acyl desaturase genes from winterness wheat

Using RT-PCR and rapid amplification of cDNA ends, two new full-length cDNAs of SAD (TaSAD1 and TaSAD2) were obtained from a hardiest winter wheat cultivar (Mironovskaya808). Sequence comparison analysis showed that the deduced amino acid sequences of TaSAD1 and TaSAD2 had high similarity to those of other reported SAD proteins. They were also different each other by some substitutions, insertions and/or deletions involving single amino acid residues or motifs. Based on evolution analysis, it was clear that all SAD genes from Poaceae were closer than those from other genus such as Arabidopsis, Glycine, Triadica, Brassica, Sesamum and Bassia. All SAD genes clustered into two major groups in Poaceae. Meanwhile, TaSAD1 and TaSAD2 were clustered into different groups. The tertiary structure prediction indicated that both TaSAD1 and TaSAD2 proteins were a compact globular protein and their model structures almost were the same.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.