Evidence for the amino acid sequence of porcine pancreatic elastase

The preparation and purification of tryptic peptides from aminoethylated Dip-elastase and [(14)C]carboxymethylated Dip-elastase, and of peptic peptides from native elastase is described. A summary of the results of chemical studies used to elucidate the amino acid sequence of these peptides is presented. Full details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50016 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 1-20. These results, together with those from previously published papers, are used to establish the complete amino acid sequence of elastase, which is a single polypeptide chain of 240 residues, molecular weight 25900, containing four disulphide bridges.     

Partial amino acid sequence of glycophorin from porcine erythrocyte membranes

Glycophorin from porcine erythrocyte membranes was digested with trypsin and chymotrypsin. Some of the peptides were isolated by conventional techniques. The amino acid sequence was determined for two isolated peptides: a chymotryptic glycopeptide of 19 residues and a tryptic peptide of 36 residues which represented the carboxy terminal of the glycophorin.     

Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.

An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.     

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A

The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A subunit I has been determined by automated Edman degradation of the cyanogen bromide fractions derived from the precursor protein. The activation peptide contains 94 amino acid residues in a unique sequence which precedes directly the amino-terminal alanine residue of carboxypeptidase A alpha. A notable feature of the activation peptide is the presence of acidic amino acid residues immediately preceding the site of activation. The amino acid sequence of the activation peptide of bovine pro-carboxypeptidase A shows extensive similarity to those of the corresponding porcine and rat enzymes.     

Nucleotide and primary amino acid sequence of porcine lactoferrin.

A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.     

Partial amino acid sequence of a human seminal plasma peptide with inhibin-like activity

An extract of human seminal plasma was found to have inhibin-like activity. The active factor was purified to homogeneity by ion exchange chromatography, molecular sieving and high performance liquid chromatography. The purified material has a mass of approximately 5 kDa and is very basic. Amino acid analysis showed the presence of approximately 35 residues while the sequencing data allowed the determination of the N-terminal 31 amino acids. There is a possibility of an additional 2–4 residues at the C-terminus, which could not be determined.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

Partial amino acid sequence of mouse beta2-microglobulin.

A highly purified preparation of mouse β₂-microglobulin has been obtained from sodium thiocyanate extracts of liver cell membranes of $\text{A}\text{J}$ strain mice and the amino acid sequence has been partially determined. The first 40 residues have been assigned except for position 34. In the sequence, the mouse protein differs only at 9 positions from human β₂-microglobulin at 8 positions from the dog homologue and at 11 positions from the rabbit homologue. The sequence has also a homology to the constant regions of mouse γG_2a, most closely to the C_H3 region. These data support the conclusion that the mouse protein is indeed the mouse homologue of human β₂-microglobulin.     

Reevaluation of the amino acid sequence of porcine follitropin

We have reevaluated the sequence of porcine follicle-stimulating hormone (pFSH) with more recent protein-sequencing methodology. This has led to revision of the earlier proposed sequence. As with almost all reported gonadotropin -subunits, NH₂-terminal heterogeneity was found in the porcine FSH -subunit (FSH), starting with residue Phe (1), Asp (3), Gly (4), or Thr (7). In the -subunit, there were found to be at least two molecular species, starting with residue Asn (1) (minor 20%) or Cys (3) (major 80%) as NH₂-terminal and ending at residue Glu (108) as COOH-terminal. The net effect of the present revisions is to increase the homology of pFSH with other reported follitropin sequences. Apparent differences in the half-cystine placements in a previous proposal for pFSH compared with other species of FSH are no longer tenable. The half-cystine placements thus remain a constant structural feature throughout the gonadotropin hormones (choriogonadotropin, follitropin, and lutropin).     

The amino acid sequence of thymopoietin II.

The amino acid sequence of bovine thymopoietin II is presented. This T cell differentiating hormone of the thymus is a single 49 amino acid polypeptide chain of 5562 daltons. There is microheterogeneity at the C terminus with approximately two thirds of the molecules lacking the C terminal arginine found on the remaining molecules. Determination of the primary structure of thymopoietin II was facilitated by a long automated sequenator run on thymopoietin II coupled to 2-isothiocyanonaphthalene-4,8-disulfonic acid (NITC), tryptic cleavage of maleated thymopoietin II to yield the overlapping C terminal peptide, and efficient manual sequencing of this peptide using benzene extractions to minimize extractive losses of peptide.     

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.     

Structural gene and complete amino acid sequence of Pseudomonas aeruginosa IFO 3455 elastase.

The DNA encoding the elastase of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited high levels of both elastase activity and elastase antigens. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consisted of 301 amino acids with a relative molecular mass of 32,926 daltons. The amino acid composition predicted from the DNA sequence was quite similar to the chemically determined composition of purified elastase reported previously. We also observed nucleotide sequence encoding a signal peptide and "pro" sequence consisting of 197 amino acids upstream from the mature elastase protein gene. The amino acid sequence analysis revealed that both the N-terminal sequence of the purified elastase and the N-terminal side sequences of the C-terminal tryptic peptide as well as the internal lysyl peptide fragment were completely identical to the deduced amino acid sequences. The pattern of identity of amino acid sequences was quite evident in the regions that include structurally and functionally important residues of Bacillus subtilis thermolysin.     

Partial amino acid sequence of two forms of human post-gamma-globulin.

We have purified two different electrophoretic forms of Post-γ-globulin defined by electrophoresis as “native slow” form and “fast” form respectively. Amino acid sequences of the first fifty-two residues of the “native slow” form and twenty-nine of the “fast” form were determined. The sequence shows that the “fast” form lacks the first nine amino acids of the “native slow” form.This observation is consistent with the existence of a “native slow” form that is degraded into other more acidic forms of Post-γ-globulin by loss of basic amino acids.