Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Partial structural analysis of a highly basic low molecular weight protein from rat testis

Automated Edman degradation of a testis-specific basic protein isolated from the rat gave the following NH₂-terminal sequence of amino acids:

Cleavage of the native protein with cyanogen bromide produced two fragments which were purified by gel filtration. Amino acid analysis of the smaller fragment revealed it to be the NH₂-terminal undecapeptide resulting from cleavage at Met₁₁. The partial sequence analysis of the intact protein coupled with compositional analyses of these cyanogen bromide peptides indicate that the basic testis protein contains 24 basic amino acids and a single methionine in a sequence of 54 amino acids.     

Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen

The Ole e 6 allergen from olive tree pollen has been isolated by combining gel permeation and reverse-phase chromatographies. It is a single and highly acidic (pI 4.2) polypeptide chain protein. Its NH₂-terminal amino acid sequence has been determined by Edman degradation. Total RNA from the olive tree pollen was isolated, and a specific cDNA was amplified by the polymerase chain reaction using a degenerate oligonucleotide primer designed according to the NH₂-terminal sequence of the protein. The nucleotide sequencing of the cDNA rendered an open reading frame encoding a 50 amino acid polypeptide chain, in which two sets of the sequential motif Cys-X₃-Cys-X₃-Cys are present. No sequence similarity has been found between this protein and other previously described polypeptides.     

Structural evidence for a liver-specific glyceraldehyde-3-phosphate dehydrogenase.

The amino acid sequences near the amino termini of glyceraldehyde-3-phosphate dehydrogenase from bovine and porcine liver have been determined. Using classical peptide isolation techniques as well as automated Edman degradation, the NH₂-terminal 30 residues of the bovine liver enzyme were determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Asn-Ser-Gly-Lys-Val-Asp-Ile-Val-Phe-Ile. Twenty-two residues from the NH₂-terminus of the porcine liver enzyme, determined using the automated Edman degradation, were identical to the corresponding sequence from bovine liver enzyme. Both liver enzymes have Asn at position 6. The corresponding residue 6 in the muscle and yeast glyceraldehyde-3-phosphate dehydrogenases is Asp. This evidence suggests that the Asn-6 residue is specific for the liver tissues. The exchange of Asn for Asp may significantly alter the allosteric properties of muscle and liver enzymes especially the activity of the liver enzymes in gluconeogenesis.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Structural studies of Bacillus subtilis glutamine synthetase. Further purification, sulfhydryl groups, and the NH2-terminal amino acid sequence.

A new procedure for the isolation of Bacillus subtilis glutamine synthetase in a high state of purity is described. Automated Edman degradation of the reduced and carboxy-methylated protein revealed a single NH₂-terminal amino acid sequence: H₂N-Ala-Lys- Tyr-Thr-Arg⁵-Glu-Asp-Ile-Gln-Lys¹⁰-Leu-Val-Ser-Glu-Ser¹⁵-CM-Cys-Val-Thr- Tyr-Ile²⁰-Ser-Leu-Gly-Phe-Ser²⁵-Asn-Ser-Leu-Gly- -. The recovery of phenylthiohydantoin(PTH)-amino acids and the single sequence obtained are consistent with the view that the dodecameric enzyme of molecular weight 600,000 is composed of identical subunits. Earlier observations of multiple sequences (80% PTH-Ala and 20% PTH-Gly as NH₂ terminal residues) appear to have been due to impurities removed by the final purification step described herein, which involves column chromatography on hydroxyapatite. Evidence for the existence of one disulfide bond and two free cysteine residues per subunit of dodecameric glutamine synthetase was obtained by alkylation of the denatured enzyme in the presence and absence of reducing agents. This distribution of the four cysteine residues in the enzyme monomer was confirmed by titration of the enzyme denatured in sodium dodecyl sulfate with 5,5′-dithiobis(2-nitrobenzoic acid).     

Partial amino acid sequence of the preprotein form of the alpha subunit of human choriogonadotropin and identification of the site of subsequent proteolytic cleavage

Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH₂-terminal extension of 24 amino acids was found and its partial sequence is:

The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH₂-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit.     

Guinea pig acylphosphatase: The amino acid sequence

We determined the primary structure of guinea pig skeletal muscle acylphosphatase, using the high degree of homology with several vertebrate acylphosphatases to obtain correct alignment of the complete series of tryptic peptides. Their sequences were obtained mainly by Edman degradation; FAB mass spectrometry was used to identify the acyl group blocking the NH₂-terminal residue and to elucidate the structure of the NH₂-terminal tryptic peptide. The comparison among acylphosphatase sequences from skeletal muscle of several vertebrate species is presented and discussed.     

Porcine liver low Mr phosphotyrosine protein phosphatase: The amino acid sequence

Porcine low M_r phosphotyrosine protein phosphatase has been purified and the complete amino acid sequence has been determined. Both enzymic and chemical cleavages are used to obtain protein fragments. FAB mass spectrometry and enzymic subdigestion followed by Edman degradation have been used to determine the structure of the NH₂-terminal acylated tryptic peptide. The enzyme consists of 157 amino acid residues, is acetylated at the NH₂-terminus, and has arginine as COOH-terminal residue. It shows kinetic parameters very similar to other known low Mr PTPases. This PTPase is strongly inhibited by pyridoxal 5′-phosphate (K=21ΜM) like the low Mr PTPases from bovine liver, rat liver (AcP2 isoenzyme), and human erythrocyte (Bslow isoenzyme). The comparison of the 40–73 sequence with the corresponding sequence of other low Mr PTPases from different sources demonstrates that this isoform is highly homologous to the isoforms mentioned above, and shows a lower homology degree with respect to rat AcP1 and human Bfast isoforms. A classification of low M_r PTPase isoforms based on the type-specific sequence and on the sensitivity to pyridoxal 5?-phosphate inhibition has been proposed.     

The amino terminal sequences of acid proteases-human pepsin and gastricsin and the protease of Rhizopus chinensis.

The amino acid sequences near the amino termini of human pepsin (34 residues) and gastricsin (24 residues) and the acid protease from $\text{Rhizopus chinensis}$ (27 residues) have been determined using automated Edman degradation. From these results three additional observations were made. First, two structural variants have been observed for human gastricsin and for the $\text{Rhizopus}$ protease. Both cases are apparently genetic in origin. Second, a stretch of sequence in the $\text{Rhizopus}$ protease, residues 14 to 26, is highly homologous to the known sequence of porcine pepsin at the region of residues 11 to 23. Third, the sequences of the NH₂-terminal region of human pepsin and gastrisin are homologous.     

Initiation of ovalbumin synthesis in chicken oviduct magnum: Transient labeling of the NH2-terminus by methionine

The incorporation of [³⁵S]methionine into ovalbumin, a protein containing NH₂-terminal N-acetylglycine, has been studied in chicken oviduct magnum cells. The purification of [³⁵S]methionine-labeled ovalbumin from total oviduct proteins was accomplished by dialysis of a crude extract at pH 3.6 followed by chromatography on carboxymethyl cellulose. The radioactive ovalbumin eluted from the column in three peaks (P₀, P₁, and P₂-containing 0, 1, and 2 moles of phosphate, respectively, per mole of ovalbumin). The kinetics of labeling of peaks P₀ and P₁ showed that the ratio of radioactivity in NH₂-terminal methionine to total incorporation was greater at 2 min of labeling than at later times. The transient labeling of the NH₂-terminus of ovalbumin with methionine indicates that methionine is the initiator amino acid for the synthesis of this protein, which in its mature form contains NH₂-terminal N-acetylglycine.     

A new method of coupling proteins to insoluble polymers

The A chain isolated from biologically active human thrombin is thirteen residues shorter from the amino terminus than the A chain isolated from bovine thrombin. Automated Edman degradation of the reduced, alkylated A chain permitted the direct identification of 34 out of the 36 residues and established all tryptic overlaps. The NH₂-terminal residue is threonine. This residue is homologous to threonine-14 of the A chain of bovine thrombin. Human thrombin A chain has one half cystine linking it to the larger B chain. Methionine, histidine, valine and tryptophan are not present. There are nine acid residues (Glu, Asp) but none of their respective amides.     

Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Polypeptide Compositions and NH2-terminal Amino Acid Sequences of Proteins in Foxtail and Proso Millets

Seed protein of foxtail and proso millets were fractionated into polypeptides that were analyzed for their major protein, prolamin, and the NH₂-terminal amino acid sequences of the proteins were determined. The proteins extracted from foxtail and proso millets were 64.1% and 80.0% prolamin, respectively. The polypeptides of the prolamins were classified into two groups. The major polypeptides of 27-19 kDa were rich in leucine and alanine, whereas the 17-14 kDa polypeptides were rich in methionine and cysteine. Glutelin-like proteins that were extracted with a reducing reagent were high in proline content, the major polypeptides being 17 and 20 kDa. The NH₂-terminal amino acid sequence showed that the major polypeptides of prolamin were homologous to α-zein and a glutelin-like protein containing the Pro-Pro-Pro sequence, like the repetitive sequence of γ-zein. Although the prolamin consisted of a similar subunit to that of zein, polypeptides with various pI values were found among them.     

Purification and part characterization of a novel antibacterial protein Scygonadin, isolated from the seminal plasma of mud crab, Scylla serrata (Forskål, 1775)

A novel protein was isolated from the seminal plasma of the mud crab, Scylla serrata (Forskål, 1775). It exhibited an antibacterial activity against the Gram-positive bacterium Micrococcus leteus with IC90 of 0.125 mg/ml. The extraction procedure for the protein included techniques of acid extraction, ion-exchange chromatography on SP-Sepharose Fast Flow and reverse-phase liquid chromatography on Source 5R RPC. It showed a molecular mass of 10.8 kDa by SDS-PAGE. A partial 20 residue NH₂-terminal sequence was determined by Edman degradation and MS-fingerprint of the protein was conducted. Similarity search in protein databases (BLAST) revealed that the protein exhibited no significant homology to any other reported antimicrobial peptides. We propose the name Scygonadin (from the gonad of S. serrata) for this antibacterial protein.     

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.     

Human somatotropin: semisynthesis of the hormone by noncovalent interaction of the NH2-terminal fragment with synthetic analogs of the COOH-terminal fragment

Complementation of the natural NH₂-terminal fragment (consisting of 134 amino acid residues) with synthetic analogs of COOH-terminal fragments of 52 or 47 amino acids of reduced-carbamidomethylated human somatotropin gave recombinants with full growth-promoting activity as evidenced by the tibia test. Radioimmunoassay data show that the semisynthetic recombinant hormones possess nearly full immunoreactivity as compared to the native molecule.     

Pipecolic acid pathway: the major lysine metabolic route in the rat brain.

A peptide of 6,700–8,000 daltons has been isolated from an ethanolic KOH digest of bovine ligamentum nuchae elastin by a combination of ion exchange chromatography and gel filtration techniques. The peptide is non-polar and is composed almost entirely of glycine, valine, and proline (or hydroxyproline) in the ratio 2:2:1. Edman degradation of the first 17 residues gave the sequence: NH₂-Gly-Phe-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val-Pro-Gly-Val-Gly-Val. These results suggest that the peptide is made up of repeating pentapeptide units with the sequence: Pro-Gly-Val-Gly-Val.     

Amino-terminal structure of spoOA protein and sequence homology with spoOF and spoOB proteins

Summary The previously reported nucleotide sequence of the spoOA coding region of Bacillus subtilis suggested that the protein is initiated with either of two possible initiation codons, ATG and GTG, 84 base pairs apart. To determine which codon is utilized as an initiator in B. subtilis, we constructed a fusion gene in which the promoter and NH₂-terminal region of the spoOA gene was connected to the chloramphenicol acetyltransferase gene (cat gene). After introduction of the plasmid carrying the spoOA-cat fusion gene into B. subtilis cells, the fusion protein was purified by affinity chromatography. The sequence of NH₂-terminal amino acids of the fusion protein was determined and the result established that the GTG codon is utilized as an initiator in B. subtilis.Comparison of the amino acid sequences revealed a marked homology between the spoOA (NH₂-terminal half) and spoOF proteins. A less striking but significant homology was also found between the spoOA (COOH-terminal half) and spoOB proteins. This suggests the presence of a common functional domain structure for these proteins that are supposed to play key regulatory roles in sporulation.