Purification and properties of a proteinaceous metallo-proteinase inhibitor from Streptomyces nigrescens TK-23.

A novel metallo-proteinase inhibitor which is capable of inhibiting the activities of metallo-proteinases such as the thermolysin, was isolated from the culture filtrates of Streptomyces nigrescens TK-23. The inhibitor was purified batch-wise from the culture filtrate by Amberlite IRC-50 and column chromatographies on CM-Sephadex C-50 and Sephadex G-50. The purified inhibitor showed a single band on 15% polyacrylamide gel electrophoresis at pH 4.3, and at pH 7.5 on SDS-gels. The inhibitor retained 80% of its original activity after treatment of 100 degrees C for 5 min between pH and 7. The molecular weight was estimated to be 12 000 by gel filtration and SDS-polyacrylamide gel electrophoresis, and calcuated as 11 950 from its amino acid composition. The isoelectric point was pH 10.3. The inhibitor showed a high content of hydrophobic amino acids, did not contain tryptophan, and had two disulfide bridges. It also showed specific inhibitory activity for metallo-proteinases but not for serine-, thio- and carboxyl-proteinases.     

Amino acid sequence of protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25

The amino acid sequence of a protein alpha-amylase inhibitor from Streptomyces griseosporeus YM-25 (Haim II), which consists of 77 amino acid residues, including two disulfide bridges, was determined by conventional methods. One of the disulfide bridges was found to be located between Cys(6) and Cys(22), and the other between Cys(40) and Cys(67) from the results of structure analyses of the two cystine-containing peptides obtained from the thermolysin digest of the native inhibitor.     

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.     

Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified by ammonium sulfate precipitation, gel filtration, and anion and cation exchange chromatography and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Their molecular weights were determined by electrospray mass spectrometry as 6916, 6807, 7676, 7944, 7848, and 7844 D, respectively, and the sequences of the first 20 N-terminal amino acid residues of these six inhibitors were found to be identical. The complete amino acid sequence of PSTI IVa was determined. This protein comprises a total of 72 residues and has 14 cysteines, all involved in disulfide bridges. Comparison of the sequence of PSTI IVa with those of other leguminous Bowman-Birk type inhibitors revealed that PSTI could be classified as a group III inhibitor, closely related toVicia faba andVicia angustifolia inhibitors.     

Amino acid sequence of protein B23 phosphorylation site

A major phosphopeptide labeled in vivo, was identified in nucleolar protein B23 (Mr/pI = 37,000/5.1) after tryptic digestion. This peptide was purified by high performance liquid chromatography using reverse-phase (C8 and C18) columns. The phosphopeptide contains 20 amino acids including 1 phosphoserine, 7 glutamic acids, and 4 aspartic acids. The amino acid sequence is: His-Leu-Val-Ala-Val-Glu-Glu-Asp-Ala-Glu-Ser(P)-Glu-Asp-Glu-Asp- Glu-Glu-Asp-Val-Lys. This amino acid sequence is similar to that of nucleolar phosphoprotein C23 (8 consecutive amino acids were identical), and to the regulatory subunit (RII) of cAMP-dependent protein kinase (7 consecutive amino acids were identical, which is phosphorylated by casein kinase II (Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982) J. Biol. Chem 257, 10440-10445). The regions near these phosphorylation sites are enriched with glutamic and aspartic acids, suggesting that this acidic amino acid cluster may be essential for kinase recognition.     

Amino acid sequence of rat epidermal thiol proteinase inhibitor

The complete amino acid sequence of rat epidermal thiol proteinase inhibitor was determined. The unique 103-residue sequence was derived by analysis of two peptides generated by limited proteolysis of the native inhibitor with Staphylococcus aureus V8 protease and of three cyanogen bromide fragments. The protein has a high degree of sequence homology to either rat liver or human leucocyte inhibitor but is not identical and may represent a new type of low molecular weight thiol proteinase inhibitors.     

Amino acid sequence of the protease inhibitor BWI-4a from buckwheat seeds

The complete amino acid sequence of protease inhibitor BWI-4a from buckwheat (Fagopyrum esculentum Moench) seeds, consisting of 67 amino acid residues with a single disulfide bond, has been established by Edman degradation in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Its N terminus is blocked by a pyroglutamic acid residue. Mass spectrometric analysis revealed that inhibitor BWI-4a is present in buckwheat seeds in two isoforms with a single amino acid substitution of Ala40 for Gly40. The reactive site of the inhibitor contains an Arg43-Asp44 bond. Analysis of the amino acid sequence suggests that the buckwheat seed protease inhibitor is a member of the potato proteinase inhibitor I family.     

Amino acid sequence of a probable amylase/protease inhibitor from rice seeds

The primary structure of a 9-kDa basic protein from rice seeds was determined by gas-phase sequencing of the intact protein and peptides derived from it by digestion with trypsin, chymotrypsin, and endopeptidase Lys-K. The protein consists of a single polypeptide chain of 91 amino acid residues with a calculated molecular mass of 8909 Da. It is rich in alanine, serine, glycine, and cysteine. The eight cysteines form four disulfide bonds. There is no methionine, histidine, phenylalanine, or tryptophan. The sequence is highly homologous with an alpha-amylase inhibitor, I-2, from seeds of Indian finger millet [F. A. P. Campos and M. Richardson (1984) FEBS Lett. 167, 221-225] and a 10-kDa barley seed protein, also called a probable amylase/protease inhibitor [B. Svensson et al. (1986) Carlsberg Res. Commun. 51, 493-500; J. Mundy and J. C. Rogers (1986) Planta 169, 51-63]. In analogy with the barley protein, the purified protein is tentatively called a rice probable amylase/protease inhibitor (PAPI). The rice PAPI does not show inhibitory activities against proteases and amylases tested. The amino acid sequence is as follows: Ile-Thr-Cys-Gly-Gln-Val-Asn-Ser-Ala-Val(10)-Gly-Pro-Cys-Leu-Thr-Tyr- Ala-Arg-Gly-Gly(20)-Ala-Gly-Pro-Ser-Ala-Ala-Cys-Cys-Ser-Gly(30)-Val-Arg- Ser-Leu-Lys-Ala-Ala-Ala-Ser-Thr(40)-Thr-Ala-Asp-Arg-Arg-Thr-Ala-Cys- Asn-Cys(50)-Leu-Lys-Asn-Ala-Ala-Arg-Gly-Ile-Lys-Gly(60)-Leu-Asn-Ala-Gly- Asn-Ala-Ala-Ser-Ile-Pro(70)-Ser-Lys-Cys-Gly-Val-Ser-Val-Pro-Tyr-Thr(80)- Ile-Ser-Ala-Ser-Ile-Asp-Cys-Ser-Arg-Val-Ser(91).     

Amino acid sequence of a specific antigenic peptide of protein B23

A specific antigenic peptide was obtained from protein B23 (Mr/pI = 37,000/5.1) after 30 min of digestion with staphylococcal V8 protease (10 micrograms/ml/mg protein B23). The antigenic peptide was purified by DEAE-cellulose chromatography and high pressure liquid chromatography on a reverse-phase C18 column. The antigenic peptide contains 14.7 and 18.7 mol% of glutamic acid and lysine, respectively. Amino acid sequence analysis showed that the peptide has 68 amino acids and is located on the carboxyl-terminal sequence of protein B23. The sequence is Ser-Phe-Lys-Lys-Gln-Glu-Lys-Thr-Pro-Lys-Thr-Pro- Lys-Gly-Pro-Ser-Ser-Val-Glu-Asp-Ile-Lys-Ala-Lys-Met-Gln-Ala-Ser-Ile-Glu- Lys-Gly- Gly-Ser-Leu-Pro-Lys-Val-Glu-Ala-Lys-Phe-Ile-Asn-Tyr-Val-Lys-Asn-Cys-Phe- Arg-Met- Thr-Asp-Gln-Glu-Ala-Ile-Gln-Asp-Leu-Trp-Gln-Trp-Arg-Lys-Ser-Leu-Cooh. Extensive digestion of the antigenic peptide with V8 protease, trypsin, or chymotrypsin results in loss of the antigenic activity. Three cloned cDNAs (hpB1, hpB2, and hpB7) which code for the 82 amino acids at the COOH terminus of protein B23 and the 3' non-translating sequence were identified and characterized. All three clones have identical nucleotide sequences coding for the antigenic portion of the protein (68 amino acids at the COOH terminus), the stop codon, and the 3' non-translated region. However, mutation of 6 nucleotide bases of one clone (hpB2) caused changes in 4 amino acids in the sequence just preceding the immunoreactive region. The result suggests the presence of at least 2 immunologically similar but distinct proteins which are both recognized by the anti-B23 antibody.     

Amino acid sequence of yeast proteinase B inhibitor 1 comparison with inhibitor 2.

The amino acid sequence of proteinase B inhibitor 1 (I^B1) from bakers' yeast has been established by automated Edman degradation up to position 42. A comparison with the sequence of proteinase B inhibitor 2 (I^B2) revealed two differences: LEU-32 and GLU-34 in I^B2 are replaced by VAL-32 and LYS-34 in I^B1. Identity of the COOH-terminal region of I^B1 with that of I^B2 was proved by degradation with the carboxypeptidases A and Y. Furthermore, a chymotryptic peptide was isolated from each of the 74 residues containing inhibitors. The two fragments, ranging from position 42 to the COOH termini of the inhibitors, were found to be identical with respect to electrophoretical mobility, end groups, amino acid composition and peptide pattern after tryptic digestion. It is concluded, that the two inhibitor sequences are identical beyond position 42. I^B1 and I^B2 are isoinhibitors, because they are coded by different genes.     

Amino acid sequence of calmodulin from wheat germ

The complete amino acid sequence of calmodulin from wheat germ was determined by isolating and sequencing the cyanogen bromide and tryptic peptides. The protein consisted of 149 amino acid residues and its amino(N)-terminus was blocked with an acetyl group. Wheat germ calmodulin lacked tryptophan and contained 1 mol each of histidine, tyrosine, cysteine, and N epsilon-trimethyllysine residues per mol of the protein. A comparison of its amino acid sequence with that of bovine brain calmodulin indicated that there were eleven amino acid subsitutions other than amide assignments, two insertions and one deletion of amino acid residues in wheat germ calmodulin.     

Amino acid sequence of a 12-kDa inhibitor of protein kinase C

The complete primary structure of a bovine-brain-derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast-atom-bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11,779 Da with the following 107-amino-acid sequence: [sequence: see text] This inhibitor does not share significant primary structural identity with any other known protein.     

Amino acid sequence of derivatives of newborn rat epidermal thiol proteinase inhibitor

Three different forms of thiol proteinase inhibitor (TPI) were isolated from newborn rat epidermis, in which two forms, TPI-1 and TPI-2, inhibited a proteinase activity, but another newly detected one (designated as TPI-3), showed no inhibitory effect. The complete amino acid sequence of TPI-2 and the sequence of the first seventeen residues from the NH2-terminus of TPI-3 were determined. The sequence shows that TPI-2 lacks in the first six (or four) residues from the NH2-terminus of intact inhibitor, TPI-1, whereas TPI-3 is devoid of its fifteen amino acid residues. These results indicate a high and specific susceptibility of TPI to proteolysis. Most significantly, the NH2-terminal region of TPI appears to be essential for inhibition of proteinase activity.     

Amino acid sequence of the ribonuclease inhibitor from porcine liver reveals the presence of leucine-rich repeats

The primary structure of the ribonuclease inhibitor from pig liver has been determined by amino acid sequence analysis. The N alpha-acetylated polypeptide chain of 456 amino acids consists of 15 homologous leucine-rich repeats, characterized by leucyl residues at constant positions. Two types of alternating repeats occur, 29 (A) and 28 (B) residues long. The degree of identity between repeats of a given type ranged from 25 to 60%. Only one deletion in the B-repeat was necessary to perfectly align the leucyl residues between the two repeats. Leucine-rich repeats have previously been found in four membrane-bound proteins and one extracellular protein, and their amphiphilic character suggested that they could be involved in membrane binding. Ribonuclease inhibitor is the first example of a cytoplasmic protein containing this type of repeat. It seems likely, therefore, that leucine-rich repeats can have functions other than forming membrane binding structures.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The amino acid sequence of zinc-carboxypeptidase from Streptomyces griseus

The amino acid sequence of a zinc-carboxypeptidase from S. griseus (Cpase SG) was determined by automated Edman degradation and carboxypeptidase digestion of the S-carboxymethylated protein and by sequence analyses of peptides produced by cyanogen bromide cleavage and by lysyl endopeptidase digestion of the S-carboxymethylated protein. This enzyme is characterized by a uniquely broad substrate specificity which combines the specificities of mammalian Cpase A and Cpase B (J. Biochem. 86, 683-694, 1979). Cpase SG consists of 328 amino acid residues. The amino acid sequence of Cpase SG is partially similar to those of bovine Cpase A and Cpase B (sequence identity, 28-29%). In the sequence of Cpase SG, residues that are functionally important in mammalian Cpase A and Cpase B were all found at the corresponding positions. Residue 255 (according to the numbering system for bovine Cpase A), which, in the other Cpases, contributes to the difference in specificity between Cpase A (Ile-255) and Cpase B (Asp-255), was Asp. However, residue 254 was Ile, in contrast to Ser or Thr in all of the forms of Cpase A and Cpase B examined to date. The increase in hydrophobicity caused by the change at position 254 and the presence of negative charge at position 255 is probably one of the reasons for the broad substrate specificity of Cpase SG.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.