Primary structure of human thromboxane synthase determined from the cDNA sequence.

Polymerase chain reaction techniques have been used to isolate a cDNA clone containing the entire protein coding region of thromboxane A2 synthase (EC 5.3.99.5) from a human lung cDNA library. The cDNA clone hybridizes with a single 2.1-kilobase mRNA species in phorbol ester-induced human erythroleukemia and monocytic leukemia cell lines. A second cDNA, differing only by an insert of 163 base pairs near the 3'-end of the translated region, was also found to be present in the same library. The proteins predicted from both nucleic acid sequences include the three polypeptide sequences determined from amino acid sequencing of the purified human platelet enzyme, five potential sites for N-glycosylation, and a hydrophobic region that may serve to anchor the synthase in the endoplasmic reticulum membrane. The longer predicted protein, designated thromboxane synthase-I, contains 534 amino acids, with a Mr of 60,684, whereas the shorter protein, designated thromboxane synthase-II, contains 460 amino acids and has a Mr of 52,408. Although thromboxane synthase-II lacks the conserved cysteine that serves as the proximal heme ligand in the other cytochromes, significant sequence similarities exist among thromboxane synthase-I and -II and several P450s, particularly those in family 3. The overall amino acid identity is considerably less than 40%, making it likely that thromboxane synthase represents a previously undefined family of cytochrome P450.     

Thromboxane A2 synthesis in human erythroleukemia cells

Human erythroleukemia cells transformed arachidonic acid and prostaglandin endoperoxide H2 into thromboxane A2. Stimulation of these cells with A23187 or thrombin, however, produced no thromboxane. Similarly, cells labeled with [3H]-arachidonic acid released no detectable label upon stimulation. Data suggest that human erythroleukemia cells contain the enzymatic capacity for thromboxane formation from exogenous precursors, but lack the endogenous mechanisms for arachidonate release. The presence of thromboxane synthase messenger RNA was verified using the polymerase chain reaction. Amplification and sequence analysis of a 528 bp cDNA demonstrated virtually 100% identity to a published thromboxane synthase cDNA fragment.     

Abundant expression of thromboxane synthase in rat macrophages

The cloned cDNA for rat thromboxane (TX) synthase with a size of 1851 bp contained a 1599-bp open reading frame which encoded a 533-amino acid protein sharing 79.7% identity with human TX synthase. RNA blot analysis was carried out with rat cells and tissues. Rat peritoneal macrophages most abundantly expressed mRNA for TX synthase, and its level was not changed by in vivo stimulation of casein. Bone marrow, spleen, lung and thymus also expressed the TX synthase gene. These findings suggest the possibility that TXA₂ plays a role in the immune system.     

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and 15-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5,13-dienoic acid, suggesting that the inactivation occurred at the active site of the enzyme. The catalytic inactivation of thromboxane synthase and the greater rate of formation of HHT in thromboxane-synthesizing system may probably play important regulatory roles in the control of thromboxane synthesis.     

Cloning and expression of a cDNA for mouse prostaglandin E receptor EP3 subtype.

A functional cDNA clone for mouse EP3 subtype of prostaglandin (PG) E receptor was isolated from a mouse cDNA library using polymerase chain reaction based on the sequence of the human thromboxane A2 receptor and cross-hybridization screening. The mouse EP3 receptor consists of 365 amino acid residues with putative seven-transmembrane domains. The sequence revealed significant homology to the human thromboxane A2 receptor. Ligand binding studies using membranes of COS cells transfected with the cDNA revealed specific [3H]PGE2 binding. The binding was displaced with unlabeled PGs in the order of PGE2 = PGE1 greater than iloprost greater than PGF2 alpha greater than PGD2. The EP3-selective agonists, M&B 28,767 or GR 63799X, potently competed for the [3H]PGE2 binding, but no competition was found with EP1- or EP2-selective ligands. PGE2 and M&B 28,767 decreased forskolin-induced cAMP formation in a concentration-dependent manner in Chinese hamster ovary cells permanently expressing the cDNA. Northern blot analysis demonstrated that the EP3 mRNA is expressed abundantly in kidney, uterus, and mastocytoma P-815 cells and in a lesser amount in brain, thymus, lung, heart, stomach, and spleen.     

Differences in human laminin B2 sequences.

A cDNA clone encoding the B2 chain of laminin has been isolated from a human endothelial lambda gt11 cDNA library. The nucleotide and deduced amino acid sequences of the clone were determined and showed one amino acid substitution (Ser1519 instead of Asn) when compared to lung laminin B2 chain and one silent nucleotide change (G4200 instead of A) in relation to the human placenta laminin B2 chain. Other differences in the 3'-untranslated region were also found.     

A lung type prostaglandin F synthase is expressed in bovine liver: cDNA sequence and expression in E. coli.

Using the cDNA of bovine lung prostaglandin F synthase (EC 1.1.1.2) as a probe, we isolated a clone from a bovine liver cDNA library which differed in only eleven nucleotides from the probe. The corresponding protein contained three amino acid substitutions, including a leucine residue which is conserved throughout all aldo-keto reductases. We inserted the liver cDNA into expression vector pUC19 and expressed the recombinant liver enzyme in E.coli. The purified liver enzyme reduced prostaglandin H2 as well as prostaglandin D2 and various carbonyl compounds. The high relative activity against prostaglandin H2 in combination with a high Km value for prostaglandin D2 identified this liver enzyme as a lung type prostaglandin F synthase. However, the binding constant for NADPH of the liver enzyme was 3.5 fold higher than that of lung prostaglandin F synthase.     

Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.     

Sequence of a cDNA encoding mouse F1F0-ATP synthase g subunit.

A pregnant-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as a full-length cDNA encoding the F1F0-ATP synthase g subunit. Comparison of the deduced amino acid sequences of mouse ATP synthase g subunit with those of bovine species showed 86% identity. The high levels of ATP synthase g subunit mRNA were detected in heart and uterine tissues.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.     

Molecular cloning of gene sequences for avian fatty acid synthase and evidence for nutritional regulation of fatty acid synthase mRNA concentration

A double-stranded cDNA library was constructed using total poly(A)+ RNA from the goose uropygial gland. Clones containing sequences complementary to fatty acid synthase mRNA were initially identified by colony hybridization with a 32P-labeled cDNA transcribed from RNA enriched for fatty acid synthase mRNA. Identity of the fatty acid synthase clones was confirmed by hybrid-selected translation. Mature fatty acid synthase mRNA is approximately 16 kilobases in length. When unfed neonatal goslings were fed for 24 hr, relative synthesis of hepatic fatty acid synthase increased more than 42-fold. Concomitantly, hepatic fatty acid synthase mRNA levels increased 70-fold. Thus, nutritional regulation of the synthesis of hepatic fatty acid synthase probably occurs at the pretranslational level. The availability of a specific probe for fatty acid synthase mRNA should allow us to analyze the regulation of expression of this gene during development, by nutrition and by hormones in both liver and uropygial gland.     

Molecular cloning of the beta-subunit of human prolyl 4-hydroxylase. This subunit and protein disulphide isomerase are products of the same gene.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyses the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in peptide linkages. We report here the isolation of cDNA clones coding for the beta-subunit of prolyl 4-hydroxylase from a human hepatoma lambda gt11 library and a corresponding human placenta library. Five overlapping clones covering all the coding sequences and almost all the non-coding sequences were characterized. The size of the mRNA hybridizing with these clones in Northern blotting is approximately 2.5 kb. The clones encode a polypeptide of 508 amino acid residues, including a signal peptide of 17 amino acids. These human sequences were found to be very similar to those recently reported for rat protein disulphide isomerase (EC 5.3.4.1). The degree of homology between these two proteins was 84% at the level of nucleotide sequences or 94% at the level of amino acid sequences. Southern blot analyses of human genomic DNA with a cDNA probe for the beta-subunit indicated the presence of only one gene containing these sequences. The product of a single gene thus appears to possess two different enzymatic functions depending on whether it is present in cells in monomer form or in the prolyl 4-hydroxylase tetramer.     

Molecular cloning and expression in Escherichia coli of the cDNA coding for rat lipocortin I (calpactin II)

Lipocortins (LC) are a family of proteins that were initially described to be induced by glucocorticosteroids and to inhibit phospholipase A2 (PLA2). Using oligodeoxynucleotide probes corresponding to partial amino acid (aa) sequences of rat lipocortin I (LCI), we have isolated a cDNA clone for rat LCI from a cDNA library prepared from poly(A)+RNA of peritoneal cells of dexamethasone-treated rat. The cDNA insert (1355 bp) had an open reading frame of 1038 bp that encoded a 346-aa polypeptide (Mr 38,784). The nucleotide sequence and the amino acid sequence deduced from it showed high homology with the reported sequences of human LCI. A plasmid containing the trc promoter and cDNA sequence for 346 aa residues of the rat LCI was constructed and expressed in Escherichia coli. Antibody to human LCI crossreacted with the recombinant rat LCI, and the recombinant protein had characteristics of natural rat LCI including PLA2 inhibitory activity in vitro.     

Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction

The polymerase chain reaction was used to clone a full-length human methylmalonyl-CoA mutase cDNA from a human liver library by priming with sequences from the 5' end of a partial cDNA and sequences in the phage vector. The amino acid sequence predicted from the cDNA corresponds to the authentic amino acid sequences of peptide fragment from purified methylmalonyl-CoA mutase. The open reading frame of the cDNA encodes 742 amino acids (82,283 Da) comprising a 32 amino acid mitochondrial leader sequence and a mature protein of 710 amino acids (78,489 Da). The use of the polymerase chain reaction to "screen" the cDNA library represents a novel application of this technique. The full length will enable analysis of mutations underlying inherited methylmalonic acidemias caused by deficiency of the methylmalonyl-CoA mutase apoenzyme.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping

A cDNA coding for a P450 expressed in human lung was isolated from a lambda gt11 library constructed from human lung mRNA using a cDNA probe to rat P450 IVA1. The cDNA-deduced amino acid sequence of this P450, designated IVB1, consisted of 511 amino acids and had a calculated molecular weight of 59,558. The IVB1 amino acid sequence bore 51%, 53%, and 52% similarities to rat IVA1, IVA2, and rabbit P450p-2, respectively. Comparison of the primary amino acid sequence of human IVB1 with rat IVA and rabbit p-2 P450 sequences revealed a region of absolute sequence identity of 17 amino acids between residues 304 and 320. However, the functional significance of this conserved sequence is unknown. Human IVB1 also appears to be related to P450 isozyme 5 that has been extensively characterized in rabbits. The IVB1 cDNA was inserted into a vaccinia virus expression vector and the enzyme expressed in human cell lines. The expressed enzyme had an absorption spectrum with a lambda max at 450 nm when reduced and complexed with carbon monoxide, typical of other cytochrome P450s. Unlike rabbit P450 isozyme 5, however, human IVB1 was unable to activate the promutagen 2-aminofluorene. Human lung microsomal P450s were also unable to metabolize this compound despite the presence of IVB1 mRNA in three out of four human lungs analyzed. In contrast to its expression in lung, IVB1 mRNA was undetectable in livers from 14 individuals, including those from which the lungs were derived. IVB1-related mRNA was also expressed in rat lung and was undetectable in untreated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)