Complete amino acid sequence of bovine neurophysin-I. A major secretory product of the posterior pituitary

Bovine neurophysin-I (bNP-I) is the first neurophysin protein which contains histidine and possesses an acidic COOH-terminal segment for which the complete amino acid sequence is presented: NH2-Ala-Val-Leu-Asp-Leu-Asp-Val-Arg-Thr-Cys-Leu-Pro-Cys-Gly-Pro-Gly-Gly-Lys-Gly-Arg-Cys-Phe-Gly-Pro-Ser-Ile-Cys-Cys-Gly-Asp-Glu-Leu-Gly-Cys-Phe-Val-Gly-Thr-Ala-Glu-Ala-Leu-Arg- Cys-Gln-Glu-Glu-Asn-Tyr-Leu-Pro-Ser-Pro-Cys-Gln-SerGly-Gln-Lys-Pro-Cys-Gly-Ser- Gly-Gly-Arg-Cys-Ala-Ala-Ala-Gly-Ile-Cys-Cys-Ser-Pro-Asp-Gly-Cys-His-Glu-Asp-Pro-Ala-Cys-Asp-Pro-Glu-Ala-Ala-Phe-Ser-Leu-COOH. Determination of the structure was greatly facilitated by new procedures used for the isolation of bNP-I and of its tryptic peptide fragments. bNP-I isolated from freshly frozen bovine posterior pituitaries is composed of 93 residues, but some preparations contain neurophysin protein with NH2- and COOH-terminal truncated sequences. bNP-I differs from bovine neurophysin-II, the second major neurophysin of cow, in 20 residue positions, and several of the differences cannot be accounted for by single nucleotide replacements in the genes coding for these two neurophysin proteins. The results reported in this study support our earlier hypothesis that neurophysin-gene duplication preceded species divergence.     

Studies on the chemical modification of the tyrosine residue in bovine neurophysin-II

1. Bovine neurophysin-II contains 1mol of tyrosine residue/10000g of protein. This residue could be readily nitrated with tetranitromethane. On hydrolysis and amino acid analysis 1mol of 3-nitrotyrosine was found/10000g of protein. Starchgel electrophoresis at pH8.5 showed that nitration had converted the native protein into a single, more acidic species. The increase in acidity was consistent with the observed fall in pK of the tyrosine hydroxyl from 9.2 in native neurophysin to 7.3 in the nitrated protein. Further, the absence of any intermediate species, even under conditions of minimum substitution, confirmed that the molecular weight of the monomer is 10000. 2. O-Acetylation of the tyrosine residue was carried out with N-acetylimidazole, in conjunction with the reversible blocking of amino groups by citraconylation. The degree of O-acetylation, determined spectroscopically, was 0.9mol of O-acetyltyrosine/10000g of protein. 3. The hormone-binding ability of modified protein was tested by equilibrium dialysis and was found to be unchanged by either nitration or O-acetylation of the tyrosine residue. 4. Interaction of neurophysin-II and [8-arginine]-vasopressin gave rise to a characteristic difference spectrum with a peak at 286.8nm and shoulder at 279.6nm. Part of this hyperchromicity is thought to result from entry of the tyrosine residue at position 2 in the hormone into the hydrophobic environment of the binding site. With nitrated neurophysin-II a second peak appeared at 436nm, showing that the tyrosine of the protein is also perturbed. The very large red shift (84nm) in this region suggests that the 3-nitrotyrosyl residue not only enters a more hydrophobic environment on protein-hormone interaction, but is caused to ionize more fully by the approach of some positively charged group.     

Fluorescence studies of native and modified neurophysins. Effects of peptides and pH.

The effect of neurophysin-hormone interaction on the environment of the single tyrosine of bovine neurophysin (Tyr-49) and on that of the tyrosine of oxytocin and vasopressin was studied by fluorescence; tyrosine-free peptides were used to determine effects on Tyr-49, and acetylated neurophysin was used to determine effects on the hormone tyrosine. Binding increases the fluorescence intensity of Tyr-49 by 130% while the fluorescence of the hormone tyrosine is almost completely quenched. Correlation of these results with those obtained on binding oxytocin or vasopressin to native neurophysin indicates that in the hormone complexes less than half of the fluorescence of Tyr-49 is lost by F?rster energy transfer to the quenched hormone tyrosine. These results support spin-label studies in indicating that the distance between Tyr-49 and the tyrosine of hormone bound to the strong hormone binding site is greater than 5 A. In the absence of peptides, the fluorescence of Tyr-49 increases by 40% on lowering the pH from 6.2 to 2. Titration of the acid fluorescence transition in bovine neurophysins-I and -II, and in bovine neurophysin-II treated with carboxypeptidase B to remove the Arg-Arg-Val sequence at the carboxyl terminus, indicates that this transition is due to titration of a side-chain carboxyl with an intrinsic pK of 4.6. The effects of guanidine, glycerol, and disulfide cleavage on the magnitude of the acid transition indicate that the conformational information necessary for the transition resides within the amino acid sequence adjacent to Tyr-49. Accordingly, the fluorescence acid transition is attributed to decreased quenching by Glu-46 or Glu-47 upon protonation. Glycerol is shown to perturb the glutamate-tyrosine interaction in the absence of general conformational effects. Comparison of the fluorescence low-pH transition with that of the low-pH circular dichroism transition of nitrated neurophysins suggests that the fluorescence and CD transitions reflect related, but not necessarily identical, phenomena. In an appendix, evidence is presented which suggests that the products of carboxy-peptidase digestion of bovine neurophysin-II are the same as two minor bovine neurophysin components, one of which is neurophysin-C.     

Cloning and nucleotide sequence of a bovine pancreatic preprocarboxypeptidase A cDNA

A full-length cDNA clone encoding bovine pancreatic preprocarboxypeptidase A was isolated and sequenced. The 1405-base pair insert contains a 26-nucleotide 5'-noncoding region, a 1260-nucleotide open reading frame and a 76-nucleotide 3'-noncoding fragment plus a poly(A) tail of at least 43 nucleotides. The open reading frame encodes a protein of 419 amino acids, including the 16 amino acid signal peptide. The mature enzyme (309 residues) has two additional C-terminal amino acids, as compared with the amino acid sequence of the protein which was reported more than 20 years ago. In addition, four residues deduced from the nucleotide sequence differed from those identified in the reported amino acid sequence from their net charge: Asp-89, Asp-114, Gln-122, and Asp-185 instead of Asn-89, Asn-114, Glu-122, and Asn-185, respectively. A high degree of identity exists between the nucleotide sequences (81.3%), on the one hand, and the amino acid sequences (78.3%), on the other hand, of bovine preprocarboxypeptidase A and rat preprocarboxypeptidase A1.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.     

Amino acid sequences of cytochrome b5 from human, porcine, and bovine erythrocytes and comparison with liver microsomal cytochrome b5

The amino acid sequences of human, porcine, and bovine erythrocyte cytochromes b5 which are soluble and present in the cytosol have been determined. In addition, the partial sequences of microsome-bound liver cytochrome b5, namely the sequence of the N-terminal region and joint region between the heme-containing and membranous part, have been established for human and porcine sources. All the cytochromes b5 from erythrocyte and liver contained N-acetylated N-termini. Of the 97 amino acid residues of erythrocyte cytochrome b5, residues 1-96 were identical with those of the liver protein of the same species. However, residue 97 (C-terminal residue) was proline for human erythrocyte cytochrome b5 and serine for the porcine protein, while residues 97 (joint region) of human and porcine liver cytochromes b5 were threonine. These findings indicate that the two forms of cytochrome b5 are encoded by two different but closely related mRNAs.     

Identification and observation of alkyl proton resonances of the amino-terminal residues of bovine neurophysins. Evidence for conformational differences between neurophysin-I and neurophysin-II.

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation of these spectra has allowed identification of the -CH3 proton resonances of the amino-terminal alanine of both proteins and of the -CH3 resonances of methionine-2 of neurophysin-II. The alanine -CH3 resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH3 resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated alpha-amino and an unprotonated side chain carboxyl. The results suggest that differeces in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. 'reporter' signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 A from residue 3 of peptides bound to the strong neurophysin hormone-binding site.     

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.     

Ostrich MSEL-neurophysin belongs to the class of two-domain "big" neurophysin as indicated by complete amino acid sequence of the neurophysin/copeptin

Mammalian neurohypophyseal hormones, oxytocin and vasopressin, are known to be synthesized as part of two larger precursors containing, respectively, a VLDV-neurophysin and a MSEL-neurophysin together with its associated glycopeptide. Starting from ostrich neurohypophyses, a "big" neurophysin was isolated and chemically characterized. Following sequence determination of the CNBr-derived fragments and of peptides obtained from trypsin and V8-protease digestion of the oxidized protein, this "big" neurophysin was found to contain an MSEL-neurophysin moiety (94 residues) still covalently associated with the COOH-terminal glycopeptide (38 residues, copeptin). This study demonstrates that the ostrich MSEL-neurophysin sequence closely resembles all known MSEL-neurophysin sequences and that, furthermore, it does not contain the single amino acid insertion shown previously in the ostrich VLDV-neurophysin. It is also shown that the stretch of amino acids, linking the MSEL-neurophysin and the copeptin, is clearly different from its mammalian homologues and lacks the Arg residue normally recognized by the cleaving enzyme. This study also demonstrates that the ostrich copeptin is more closely related to the amphibian copeptin sequence than to its mammalian homologue, leading to the hypothesis that two families of copeptin molecules might exist. Thus, the ostrich MSEL-neurophysin-copeptin molecule is the first "big" neurophysin reported in birds and, together with the guinea pig and amphibian homologues, represents the third example of partial or no neurophysin-copeptin cleavage.     

Carbon-13 nuclear magnetic resonance studies of the binding of selectively 13C-enriched oxytocins to the neurophypophyseal protein, bovine neurophysin II

Complex formation between bovine neurophysin II and oxytocin molecules containing 85% 13C enrichment in specific amino acid residues was studied using 13C nuclear magnetic resonance spectroscopy. Chemical shift and relaxation time values of the analogue [13C-Leu3]oxytocin, [13C-Gly9]oxytocin, and the doubly labeled [13C-Ile3 Gly9]oxytocin were obtained for the hormones in the absence and presence of neurophysin. The results showed that certain 13C nuclear magnetic resonance parameters of residue 3 but not of residue 9 of oxytocin are altered upon binding to neurophysin. These observations suggest that residue 3 but not residue 9 is involved in the protein-hormone interaction and they demonstrate the general applicability of selective 13C enrichment for the study of peptide-protein interactions.     

Processing of the oxytocin precursor: isolation of an exopeptidase from neurosecretory granules of bovine pituitaries

The bovine pro-oxytocin precursor consists of the nonapeptide hormone and its neurophysin carrier protein that contains at its C-terminus a rudimentary processing signal, the single basic amino acid residue histidine. An exopeptidase has been isolated from neurosecretory granules of the bovine posterior pituitary that releases the supernumary histidine residue from the pro-hormone precursor. Based on its sensitivity to inhibitors and activators the enzyme has carboxypeptidase B-like properties with a pH optimum between 5.0 and 5.5.     

Characterization by cDNA cloning of the mRNA for seminalplasmin, the major basic protein of bull semen

A cDNA library derived from poly(A)+RNA of bull seminal vesicle tissue was screened with synthetic DNA probes specific for seminalplasmin (SAP), the major basic protein of bull semen. From a number of positive clones, pBSV12, containing a 577-bp insert, was identified and sequenced. The derived amino acid sequence comprises the known amino acid sequence of SAP with an amino terminal representing a putative signal sequence; at the carboxyl terminus the sequence contains an additional lysine residue. Present experimental data do not distinguish between two potential SAP precursor molecules, each starting with a methionine residue and differing by 10 amino acid residues in the leader peptide. Comparative Northern analysis reveals a SAP-specific mRNA of 700 bp, which lacks RNA from bovine testis as well as from seminal vesicle tissue of a bull calf; hence, expression of the SAP gene appears to be under androgen and/or developmental control. Southern analysis indicates that one gene appears to specify SAP. SAP-like DNA sequences were detected in ovine and porcine genomic DNA.     

Collagen type IX: Evidence for covalent linkages to type II collagen in cartilage

A major site of pyridinoline cross-linking in bovine type IX collagen was traced to a tryptic peptide derived from one of the molecule's HMW chains. This peptide gave two amino acid sequences (in 2/1 ratio) consistent with it being a three-chained structure. The major sequence matched exactly that of the C-telopeptide of type II collagen from the same tissue. A second HMW chain that contained pyridinoline cross-links also gave two amino-terminal sequences, one from its own amino terminus, the other matching exactly the N-telopeptide cross-linking sequence of type II collagen. We conclude that type IX collagen molecules are covalently cross-linked in cartilage to molecules of type II collagen, probably at fibril surfaces.     

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by B?hmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.     

Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism

Hybrid molecules containing DNA sequences complementary to bovine pituitary mRNA were constructed in the Pst I site of pBR322 by the dC . dG tailing technique. Recombinant plasmids containing bovine prolactin (bPRL) sequences were amplified in bacteria and identified by hybridization to purified [32P]bPRL cDNA sequences. Nucleotide sequence analysis was performed on the inserts from two of the positive clones. One clone, pBPRL72, contained a 982-base pair insert that included 67 nucleotides of the 5'-untranslated region, the complete coding region of the preprolactin protein (690 nucleotides), and the entire 3'-untranslated region (150 nucleotides) of bPRL mRNA. The nucleotide sequence analysis of clone pBPRL72 predicted the sequence of a 30-amino acid signal peptide and confirmed the published amino acid sequence of the protein with one exception. A comparison of the pBPRL72 cDNA sequence with a second bPRL clone, pBPRL4, revealed four silent nucleotide differences. Three of the base changes occurred in the third position of amino acid codons, and one occurred in the 3'-noncoding region. The sequence polymorphism suggests the existence of alleles or multiple loci for bPRL that do not alter the protein structure.     

Bovine and feline gastrin cDNA sequences and the amino acid and nucleotide sequence homologies among mammalian species.

The complete nucleotide sequences of cDNAs encoding bovine and feline preprogastrins have been cloned from the antral mucosa mRNA. The gastrin mRNA of each animal encodes a preprogastrin of 104 amino acids consisting of a signal peptide, a prosegment of 37 amino acids, and a gastrin 34 sequence, followed by a glycine (the amide donor). The cleavage following a pair of lysine residues yields gastrin 17. We found that pairs of arginine residues flanking gastrin 34, the typical processing site sequence of all other preprogastrins and many peptide hormones, were arginines in the bovine preprogastrin, but the first basic amino acid pair had changed to Arg-Trp (57-58 residues) instead of Arg-Arg in the feline preprogastrin. Comparison of these amino acid and nucleotide sequences with published mammalian sequences showed extensive homology in the coding (63 to 73% amino acid identity) and in the untranslated regions (67 to 89% identity). Prosequence, the most variable region, shows greater amino acid difference between bovine and human preprogastrin (54% identity), and between bovine and rat preprogastrin (54% identity) than between other species (62 to 82% identity).     

Cleavage of proteoglycan aggregate by leucocyte elastase.

The partial degradation of proteoglycan aggregate by human leucocyte elastase yielded products that banded with Mr 190,000, 140,000, 88,000, and 71,000 when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis. Analysis of these bands revealed that the 190,000- and 140,000-Da bands contained chondroitin and keratan sulfate stubs and had N-terminal amino acid sequences corresponding to a sequence starting at residue 398 of the core protein of rat or human aggrecan. With increased time of digestion, the staining intensities of the 190,000-, 140,000-, and 88,000-Da bands decreased relative to the 71,000-Da band. Analysis of the 88,000- and 71,000-Da bands showed that they contained peptides substituted only with keratan sulfate stubs and that each band contained two peptides with different N-terminal sequences. One of these corresponded to a sequence that started at residue 398 of rat or human aggrecan and the other to the N-terminal sequence of bovine aggrecan. Under conditions of complete digestion, bands of 71,000 and 56,000 Da which contained only keratan sulfate stubs were observed on SDS-polyacrylamide electrophoresis. The 71,000-Da band was shown to have a single sequence similar to that starting at residue 398 of human and rat aggrecan and thus represents the globular domain 2 (G2) of the core protein of aggrecan. The 56,000-Da band was shown to have a sequence similar to that of the N-terminal sequence of bovine aggrecan indicating that this peptide corresponds to the globular domain 1 (G1) of the molecule. These results suggest that leucocyte elastase cleaves the core protein of aggrecan between valine 397 and isoleucine 398, which are located in the interglobular domain linking the G1 and G2 domains of the core protein of aggrecan. Further digestion of the proteoglycan aggregate with elastase resulted in the cleavage of the core protein within the chondroitin sulfate attachment domains.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Protein sequence comparison based on the wavelet transform approach

A protein's chemical properties, the chain conformation, the function of the protein and its species specificity are determined by the information contained in the amino acid sequence. Proteins of similar functions have at some level sequential identical amino acid sequences. The closer the phylogenetic relationship, the more similar are the sequences. To find the similarities between two or more protein sequences is of great importance for protein sequence analysis. The differences in the amino acid sequences permit the construction of a family tree of evolution. In this work, a comparison method was devised that is capable of analysing a protein sequence 'hierarchically', i.e. it can examine a protein sequence at different spatial resolutions. Based on a wavelet decomposition of protein sequences and a cross-correlation study, a sequence-scale similarity concept is proposed for generating a similarity vector, which renders the comparison of two sequences feasible at different spatial resolutions (scales). This new similarity concept is an expansion of the conventional sequence similarity, which only takes into account the local pairwise amino acid match and ignores the information contained in coarser spatial resolutions.