N-terminal Amino Acid Sequence Data Research Articles

Nucleotide and deduced amino acid sequences of two distinct cDNAs for rat phosphoribosylpyrophosphate synthetase.

The rat Yoshida sarcoma (YS) cDNA library was screened using oligonucleotide probes designed from peptide sequences of rat phosphoribosylpyrophosphate synthetase, and two distinct clones were obtained. Nucleotide sequencing revealed that both clones encode 317 amino acids containing the peptide sequences. The deduced amino acid sequences of the two differ only by 13 residues (96%) identity), whereas the nucleotide sequences are relatively divergent (81% identity in the coding regions). These results, together with N-terminal amino acid sequencing data, suggest the existence of two different subunits of this enzyme, designated as PRS I and II (their genes as PRPS1 and PRPS2). Transcripts of 2.3 and 3.7 kilobases were detected in YS cells and rat liver by Northern blot analysis, using PRS I and II cDNAs respectively, as probes. In the liver, the expression of both genes increased after partial hepatectomy.

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition.

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.

Complementary DNA sequences of ovarian follicular fluid inhibin show precursor structure and homology with transforming growth factor-β

Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5′ cap and the 3′-terminal poly(A) tract. After the 5′-terminal cap there are 59 nucleotides of 5′ nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 unstranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3′ untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of posttranslational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3′ end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.

Further characterization and N-terminal sequence of cobra venom phospholipase A 2

N-terminal amino acid sequence data on cobra venom phospholipase A2 (Naja naja naja) demonstrate the absence of histidine at positions 10 and 20, in contrast to most other Naja enzymes. The presence of three tryptophans was demonstrated by p-toluenesulfonic acid hydrolysis, and the location of two of these at positions 18 and 19 parallels other Naja phospholipase A2 sequences. Based on the amino acid composition of the enzyme, the theoretical E278 0.1% should be 2.2, which is consistent with dry weight determinations, refractometry and amino acid analysis. Unusually high reactivity of the enzyme toward the protein assay of Lowry et al. gives an erroneously low E278 0.1% value of 1.45 when compared to bovine serum serum albumin as a protein standard. Using an E278 0.1% of 2.2, this enzyme is inactivated by the active site reagent p-bromophenacyl bromide with a stoichiometry of 1 mol reagent per mol enzyme. Chromatography on Affi-Gel Blue provides further purification of this phospholipase A2.

Chemical Characterization of Murine Ia Antigens

Abstract Several products of the I region of the mouse H-2 complex have been characterized by micropeptide analysis, microsequence analysis, and two-dimensional gel (IEF/SDS-PAGE) analysis. Ia antigens have been isolated using the biosynthetic radiolabeling technique together with highly specific alloantisera and indirect immunoprecipitation. Chromatography of tryptic peptides prepared from the α and β polypeptides of Ia antigens encoded by the I-A and I-EC subregions will be compared from both spleen and epidermal cell preparations. These data, in conjunction with partial N-terminal amino acid sequence data on Ia antigens from the I-Ab, I-Ak, I-Ad, I-As, I-ECk and I-ECd subregions and haplotypes will be discussed in terms of homology relationships and haplotype-associated differences in the structures of these Ia polypeptides. Two-dimensional gel patterns of isolated α and β polypeptides will also be presented. The implications of these data will be discussed as they relate to the genetic organization of the I-region.

Studies on the small subunit of Fraction I protein from pisum sativum L. and vicia faba L.

We have measured the molecular weight of the small subunit of Fraction I protein from pea and broad bean by sodium dodecyl sulphate polyacrylamide gel electrophoresis, Sephadex gel-filtration and amino acid composition data. The results suggest a molecular weight of 12 000–14 500, although measurements by gel-filtration in alkali suggest a molecular weight of approximately 22 000. N-terminal amino acid sequence data and C-terminal determinations show that the protein consists of a single type of polypeptide chain, although the anomalously high molecular weight obtained on gel-filtration in alkali does not preclude the existence of the polypeptides as dimers under certain conditions.