Purification and molecular characterization of two inhibitors of yeast proteinase B.

A rapid purification procedure for large scale preparations of yeast proteinase B inhibitors 1 and 2 (IB1 and IB2) is described. By disc gel electrophoresis, amino acid analysis, and end-group determinations, each of the inhibitors is homogeneous. Both inhibitors are polypeptides with molecular weights of 8,500, containing 74 residues. No components other than amino acids could be detected. There is no significant difference in the amino acid compositions of the two inhibitors as analyzed after acid hydrolysis. Both polypeptides are characterized by the total absence of arginine, tryptophan, and sulfur-containing amino acid residues. The proteinase B inhibitors of yeast, therefore, differ fundamentally from proteinase inhibitors of many other organisms, which generally contain a large number of disulfide bridges. Both proteinase B inhibitors have threonine as the NH2-terminal residue and -Val-His-Thr-Asn-COO- as the COOH-terminal sequence. Comparison of peptide maps after tryptic digestion reveals that the two inhibitors differ definitely in only a few tryptic peptides. The inhibitors are rapidly inactivated by digestion with carboxypeptidase A from bovine pancreas at pH 8.5. Inactivation occurs stoichiometrically with the release of threonine, the penultimate residue at the COOH-terminal end of both inhibitors.     

Dental enamel matrix: Sequences of two amelogenin polypeptides

The amino acid sequences of a leucine-rich amelogenin polypeptide (LRAP) and a tyrosine-rich amelogenin polypeptide (TRAP), isolated from foetal bovine enamel matrix, were determined. Both LRAP and TRAP occurred in two forms; in each case, one of the molecular species appeared to be shortened at the COOH terminus by 2 and 4 residues, respectively. A striking finding was that LRAP and TRAP had identical sequences for the first 33 residues but were almost completely different for the remaining 12 amino acids.     

Purification and characterization of a low and a high molecular weight form of epidermal growth factor from rat urine

Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.     

Comparative studies on the primary structure of acetylcholinesterases from bovine caudate nucleus and bovine erythrocytes

1. Comparison of partial amino acid sequences of G2-acetylcholinesterase (AChE) from bovine erythrocytes and G4-AChE from bovine caudate nucleus revealed no differences in primary structure between the two enzymes. The first 33 residues of the N-terminal sequences were identical. 2. In addition, the amino acid sequences of four peptides generated by tryptic and cyanogen bromide cleavage were identical for bovine erythrocyte and brain AChE, suggesting one identical major coding exon for the adult bovine AChE forms. Comparison of these sequences with that of fetal bovine serum AChE (Doctor et al., 1988), showed differences in residues 16, 181, 212, and 216. 3. Deglycosylation studies of the two adult enzyme forms revealed that the core protein of erythrocyte AChE has an approximately 4 kDa lower molecular mass than brain AChE. This most probably reflects differences in the C-terminal sequences of the two enzymes.     

Bovine proacrosin from cauda epididymal sperm: purification, characterization and partial sequencing at N-terminus.

1. In the present study, we isolated the two forms of proacrosin from acid extracts (pH 3.0) of cauda epididymal bovine spermatozoa by ammonium sulfate fractionation, gel filtration on Sephadex G-150 and affinity chromatography on Concanavalin A Sepharose 4B. The overall purification was 13-fold with respect to crude acid acrosomal extract. 2. The apparent molecular weight of the proacrosins determined by SDS-PAGE were 44,000 and 38,000. Both forms have proteinase activity on gelatin-SDS-polyacrylamide gel electrophoretic zymography. 3. The M(r) = 38,000 component was isolated by reverse phase HPLC. Thirty-nine amino acid residues at the N-terminus have about 72 and 77% sequence similarity with boar and human proacrosin, respectively. 4. The amino acid sequence of 14 amino acids at the N-terminus of the high molecular weight component (M(r) = 44,000) was determined after electroblotting on a polyvinylidene difluoride membrane. This portion of the molecule is identical with that of the low molecular weight component. 5. Proacrosin autoactivation followed the sigmoidal activation curve.     

Effect of poly(ADP-ribose) formation on DNA synthesis in chick-embryo-liver nuclei. Possible mechanism of template activation.

The complete amino acid sequence of bovine phospholipase A2 (EC 3.1.1.4) was determined. This enzyme has a molecular weight of 13 782 and consists of a single polypeptide chain of 123 amino acids cross-linked by seven disulfide bridges. The main fragmentation of the polypeptide chain was accomplished by digesting the reduced and thialaminated derivative of the protein with trypsin, staphylococcal protease and cyanogen bromide. A number of chymotryptic peptides were used for alignment and to obtain overlaps of at least two residues. The sequence of the peptides was determined by Edman degradation by means of direct phenylthiohydantoin identification in combination with identification as dansyl amino acids. Although 71% of all residues of phospholipase A2 from bovine, porcine and equine sources are conserved, bovine phospholipase A2 differs from the others by the total number of residues and by substitutions at 20 (porcine) and 33 (equine) positions.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Primary structure of a protease isoinhibitor from bovine spleen. A possible intermediate in the processing of the primary gene product

Sequence studies on the protease isoinhibitor I isolated from bovine spleen have revealed that it consists of two molecular variants which differ only in the presence of an additional COOH-terminal residue, asparagine, in the less abundant form. The complete amino acid sequence shows that they are composed of 65 or 66 residues and predicts Mr of 7223 or 7338, respectively. The sequences correspond exactly to the 58-residue polypeptide chain of spleen isoinhibitor II plus NH2- and COOH-terminal extensions of 2 and 5 or 6 amino acid residues, respectively. Moreover the entire sequences are located within the 100-residue structure deduced from the mRNA and DNA sequences of the putative precursor. These data support the idea that the molecular variants of isoinhibitor I are either mature proteins with distinct functional roles, or intermediates in the multistage processing of the primary product of gene expression, which eventually leads to the mature protein, i.e. inhibitor II.     

The isolation, partial characterization, and biosynthesis of the paragonial substances, PS-1 and PS-2 of Drosophila funebris

In methanolic extracts of accessory glands (paragonia) from Drosophila funebris, two specific, ninhydrin-positive substances, PS-1 and PS-2, were found. PS-1 and PS-2 were isolated by column chromatography. PS-1 consists of 27 amino acid residues. Two forms of PS-1 are present in the ratio of 7:3 which differ only in the content of valine and leucine. Fractions containing partially purified valine-PS-1 and leucine-PS-1, respectively, have the same biological activity. All males in the population synthesize both forms of PS-1. PS-2 is a low molecular weight substance containing glycine and ammonia as ninhydrin-positive components and carbohydrate as indicated by several sugar tests. In vitro studies showed that copulation provides the stimulus for enhanced synthesis of paragonial substances.     

Isolation and properties of goat beta 2-microglobulin

Goat beta 2-microglobulin was isolated and purified from colostrum. Comparisons of the amino acid composition and amino-terminal sequence of the goat protein with the bovine and human homologues, indicates a high degree of similarity. Both goat and bovine beta 2-microglobulins differ slightly in composition from the human molecule, most notably in threonine and proline values. For the first 32 residues, bovine and goat differ only at two positions, one of which is a valyl/isoleucyl substitution consistent with the amino acid compositions. The equivalent goat/human sequence comparison shows seven differences. Immunological studies, using the ELISA method, also confirm the close relatedness of goat and bovine beta 2-microglobulin and their more distant relatedness to the human homologue.     

Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11.5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.     

Complete amino acid sequence of BSP-A3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding domain of fibronectin.

Bovine seminal plasma was shown to contain three similar proteins, called BSP-A1, BSP-A2 and BSP-A3. Both BSP-A1 and BSP-A2 were shown to be molecular variants of a recently characterized peptide called PDC-109. They seem to differ only in their degree of glycosylation and otherwise seem to possess an identical amino acid composition. The work in the present paper deals with the complete characterization of the third member of this series, namely BSP-A3. The complete amino acid sequence revealed that it is composed of 115 amino acids and predicts a Mr of 13,403. An analysis of the primary structure of BSP-A3 revealed a high degree of internal homology, with two homologous domains composed of 39 (residues 28-66) and 43 (residues 73-115) amino acids. An exhaustive computer-bank search for the similarity of this sequence to any known protein, or segment thereof, revealed two significant homologies. The first is between PDC-109 and BSP-A3, which is so high that we can confidently predict that both proteins evolved from a single ancestral gene. The collagen-binding domain of bovine fibronectin (type II sequence) was also found to be highly homologous to both BSP-A3 and PDC-109.     

Structural analysis of bovine pancreatic thread protein

Pancreatic thread protein (PTP) forms double helical threads in the neutralpH range after purification, undergoing freely reversible,pH-dependent globule-fibril transformation. The purified bovine PTP consists on SDS gels of two carbohydrate-free polypeptide chains (Grosset al., 1985). Plasma desorption mass spectrometry and amino acid sequence analysis now confirm that bovine PTP contains two disulfide-bonded polypeptides, an A chain of 101 amino acid residues with a molecular weight of 11,073 and a B chain of 35 residues with a molecular weight of 3970. The intact protein exhibits a molecular weight of 15,036, agreeing >99.9% with the molecular weight calculated from the sequence. The B chain sequence was determined by gas-phase Edman degradation of the intact polypeptide. The A chain sequence was determined from overlapping peptides generated by cleavage at lysyl, tryptophanyl, and aspartyl-prolyl residues. Based upon the bovine PTP cDNA structure, the two chains of the protein result from cleavage of a single polypeptide with removal of a dipeptide between the NH₂-terminal A chain and COOH-terminal B chain. Comparison of bovine PTP with other proteins reveals significant structural relatedness with the single-chain homologues from human and rat pancreas and with the motif associated with Ca²⁺-dependent carbohydrate recognition domains. The physiological role of PTP has not yet been resolved. The protein is present in very high concentration in pancreatic secretion and it has been detected in brain lesions in Alzheimer's disease and Down syndrome and in regenerating rat pancreatic islets. The present results provide a firm protein base for ongoing molecular, physical-chemical, and structure-function studies of this unusual protein.     

Purification and partial sequencing of human placental alkaline phosphatase

Two forms of human placental alkaline phosphatase have been purified to homogeneity utilizing high performance liquid chromatography. Both have the same amino acid composition but they differ in their carbohydrate substituents. Sequence data indicate that the two forms are identical for the first forty two residues from the amino terminus are presented.     

A form of human basic fibroblast growth factor with an extended amino terminus

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986).     

Complete amino acid sequence of PO protein in bovine peripheral nerve myelin

The P0 protein is a major structural glycoprotein of molecular weight 28,000 in peripheral nerve myelin. The complete amino acid sequence of bovine P0 protein was determined. The polypeptide chain consists of 219 amino acid residues and includes a highly hydrophobic domain (residues 125-150) in the middle, which probably represents a transmembrane segment. The amino terminal domain (residues 1-124) is relatively hydrophobic, but contains a negatively charged carbohydrate chain at Asn93. This domain is most likely located on the extracellular side of the membrane and may contribute to formation of the myelin intraperiod line by hydrophobic and electrostatic interactions. On the other hand, the basic carboxyl-terminal domain (residues 151-219) may protrude from the cytoplasmic side of the membrane and is probably involved together with basic proteins in the formation of the major myelin dense line through electrostatic interaction with acidic lipids in the membrane. The few interspecies amino acid variations between the bovine P0 and the rat P0 sequences, deduced from the cDNA (Lemke, G., and Axel, R. (1985) Cell 40, 501-508), indicate that the P0 protein is conserved across species.     

Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart. Use of FAB-MS for molecular mass determination

We have isolated a 6.8 kDa proteolipid from an acidic chloroform/methanol extract of bovine cardiac muscle. The molecular mass of the polypeptide was measured by fast atom bombardment-mass spectrometry (FAB-MS) (m/z6834.1). Its amino acid sequence was partly determined by direct sequencing and completed by characterization of cyanogen bromide and tryptic fragments (sequencing, FAB-MS and amino acid analysis). The polypeptide consists of 60 amino acid residues. Polyclonal antibodies raised in rabbit allowed its localization by electroimmunoblotting in mitochondria.     

Characterization of multiple forms of prostatropin (prostate epithelial cell growth factor) from bovine brain

Two molecular forms of prostatropin distributed among five chromatographic peaks have been isolated from bovine brain by heparin-Sepharose affinity and reverse phase high performance liquid chromatography. One form has an apparent molecular weight of 16000 and an amino terminal sequence of asn-tyr-lys-lys-pro-lys-leu-leu-tyr-x-ser-asn-gly. The other form has an apparent molecular weight of 18000 and a blocked amino terminus. Both forms are similar in amino acid composition. The sequence of a proteolytic fragment from the blocked form overlaps the NH2-terminal sequence of the unblocked form, therefore, the smaller form may be derived from the larger form through proteolytic processing. Both forms contain regions identical in sequence to brain-derived, heparin-binding growth factors that have been isolated on the basis of mitogenic activity for fibroblasts and endothelial cells. Both forms of prostatropin exhibit potent mitogenic activity for normal and tumor prostate epithelial cells.     

Amino acid sequence of subunit V of bovine heart cytochrome oxidase, the heme alpha-containing subunit.

The complete amino acid sequence of the heme alpha-containing subunit V of bovine heart cytochrome oxidase was determined to be: H2N-Ser-His-Gly-Ser-His-Glu-Thr-Asp-Glu-Glu-Phe-Asp-Ala-Arg-Trp-Val-Thr-Tyr-Phe-Asn-Lys-Pro-Asp-Ile-Asp-Ala-Trp-Glu-Leu-Arg-Lys-Gly-Met-Asn-Thr-Leu-Val-Gly-Tyr-Asp-Leu-Val-Pro-Glu-Pro-Lys-Ile-Ile-Asp-Ala-Ala-Leu-Arg-Ala-Cys-Arg-Arg-Leu-Asn-Asp-Phe-Ala-Ser-Ala-Val-Arg-Ile-Leu-Glu-Val-Val-Lys-Asp-Lys-Ala-Gly-Pro-His-Lys-Glu-Ile-Tyr-Pro-Tyr-Val-Ile-Gln-Glu-Leu-Arg-Pro-Thr-Leu-Asn-Glu-Leu-Gly-Ile-Ser-Thr-Pro-Glu-Glu-Leu-Gly-Leu-Asp-Lys-Val-COOH. The subunit V is a single polypeptide which consists of 109 amino acid residues. The protein contains 48.6% hydrophobic residues and 34.0% hydrophilic residues and it is an acidic protein having a net charge of -3 at neutral pH. The molecular weight of subunit V was calculated to be 12,436 and that for the heme alpha-containing polypeptide was 13,295.