The nucleotide sequence of rat liver glycogen phosphorylase cDNA.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase/oxidoreductase)

A human liver cDNA expression library in lambda-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase/oxidoreductase (EC 5.3.4.1/1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3'-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Characterization of a cDNA for human glutathione-insulin transhydrogenase (protein-disulfide isomerase / oxidoreductase)

A human liver cDNA expression library in λ-phage gt11 was screened with monoclonal antibodies to rat liver protein-disulfide isomerase / oxidoreductase (EC 5.3.4.1 / 1.8.4.2), also known as glutathione-insulin transhydrogenase (GIT). The nucleotide sequence of the largest cDNA insert (hgit-1) was determined. It contained approx. 1500 basepairs, representing an estimated 65% of the glutathione-insulin transhydrogenase message. The amino-acid sequence deduced from this cDNA insert contains a 7-amino-acid long polypeptide determined by sequencing the active-site fragment isolated from the rat GIT protein. A comparison of the nucleotide sequence of hgit-1 and a previously reported nucleotide sequence of rat glutathione-insulin transhydrogenase cDNA shows that the human hgit-1 clone corresponds to the middle of the transhydrogenase message at amino-acid residue number 275 of the rat protein, and codes for 206 amino-acid residues, including one of the two active-site regions of glutathione-insulin transhydrogenase, a stop codon (TAA), a long 3′-noncoding region of over 800 bases, a polyadenylation signal (AATAA), and a 29 base poly(A) tail. There exists high homology between the human and rat enzymes (94% in the overall amino-acid sequence, with 100% in the active site region and 81% in the nucleotide sequence within the coding portion of hgit-1). As with the rat enzyme, the human enzyme shows some identity with another dithiol-disulfide-exchange protein, Escherichia coli thioredoxin. Like rat cDNA, the human hgit-1 cDNA hybridized to rat mRNA of 2500 bases on a Northern blot. The relative quantitative abundance of GIT mRNA in nine rat tissues studied using hgit-1 as a hybridization probe was found to be in the same order as previously found with the rat cDNA. Thus, the above studies indicate that glutathione-insulin transhydrogenase is a highly conserved protein and that the human hgit-1 cDNA is suitable for use as a probe for further studies on gene regulation of this enzyme.     

Purification and properties of recombinant rat catalase produced in Escherichia coli

Catalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.     

Purification, characterization, and biological compartmentalization of rat fetal antigen 1

This study has established the rat as an animal model for the analysis of the biological role of fetal antigen 1 (FA1), a protein previously described in humans and mice. FA1 was purified from rat amniotic fluid by immunospecific affinity chromatography. Immunochemical identity between mouse and rat FA1 was established by crossed tandem immunoelectrophoresis. Molecular size was analyzed by mass spectrometry (33 kDa). The amino acid composition was determined, and the amino acid sequence was analyzed. The overall amino acid composition and sequence of the 28 first N-terminal amino acids were identical to the corresponding parts of rat preadipocyte factor 1 and rat adrenal zona glomerulosa protein. Extensive sequence similarity was found between rat and mouse FA1 (86%) and between rat and human FA1 (82%). The concentration of FA1 in fetal serum, maternal serum, urine, and amniotic fluid in rats was determined using an ELISA. The highest concentrations were found in fetal serum and amniotic fluid around Day 18 of pregnancy. This is the first report on the physicochemical characteristics and compartmentalization of rat FA1.     

Amino acid sequence homologies between rabbit, rat, and human serum retinol-binding proteins

The main transporting protein for vitamin A in rabbit serum, the retinol-binding protein (RBP), was isolated and its amino acid sequence determined. Rabbit RBP was found to be highly homologous to human RBP, whose amino acid sequence was elucidated earlier, and to rat RBP. The rat RBP sequence was obtained by combining information deduced from the nucleotide sequences of two overlapping cDNA clones with the NH2-terminal sequence of the isolated protein determined by automated Edman degradation. The identity between the three proteins is approximately 90%. The high degree of homology between RBP molecules from different species is probably explained by the fact that RBP participates in at least three types of molecular interactions: in the binding of prealbumin, in the interaction with retinol, and in the recognition of a specific cell surface receptor. All these interactions should lead to a conservation of RBP structure. The amino acid differences between rabbit, rat, and human RBP are discussed in light of the recent elucidation of the three-dimensional structure of human RBP. Hybridization of a probe isolated from a rat RBP cDNA clone to restriction enzyme-digested genomic DNA from rat and mouse suggests that RBP is encoded by a single gene.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

N-terminal amino-acid sequence of pig kidney dipeptidyl peptidase IV solubilized by autolysis.

The N-terminal amino-acid sequence of the intrinsic membrane protein dipeptidyl peptidase IV (DP IV) was determined. The protein was isolated from pig kidney and solubilized by autolysis at pH 3.8. The first 34 amino acids were sequenced and indicated approximately 78% identity to the N-terminal sequence of rat liver DP IV.     

The nucleolar protein, B-36, contains a glycine and dimethylarginine-rich sequence conserved in several other nuclear RNA-binding proteins

The amino terminal sequence of the 34 kD nucleolar protein B-36 isolated from the slime mold Physarum polycephalum has been determined. This portion of B-36 is rich in glycine, phenylalanine and the modified amino acid asymmetrical dimethylarginine (DMA) and is 65% identical to that for fibrillarin, a similar and potentially homologous 34 kD nucleolar protein from rat. The terminus of B-36 contains an interesting nine amino acid sequence, Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly, which is precisely repeated three times in the 110 kD nucleolar protein nucleolin. Similar sequences have also been reported in a yeast nucleolar protein (SSB-1) and several hnRNP proteins (rat A1 and brine shrimp GRP33). The conserved nature of this unusual sequence is suggestive of an important function which may include RNA-binding since several of these proteins share this feature.     

Amino acid sequence of rat thymus histone H2B and identification of the in vitro phosphorylation sites.

The amino acid sequence of rat thymus histone obtained in highly purified form by preparative electrophoresis, was determined. This sequence is identical to the sequence of calf thymus histone H2B. The in vitro phosphorylation of the rat histone with a cyclic AMP-dependent protein kinase isolated from rat pancreas led to the identification of four sites of phosphorylation: two major ones, at serine residues 32 and 36, and two minor ones, specific of the rat protein kinase, at serine residues 87 and 91.     

Amino acid sequence homology between rat and human C-reactive protein.

The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], 'phosphoryl choline-binding protein' [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP.     

Acyl-CoA-binding protein in the rat. Purification, binding characteristics, tissue concentrations and amino acid sequence.

Acyl-CoA-binding protein (ACBP) was purified from rat liver. The Mr was determined as 9932 +/- 10 by mass spectrometry and calculated as 9937.8 from the sequence. The protein binds acyl-CoA esters (C8-C16) with high affinity, but was unable to bind fatty acids. ACBP was found mainly (86%) in the soluble fraction, and the concentration was highest in liver, 5-6 micrograms/mg of soluble protein. The complete primary structure was determined by a combination of gas-phase Edman degradations and mass spectrometry. Extensive use of 252Cf plasma-desorption mass spectrometry facilitated the identification and verification of peptides. Comparison with the previously determined sequence of bovine acyl-CoA-binding protein revealed a very strong sequence similarity (83%), and all of the differences could be accounted for by single base changes.     

Alpha B-crystallin in skeletal muscle: purification and localization.

Atrophy of rat soleus muscles by hindlimb suspension is characterized by an early dramatic decrease in a soluble 22-kDa protein. The 22-kDa protein was purified from rat red skeletal muscle and rat lens by three different methods of chromatography. The partial amino acid sequence (65% of total amino acids) determined for muscle 22-kDa protein was identical with that of rat lens crystallin. The HPLC elution patterns of lysylendopeptidase fragments of 22-kDa protein from the two sources were identical. Polyclonal antibodies to rat muscle and bovine lens alpha B-crystallin with the two proteins on immunoblotting. alpha B-Crystallin protein was expressed and synthesized efficiently in slow skeletal muscle and poorly in fast muscle. Thus, the decreased 22-kDa protein of slow muscle in the suspension treatment was confirmed to be alpha B-crystallin. Immunoblotting confirmed that most of the alpha B-crystallin was solubilized, though some was tightly bound to myofibrils. This bound portion was localized in Z-bands of isolated myofibrils by immunocytochemical light and electron microscopy. Muscle alpha B-crystallin is tentatively proposed to be a myofibril-stabilizing protein, based upon its extraction characteristics, localization, and amino acid sequence.     

The sequence of a human mitochondrial glycerol-3-phosphate dehydrogenase-encoding cDNA

A 2618-bp cDNA that encodes the human mitochondrial glycerol-3-phosphate dehydrogenase has been isolated from a HeLa cell cDNA library and the nucleotide sequence determined. An open reading frame encodes a protein of 727 amino acids that is 96% similar to the rat protein and, like the rat protein, contains sites homologous to the Ca²⁺ -binding sites of calmodulin, as well as FAD- and putative glycerol-phosphate-binding sites.     

Amino acid sequence of rat liver cathepsin L

The complete amino acid sequences of the heavy and light chains of rat liver cathepsin L (EC 3.4.22.15) were determined at the protein level. The heavy and light chains consisted of 175 and 44 amino acid residues, respectively, and their Mr values without glycosyl groups calculated from these sequences were 18941 and 5056, respectively. The amino acid sequence was also determined from the N-terminal sequences of the heavy and light chains, and the sequences of cleavage fragments of the heavy chain with lysylendopeptidase and cyanogen bromide. The fragments were aligned by comparison with the amino acid sequence deduced from the sequence of cDNA of rat preprocathepsin L. The sequence of rat liver cathepsin L determined at the protein level was identical with that deduced from the cDNA sequence except that in the heavy chain, residues 176-177 (Asp-Ser) were not present at the C-terminus and alanine was replaced by proline at residue 125. Asn-108 in the heavy chain is modified with carbohydrate.     

Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families

The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.     

Isolation and characterization of the human tyrosine aminotransferase gene.

Structure and sequence of the human gene for tyrosine aminotransferase (TAT) was determined by analysis of cDNA and genomic clones. The gene extends over 10.9 kbl and consists of 12 exons giving rise to a 2,754 nucleotide long mRNA (excluding the poly(A)tail). The human TAT gene is predicted to code for a 454 amino acid protein of molecular weight 50,399 dalton. The overall sequence identity within the coding region of the human and the previously characterized rat TAT genes is 87% at the nucleotide and 92% at the protein level. A minor human TAT mRNA results from the use of an alternative polyadenylation signal in the 3' exon which is present but not used at the corresponding position in the rat TAT gene. The non-coding region of the 3' exon contains a complete Alu element which is absent in the rat TAT gene but present in apes and old world monkeys. Two functional glucocorticoid response elements (GREs) reside 2.5 kb upstream of the rat TAT gene. The DNA sequence of the corresponding region of the human TAT gene shows the distal GRE mutated and the proximal GRE replaced by Alu elements.     

Identification of the cDNA clone which encodes the 58-kDa protein containing the amino acid sequence of rat liver non-specific lipid-transfer protein (sterol-carrier protein 2). Homology with rat peroxisomal and mitochondrial 3-oxoacyl-CoA thiolases

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein cross-reactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.     

Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in lambda gtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. However, the quantity of nucleophosmin mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, when compared with normal rat liver. Dot blot analysis also showed a nucleophosmin mRNA ratio of 64:5:1 in the three types of rat liver. When the protein levels were compared with Western blot immunoassays, Novikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver.