PHI--a new brain-gut peptide

The detection of the C-terminal amide structure in porcine intestinal extracts has led to the discovery of a 27 amino acid residue peptide designated PHI (PHI-27, peptide HI). The peptide was found to have structural homologies to vasoactive intestinal peptide (VIP) and growth hormone-releasing factor (GRF). Subsequent studies have revealed that PHI exhibits a variety of biological activities which resemble those of VIP. Moreover, it was found that the peptide is able to inhibit the binding of VIP to its receptors, and to stimulate cyclic AMP production. PHI is present in both brain and gut in high concentrations and probably acts as a neurotransmitter or neuromodulator rather than a hormone. A comparison of the amino acid sequences of porcine, human and bovine PHI indicated that human PHI differs from the porcine peptide in two positions (12 and 27), and bovine PHI differs in one position (10). The amino acid sequence (deduced from the cDNA sequence) of the VIP precursor recently obtained from human neuroblastoma cells also contains an identical sequence to the newly-isolated human PHI from human colonic extracts. PHI has thus been shown to be co-synthesized with VIP in the same precursor molecule.     

The secretin precursor gene. Structure of the coding region and expression in the brain

Secretin is a 27-amino acid gastrointestinal hormone that stimulates the secretion of bicarbonate-rich pancreatic fluid. We isolated and analyzed the coding region of the gene for the rat secretin precursor. The entire coding region spans 692 base pairs and is divided into four regions corresponding to the signal peptide and NH2-terminal peptide, the secretin peptide and processing signal sequences, a part of the COOH-terminal peptide, and the remainder of the COOH-terminal peptide, which are interrupted by three short introns (81, 105, and 104 base pairs). The organization is similar to those of the genes for other members of the secretin family, glucagon and VIP/PHI-27 precursors, supporting the assumption that the genes for the secretin family peptide precursors originated from a common ancestral gene. We also demonstrated that the secretin precursor gene is widely expressed in the brain and in the hypophysis. The regional expression pattern of the secretin precursor gene in the brain is quite different from those of the glucagon and VIP/PHI-27 precursor genes. The secretin precursor gene is highly expressed in the medulla oblongata and pons of the brain and the hypophysis, the expression levels of which are comparable to those in the duodenum. The secretin precursor mRNA in the brain and the hypophysis has the same coding sequence as that in the duodenum, indicating that secretin in the brain and the hypophysis is produced from the same secretin precursor protein as that in the duodenum. This is the first evidence to be reported that the secretin precursor gene is definitely expressed in the brain.     

Isolation and nucleotide sequence of a cDNA clone encoding rat mitochondrial malate dehydrogenase.

We have determined the complete sequence of the rat mitochondrial malate dehydrogenase (mMDH) precursor derived from nucleotide sequence of the cDNA. A single synthetic oligodeoxynucleotide probe was used to screen a rat atrial cDNA library constructed in lambda gt10. A 1.2 kb full-length cDNA clone provided the first complete amino acid sequence of pre-mMDH. The 1014 nucleotide-long open reading frame encodes the 314 residue long mature mMDH protein and a 24 amino acid NH2-terminal extension which directs mitochondrial import and is cleaved from the precursor after import to generate mature mMDH. The amino acid composition of the transit peptide is polar and basic. The pre-mMDH transit peptide shows marked homology with those of two other enzymes targeted to the rat mitochondrial matrix.     

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.     

Structural specificity of peptides influencing neuronal survival during development

Vasoactive intestinal peptide (VIP) has been shown to increase the survival of developing neurons grown in dissociated spinal cord cultures. This result was evident when synaptic activity was blocked with tetrodotoxin (TTX) during a critical period of development (days 7-21 after plating). Other neuropeptides, with a close sequence homology to VIP, have now been tested for their effects on neuronal survival in culture. Within the critical period, the survival of spinal cord neurons was significantly decreased (30-35%) after incubation with 1 nM peptide histidyl-isoleucine amide (PHI-27) or 0.1 nM growth hormone releasing factor (GRF). Neuronal cell death produced by these peptides did not exceed that observed from tetrodotoxin treatment alone. Secretin had no detectable effect on neuronal survival at any of the concentrations tested. In tetrodotoxin-treated cultures, PHI-27 and GRF prevented the neuronal cell death produced by TTX, but only at concentrations greater than 0.1 microM. In contrast, VIP significantly increased neuronal survival at concentrations less than 0.01 nM. The presence of 0.1 nM PHI-27 significantly decreased the effectiveness of VIP in preventing TTX-mediated neuronal cell death. Addition of PHI-27 or VIP, with or without TTX, to one month-old cultures produced no significant change in the number of neurons compared to control cultures. These studies indicate that the survival-promoting effect of VIP is highly structure-dependent and that this action appears to be confined to a critical period of development.     

Sequence of the precursor to rat ornithine aminotransferase deduced from a cDNA clone

The nucleotide sequence of ornithine aminotransferase mRNA from rat liver, including the entire coding and 3' untranslated regions, was determined from two overlapping cDNA clones. The mRNA encodes a precursor polypeptide of 439 amino acid residues with a molecular weight of 48,332. The deduced amino acid composition of the proposed mature enzyme sequence (residues 35 through 439) was in good agreement with that reported for the purified protein. The amino-terminal segment of the precursor corresponding to residues 1 through 34 has an overall positive charge, containing 6 basic residues and only a single acidic residue, and is postulated to be the mitochondrial leader sequence. The first 22 amino acid residues of the proposed leader sequences share 54% homology with the leader peptide of rat ornithine transcarbamylase precursor and more limited homology to the leader peptides of other nuclear-encoded mitochondrial matrix proteins. Homology was also observed between residues 286 through 362 ornithine aminotransferase precursor and a region containing the pyridoxyl phosphate binding domain of mitochondrial aspartate aminotransferase.     

Molecular cloning and DNA sequence analysis of the human guanine nucleotide-binding protein Go alpha

The nucleotide sequence of human Go alpha was determined from a partial human brain cDNA clone and the sequence of the first two 5' coding exons of a human genomic Go alpha clone. Comparison of this sequence with bovine and rat Go alpha shows greater than 90% homology at the nucleotide and deduced amino acid level. There is 100% identity at the amino acid level for the cholera and pertussis toxin-catalyzed ADP ribosylation sites, the putative guanine nucleotide binding, and the GTP hydrolysis sites.     

Amino acid sequence homology between rat alpha-fetoprotein and albumin at the COOH-terminal regions

The nucleotide sequence of the 3'-terminal untranslated region and a portion of the coding region of rat alpha-fetoprotein mRNA has been determined from a cloned double-stranded cDNA. the amino acid sequence of the COOH-terminal portion of alpha-fetoprotein was inferred from the nucleotide sequence and compared to the amino acid sequence of the corresponding portion of rat, bovine, and human albumin. A striking homology in amino acid sequence between alpha-fetoprotein and albumin was observed. These results confirm earlier suggestions that the two proteins are closely related in structure and probably arose from a common ancestral gene.     

Cloning and sequencing of the cDNA for human monocyte chemotactic and activating factor (MCAF)

cDNA clones having a nucleotide sequence encoding a human monocyte chemotactic and activating factor (MCAF) were isolated and sequenced. The amino acid sequence deduced from the nucleotide sequence reveals the primary structure of the MCAF precursor to be composed of a putative signal peptide sequence of 23 amino acid residues and a mature MCAF sequence of 76 amino acid residues. The amino acid sequence of MCAF showed 25-55% homology with other members of an inducible cytokine family, including macrophage inflammatory protein and some putative polypeptide mediators known as JE, LD78, RANTES and TCA-3. This suggests that MCAF is a member of family of factors involved in immune and inflammatory responses.     

Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.     

Chemical characterization of vasoactive intestinal polypeptide-like immunoreactivity in ovine hypothalamus and intestine.

Two forms of pituitary adenylate cyclase activating polypeptides with 38 (PACAP38) and 27 residues (PACAP27) respectively were recently isolated from ovine hypothalamic tissues. The N-terminal 28 amino acids sequence of PACAP was found to have 68% homology with porcine vasoactive intestinal peptide (VIP). In order to determine whether the primary structure of VIP of ovine hypothalamus is identical with porcine VIP or similar to PACAP, VIP immunoreactivity as determined by radioimmunoassay for porcine VIP was isolated in a pure form from ovine hypothalamic extracts. VIP was also isolated from ovine intestine. Amino acid analysis as well as amino acid sequence analysis showed that ovine hypothalamic and intestinal VIP were identical to porcine VIP, but different from PACAP.     

Production of VIP- and PHM (human PHI)-related peptides in human neuroblastoma cells

We demonstrated the production and release of a peptide structurally identical with porcine and bovine VIP-28 in human neuroblastoma NB-OK-1 cell line. In the cells, VIP-like immunoreactive (IR-VIP) components of 8 K dalton (Kd), 11 Kd, 18 Kd and 30 Kd were also detected and the 8 Kd and 18 Kd components were apparently released into the culture medium, indicating the possibility of less extended or limited processing of the VIP precursor in the cultured cells of tumor origin. The cells were also shown to produce, simultaneously with the VIP-28, a PHI/PHM-like immunoreactive (IR-PHI/PHM) component which coeluted with synthetic PHM-27, not PHI-27, in reverse-phase high performance liquid chromatography (HPLC). In addition to the PHM-27-like component, another IR-PHI/PHM component was detected in the cell extract which eluted in HPLC immediately before synthetic PHM-27 and crossreacted with PHI-27 amino-terminal specific antiserum but not with PHI-27 central-portion specific or PHM-27 carboxyl-terminal specific antiserum. The presence in NB-OK-1 cells of this IR-PHI/PHM component related to the amino-terminal portion of PHI/PHM suggested possible alternative(s) of post-translational processing of the VIP precursor in the cells in terms of the production of PHM-27-related peptides.     

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.     

Rat pre-prosomatostatin. Structure and processing by microsomal membranes

The tetradecapeptide hormone somatostatin arises from proteolytic processing of a large precursor, pre-prosomatostatin. Studies of other hormone precursors predict that the NH2 terminus of pre-prosomatostatin comprises a leader, or signal, region which is cleaved during its translation. Such co-translational cleavage would generate prosomatostatin. In these studies, we present the complete sequence of rat pre-prosomatostatin, deduced from the nucleotide sequence of cDNAs derived from a somatostatin-rich medullary thyroid carcinoma. These findings indicate that rat pre-prosomatostatin contains 116 amino acids (12,737 daltons). Cell-free translations of medullary thyroid carcinoma mRNA with dog pancreas microsomal membranes were performed to identify the cleavage point of the leader region from prosomatostatin. Partial microsequencing data indicates that the cleavage occurs between the glycine and alanine at positions 24 and 25 of pre-prosomatostatin. Thus, rat prosomatostatin contains 92 amino acids (10,388 daltons). Comparison of the amino acid sequences of the rat and human pre-prosomatostatins reveals only four amino acid substitutions. In view of the high degree of homology between rat and human pre-prosomatostatin, we expect a similar cleavage site and NH2-terminal structure for human prosomatostatin. The high level of conservation between rodents and humans of the entire pre-prosomatostatin molecule further suggests the possibility of biologic functions of the NH2-terminal portions of prosomatostatin.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Primary structure of rat liver D-beta-hydroxybutyrate dehydrogenase from cDNA and protein analyses: a short-chain alcohol dehydrogenase.

The amino acid sequence of D-beta-hydroxybutyrate dehydrogenase (BDH), a phosphatidyl-choline-dependent enzyme, has been determined for the enzyme from rat liver by a combination of nucleotide sequencing of cDNA clones and amino acid sequencing of the purified protein. This represents the first report of the primary structure of this enzyme. The largest clone contained 1435 base pairs and encoded the entire amino acid sequence of mature BDH and the leader peptide of precursor BDH. Hybridization of poly(A+) rat liver mRNA revealed two bands with estimated sizes of 3.2 and 1.7 kb. A computer-based comparison of the amino acid sequence of BDH with other reported sequences reveals a homology with the superfamily of short-chain alcohol dehydrogenases, which are distinct from the classical zinc-dependent alcohol dehydrogenases. This protein family, initially discerned from Drosophila alcohol dehydrogenase and bacterial ribitol dehydrogenase, is now known to include at least 20 enzymes catalyzing oxidations of distinct substrates.     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

Prothymosin alpha and parathymosin: amino acid sequences deduced from the cloned rat spleen cDNAs

A rat spleen cDNA library was screened for clones carrying the cDNAs for prothymosin alpha and parathymosin. Sequence analysis of a clone carrying the entire coding region for prothymosin alpha confirmed and completed the amino acid sequence for this polypeptide and established the number of amino acid residues as 111. Rat prothymosin alpha differs from human prothymosin alpha at six positions, including four substitutions and two insertions. The nucleotide sequences of the cDNAs for the rat and human polypeptides are more than 90% identical in the open reading frames, with significant homology extending into the 5' and 3' flanking regions. From the same library, we also isolated a clone carrying 80% of the coding region for rat parathymosin. The number of amino acid residues in rat parathymosin is 101, based on the sequence deduced from the cDNA insert and earlier information on the sequence in the amino-terminal portion of this polypeptide. Despite their similarity in size and amino acid composition, rat prothymosin alpha and rat parathymosin show only limited sequence homology, primarily in the segment including residues 14 through 25, where 10 of 12 positions are identical in the two polypeptides. this is also the region of significant sequence similarity to a 12-amino-acid segment in the p17 protein of the human immunodeficiency disease associated virus (HTLV-IIIB).     

Cloning and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.