Primary structure of the "fast" component of avenin (Avena sativa L

A procedure for the isolation and sequence analysis of the "fast" avenin component (N9) from the oat (Avena sativa L., cv. Narymsky 943) is described. Component N9 was prepared by an ion-exchange high-performance liquid chromatography on a strong cation exchange column type Mono S (Pharmacia, Sweden) in 4 M urea, pH 3.5, with a linear gradient of NaCl. A polypeptide chain of avenin N9 was reconstructed by the CNBr and tryptic peptides on a model 470A protein gas-phase sequencer (Applied Biosystems, USA). A good yield of tryptic peptides were obtained by an enzymatic hydrolysis of avenin N9 preliminary immobilized on Thiopropyl-Sepharose 6B (Pharmacia, Sweden) at cysteine residues. Avenin N9 consists of 182 amino acid residues end exhibits the features common for all the known prolamins.     

Tryptic peptides from the beta polypeptide chain of AII component of chicken hemoglobin.

The aminoethylated beta polypeptide chain in AII component from the hemoglobin of adult chicken was digested with trypsin [EC 3.4.21.4] and the resulting peptides were separated and purified by ion exchange chromatography, paper chromatography, and gel filtration. Eighteen tryptic peptides, which were nonoverlapping, accounted for all of the amino acid residues in the beta polypeptide chain. The amino acid sequences of the tryptic peptides were established by a combination of enzymatic digestion and subtractive Edman degradation.     

Amino acid sequence of human plasma apolipoprotein C-III from normolipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C-III (apoC-III) isolated from normal subjects is described. ApoC-III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC-III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC-III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high-performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC-III isolated from normolipidemic subjects is identical to the apoC-III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC-III derived from a patient with type V hyperlipoproteinemia.     

Amino- and carboxy-terminal sequence of mouse J chain and analysis of tryptic peptides

Mouse J chain was isolated from an IgM-producing hybridoma by gel filtration and ion-exchange chromatography. The sequence of the amino-terminal 25 residues was determined. At these positions, the results agree with the amino acid sequence deduced from the cDNA sequence determined previously by Koshland and co-workers and indicate that a leader sequence terminating in glycine is removed to form the mature J chain. Tryptic peptides of J chain were isolated by high pressure liquid chromatography and their amino acid compositions were compared with those expected from the cDNA sequence. The amino acid sequence of the carboxy-terminal peptide and a mixture of two other peptides was determined. The results were consistent with the cDNA sequence except that we found valine, not leucine, at position 67, and arginine, not glycine, at position 117. The presence of aspartic acid at the carboxy-terminus, as predicted from the cDNA, indicates that processing does not occur at this end of the polypeptide chain. Upon amino acid analysis, glucosamine was found in tryptic peptides 47-57 and 47-58. J chain was also cleaved at aspartylproline bonds with formic acid and the unfractionated digest was subjected to automated Edman degradation. The mixed sequence was consistent with the sequence deduced from the cDNA at positions 1 to 13, 28 to 40, 52 to 64, and 73 to 85. In conjunction with the results obtained previously by analysis of cDNA, these data show that mouse J chain is a polypeptide containing 137 amino acid residues, 93 of which are identical to residues in human J chain.     

Construction and characterization of cDNA encoding the alpha 2 chain of chicken type IX collagen

We have isolated and characterized a cDNA encoding the carboxy-terminal half of one of the polypeptide subunits of a novel disulfide-bonded collagen found in hyaline cartilage. This collagen has been given the type assignment type IX, and it has several unusual characteristics. First, the polypeptide subunits are shorter than alpha-chains of the fibrillar collagens types I, II, and III. Second, type IX molecules are heterotrimers of three genetically distinct polypeptide subunits. Third, type IX molecules contain three triple-helical collagenous domains interspersed with noncollagenous domains. When chicken cartilage collagens are extracted with pepsin, type IX collagen is cleaved and gives rise to the triple-helical fragments HMW and LMW. The identification of the cDNA reported here is based on a comparison of the amino acid composition of tryptic peptides derived from LMW with the composition of tryptic peptides predicted from the nucleotide sequence of the cDNA. We also show that the amino-terminal sequence of one of the subunits of LMW is identical with the sequence predicted from the nucleotide sequence of the cDNA. Finally, we demonstrate that the amino-terminal amino acid sequence of a tryptic peptide isolated from one of the subunits of HMW is identical with a sequence predicted from the cDNA. We have given the polypeptide chain encoded by the cDNA reported here the name alpha 2(IX), and we show that it is homologous to the alpha 1(IX) chain previously characterized by us.     

Primary structure of macromomycin, an antitumor antibiotic protein

The antitumor protein macromomycin is a single chain polypeptide of 112 amino acid residues cross-linked by two intramolecular disulfide bonds. The protein was reduced and S-alkylated with 2-mercaptoethanol in 8 M urea followed by treatment with iodoacetic acid. Tryptic digestion of tetra-S-carboxymethyl macromomycin gave four tryptic peptides which were fractionated by gel permeation on Sephadex G-50. The amino acid sequence of the tryptic peptides and the overlap sequences were determined by a combination of automated Edman degradation analysis, gas chromatographic mass spectrometry, and fast atom bombardment mass spectrometry. A comparison of the structures of macromomycin, actinoxanthin, and neocarzinostatin suggests that they belong to a family of related proteins.     

Synthesis of Penicillins and Cephalosporins by Penicillin Acylase Entrapped in Fibres

Tryptic peptides from two cyanogen bromide (CNBr) fragments CB II and CB III of the Ala chain of ricin D were sequenced by manual Edman degradation. Chymotryptic or peptic peptides from the two fragments were isolated by Dowex 1 x 2 column chromatography to obtain overlaps for the tryptic peptides, and the complete amino acid sequences of fragments CB II and III were established. The amino acid residues in fragments CB II and CB III accounted for 75 and 45 residues, respectively, of 260 residues in the Ala chain.

These sequences together with the sequence of fragment CBI described in the preceding paper established the complete sequence of the 260 amino acid residues in the Ala chain. Some structural characteristics of the protein are also discussed.     

The complete amino acid sequence of rubredoxin from the green phototrophic bacterium Chlorobium thiosulphatophilum strain PM

A complete amino acid sequence for the rubredoxin from the photosynthetic bacterium Chlorobium thiosulphatophilum is proposed. The sequence, a single polypeptide chain of 53 amino acids, was deduced from the sequences of peptides obtained by chymotryptic, tryptic, thermolytic or mild acid digestion. The rubredoxin shows a high degree of sequence homology with rubredoxins from non-photosynthetic bacteria, and the evolutionary implications of this are considered.     

Molecular cloning, nucleotide sequence, and tissue distribution of malonyl-CoA decarboxylase

Malonyl-CoA decarboxylase was purified from goose uropygial gland, reduced, carboxymethylated, and digested with trypsin. Several peptides were purified by high performance liquid chromatography and their amino acid sequences determined. Oligonucleotide probes were prepared based on their amino acid sequences. Size-selected RNA from the goose uropygial gland was used to construct cDNA libraries in lambda gt11 and pUC9 vectors. Immunological screening of the lambda gt11 cDNA library yielded one clone, lambda DC1, which contained a 2.2-kilobase pair insert; hybridization with the synthetic oligonucleotide probes confirmed its identity as malonyl decarboxylase. Screening of the pUC9 cDNA library with the insert of lambda DC1 as a probe detected one clone, pDC2, with an insert of 2.9 kilobase pairs. The nucleotide sequences of the two cDNAs revealed an open reading frame encoding a polypeptide of 462 amino acids. The deduced amino acid sequence was confirmed as malonyl-CoA decarboxylase by matching it to the amino acid sequences of three tryptic peptides derived from mature enzyme. Northern blot analysis of mRNA from goose brain, kidney, liver, lung, and gland revealed malonyl-decarboxylase mRNA of 3000 nucleotides. Since clone pDC2 contains a 2928-nucleotide insert, it represents nearly the full length of mRNA. Brain, kidney, lung, and liver contained less than 1% of the malonyl-CoA decarboxylase mRNA in the gland. Southern blot analysis of genomic DNA showed a single band in both liver and gland, suggesting that malonyl-CoA decarboxylase is a single copy gene.     

Primary structure of water buffalo beta-casein tryptic and CNBr peptides

The partial amino acid sequence (70%) of water buffalo beta-casein has been determined by aligning the sequences of tryptic and CNBr peptides along the polypeptide chain of bovine beta-casein. Only five amino acid substitutions are observed. Moreover, as in all the beta-caseins so far investigated, a morphine-like peptide is present.     

Structure of the cDNA encoding transcobalamin I, a neutrophil granule protein

Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells.     

Isolation and Amino Acid Sequences of Small Tryptic Peptides from the Oxidized Ala Chain of Ricin D

Twelve tryptic peptides as well as free arginine were isolated from the performic acid-oxidized Ala chain of ricin D by gel filtration on Sephadex G–25 and Dowex 1×2 column chromatography followed by paper chromatography. Total number of the amino acid residues in these peptides accounted for 90 out of 263 residues in the Ala chain of ricin D.

The amino acid sequences of nine peptides were determined by manual Edman degradation.     

Sites of methyl esterification on the aspartate receptor involved in bacterial chemotaxis

The methyl esterification of the aspartate receptor involved in chemotaxis has been studied in order to clarify the role of receptor modification. Receptors were methyl esterified in an in vitro system using S-adenosyl-L-[methyl-3H]methionine as a methyl donor. Methyl esterified receptors were digested with trypsin and radioactive tryptic peptides were purified using high performance liquid chromatography. Comparing the amino acid composition of the modified peptides with the DNA sequence of the receptor gene, two regions of the polypeptide chain which contain methyl esterified residues were identified. The regions are homologous and contain a strongly conserved 13 amino acid sequence. One region, containing up to three modified residues, is near the middle of the protein; the other, containing one modified residue, is near the carboxyl terminus.     

Partial amino acid sequence of rat topoisomerase I: comparison with the predicted sequences for the human and yeast enzymes

The partial amino acid sequence of rat topoisomerase I was determined by gas-phase microsequencing. Seven tryptic peptides closely matched the sequences deduced from human topoisomerase I cDNA (94.5% homology). Similarity to sequences deduced from baker's yeast and fission yeast genomic DNA were restricted to conserved domains which may represent important sites of interaction with DNA or with other proteins.     

The nucleotide sequence of a complete chicken delta-crystallin cDNA

The nucleotide sequence of a full length cDNA of delta-crystallin mRNA from chicken lens has been determined using a delta-crystallin cDNA clone (pB delta 11), which represents the mRNA sequence of 1530 nucleotides from the poly(A) junction but does not contain the 5'-terminal sequence of 44 nucleotides of the mRNA. The 5'-terminal sequence of the mRNA, absent in the cDNA clone, has been determined with a stretch of cDNA sequence by the primer extension procedure. The amino acid sequence deduced from the nucleotide sequence is consistent with the amino acid sequences of several tryptic peptides, the total amino acid composition, and the mol. wt. of delta-crystallin estimated by SDS-polyacrylamide gel electrophoresis. The computer-assisted analysis predicts high alpha-helical content throughout the polypeptide. Sequence analyses have revealed that gene 1 encodes the mRNA from which the cDNA clone was derived.     

Isolation of partial complementary DNA encoding human thromboxane synthase

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.     

Identification of the serine residue phosphorylated by protein kinase C in vertebrate nonmuscle myosin heavy chains

Two-dimensional mapping of the tryptic phosphopeptides generated following in vitro protein kinase C phosphorylation of the myosin heavy chain isolated from human platelets and chicken intestinal epithelial cells shows a single radioactive peptide. These peptides were found to comigrate, suggesting that they were identical, and amino acid sequence analysis of the human platelet tryptic peptide yielded the sequence -Glu-Val-Ser-Ser(PO4)-Leu-Lys-. Inspection of the amino acid sequence for the chicken intestinal epithelial cell myosin heavy chain (196 kDa) derived from cDNA cloning showed that this peptide was identical with a tryptic peptide present near the carboxyl terminal of the predicted alpha-helix of the myosin rod. Although other vertebrate nonmuscle myosin heavy chains retain neighboring amino acid sequences as well as the serine residue phosphorylated by protein kinase C, this residue is notably absent in all vertebrate smooth muscle myosin heavy chains (both 204 and 200 kDa) sequenced to date.     

The soluble c-type cytochromes from the bacterium Aquaspirillum itersonii. The complete amino acid sequence of the cytochrome c-550

A complete amino acid sequence is proposed for the cytochrome c-550 isolated from the gram-negative chemo-organotrophic bacterium Aquaspirillum itersonii. The sequence, a single polypeptide chain of 111 residues, was deduced from the sequences of peptides obtained by tryptic, thermolytic or chymotryptic digestion. The cytochrome shows a high degree of sequence homology with the cytochrome c2 from the photosynthetic bacterium Rhodospirillum rubrum, and the evolutionary implications of this are considered.