Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells.

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.

Gene and precursor structures of human C-type natriuretic peptide.

We have isolated the gene for human C-type natriuretic peptide (CNP) from a human genomic library using a cloned porcine CNP genomic DNA fragment as probe. Human CNP gene consists of at least two coding blocks and an intron, and encodes a 126-residue CNP precursor (human prepro-CNP). From a comparison of the amino acid sequences of porcine and rat prepro-CNPs, human prepro-CNP is found to be processed to generate 22-and 53-residue peptides (human CNP-22 and human CNP-53, respectively) as major endogenous CNPs in human. Interestingly, human CNP-53 has two amino acid substitutions as compared to the porcine and rat CNP-53s, whereas human CNP-22 is identical to the porcine and rat CNP-22s. Intravenous injection of human CNP-53 into anesthetized rats induces diuretic-natriuretic and hypotensive activities with same potencies as porcine CNP-53 does, although these activities were considerably lower (about 1/100) than those of human alpha-ANP.

Gene and precursor structure of porcine C-type natriuretic peptide.

Recently we isolated from porcine brain two related peptides, a 22-residue peptide (CNP-22) and its N-terminally elongated peptide (CNP-53; 53-residue), which belong to the third type of mammalian natriuretic peptide designated C-type natriuretic peptide family (CNP) (1,2). To elucidate the structure of their precursor form, we have now isolated the gene for this porcine CNP and prepared its cDNA from COS-1 cells transfected with the gene. Nucleotide sequence analyses have revealed that the gene consists of a least two exons and an intron and encodes the 126-residue CNP precursor (porcine prepro-CNP), in which a putative signal peptide and the CNP-53 sequence are located at the N- and C-terminus, respectively. The C-terminal cysteine codon of CNP-53 is directly followed by a termination codon, indicating that the C-terminus of porcine CNP is generated per se.

Primary structure of human microsomal dipeptidase deduced from molecular cloning.

Two cDNA clones corresponding to human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase (EC 3.4.13.11] were isolated from human placental and renal cDNA libraries employing rapid amplification of cDNA ends strategy. The complete amino acid sequence deduced from the cDNAs contains 411 residues, beginning with a signal peptide of 16 residues. A highly hydrophobic sequence of 16 amino acids is located at the carboxyl terminus, supporting the previous observation which suggested that mature MDP is anchored to the membrane by covalently attached phosphatidylinositol. MDP has four potential N-glycosylation sites and has no apparent sequence similarity to other metallopeptidases. Expression of immunologically cross-reactive and enzymatically active MDP was attained in COS cells transfected with the cDNA. DNA and RNA blot analyses indicated the existence of a single gene and a substantial amount of 1.8-kilobase mRNA in kidney.

Four species of cDNAs for cytochrome P450 isozymes immunorelated to P450C-M/F encode for members of P450IID subfamily, increasing the number of members within the subfamily.

A chicken antibody mono-specific to cytochrome P450C-M/F, which exists in untreated male and female rat liver and catalyses the 2- and 16 alpha-hydroxylation of estrogens (1), was used to screen a cDNA library of male Sprague-Dawley rat liver. Four cDNA clones which encoded P450 isozymes, CMF1a, CMF1b, CMF2 and CMF3, were isolated. CMF1a and CMF2 deduced consisted of 504 and 500 amino acid residues, respectively, while C-terminal 487 and 324 residues for CMF1b and CMF3, respectively, were deduced from the 5'-truncated cDNAs. The isozymes were more than 72% similar in amino acid sequences to each other and to rat P450db1, P450db2 (2), and to a mouse male specific C-P45016 alpha (3), suggesting that they belonged to a new P450 subfamily, P450IID. CMF1a and db1, and CMF2 and db2, respectively, were 99.2% and 99.0% similar in amino acid sequences, suggesting that they were virtually identical. CMF1a and CMF1b were different but 96.1% similar, and CMF3 was between 76% and 78% similar to other members of the rat P450IID family.

Inhibition of in vitro angiogenesis by lymphotoxin and interferon-gamma.

The effects of lymphotoxin (LT) and interferon (IFN)-gamma on the capillary formation was examined using an in vitro angiogenesis model system. Both LT and IFN-gamma inhibited the capillary formation in a dose dependent manner. To elucidate the mode of action, effects of the lymphokines on endothelial and myofibroblastic cells were studied. We found that the lymphokines inhibited not only the growth of endothelial cells but also the production of collagen by myofibroblastic cells. These results suggest that the pleiotropic effects of the lymphokines on different types of cells might result in the inhibition of the capillary formation.

Primary structure of nonspecific crossreacting antigen (NCA), a member of carcinoembryonic antigen (CEA) gene family, deduced from cDNA sequence.

A cDNA containing the entire coding region for a member of carcinoembryonic antigen (CEA) gene family has been cloned from cDNA library of HLC-1 cells by immunochemical screening with the antibody specific to nonspecific crossreacting antigen (NCA). The cDNA encodes a precursor form of a polypeptide consisting of a 34-residue signal sequence, a 108-residue N-terminal (N-) domain, a 178-residue domain (NCA-I domain) and a 24-residue domain rich in hydrophobic amino acids (M-domain). Each domain has a distinct but homologous amino acid sequence to that of the corresponding domain of CEA. Unlike the coding sequences, the 3'-untranslated sequences differ markedly in the NCA and CEA cDNAs facilitating the preparation of probes that will discriminate between nucleotide sequences for CEA and NCA.

Purification and characterization of a deoxyribonucleic acid dependent adenosinetriphosphatase from mouse FM3A cells: effects of ribonucleoside triphosphates on the interaction of the enzyme with single-stranded DNA.

There are at least four forms of DNA-dependent ATPase in mouse FM3A cells [Tawaragi, Y., Enomoto, T., Watanabe, Y., Hanaoka, F., & Yamada, M. (1984) Biochemistry 23, 529-533]. One of these, ATPase B, has been purified and characterized in detail. During the purification of the enzyme, we encountered the difficulties that the enzyme could not be recovered well from the single-stranded DNA-cellulose column and that the enzyme activity was distributed very broadly. The problems were resolved by the addition of ATP in the elution buffer. The ATPase has a sedimentation coefficient of 5.5 S in both high salt and low salt. The enzyme hydrolyzes rNTPs and dATP, but ATP and dATP are preferred substrates. Adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), 5'-adenylyl methylenediphosphate (AMP-PCP), and 5'-adenylyl imidodiphosphate (AMP-PNP) inhibit the enzyme activity. The enzyme is insensitive to ouabain, oligomycin, novobiocin, and ethidium bromide. A divalent cation (Mg2+ congruent to Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Poly(dT), single-stranded circular DNA, and heat-denatured DNA were very effective. Native DNA was little effective with an efficiency of 29% of that obtained with heat-denatured DNA. In addition, the enzyme showed almost no activity with poly(dA).poly(dT) although it showed very high activity with the noncomplementary combination of poly(dT) and poly(dC), suggesting that ATPase B requires single-stranded DNA for activity. ATP altered the affinity of ATPase B for single-stranded DNA. The interaction of the enzyme with DNA was studied by Sephadex G-200 gel filtration assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of dog and rabbit precursors of atrial natriuretic polypeptides deduced from nucleotide sequence of cloned cDNA.

The structure of precursors of dog and rabbit atrial natriuretic polypeptides was determined by nucleotide sequence analysis of cloned cDNA of mRNA encoding the peptides. The dog and rabbit precursors are 149 and 153 residues long having 23- and 25-residue putative signal peptides at their N-termini respectively. The 28-residue peptide with identical sequence to that of human, which has potent natriuretic activity, is located at the C-terminus of the dog precursor. The 28-residue peptide of identical sequence to that of mouse/rat is located at C-terminus of rabbit precursor followed by additional Arg-Arg sequence which is also found in rat/mouse precursors and is apparently removed during processing.

Identification of rat gamma atrial natriuretic polypeptide and characterization of the cDNA encoding its precursor.

Diuretic and smooth muscle-relaxing peptides, designated atrial natriuretic peptides (ANPs), have been identified in human and rat atrial tissues and implicated in the control of fluid volume and vascular function. Recently, cDNAs encoding the human and rat ANP precursors have been sequenced. We previously isolated from human tissue a natriuretic peptide of molecular weight (MW) 13,000 (gamma-hANP) comprising 126 amino acid residues, the largest natriuretic peptide so far identified, and showed that it is directly derived from the 151-residue human ANP precursor by the removal of a signal peptide. We now report the isolation and sequence analysis of a novel rat atrial natriuretic peptide (gamma-rANP) of MW 13,000, which derives from the rat ANP precursor. We also report the molecular cloning and nucleotide sequence analysis of the cDNA of the 152-residue rat ANP precursor, which is remarkably similar to the human 151-residue precursor (pre-hANP) except at the C-terminus. Differences in the rat and human precursor nucleotide sequences around the termination codons lead to a difference in processing pattern.

Effect of the strandedness of cofactor DNA on the activities of DNA-dependent ATPases B and C3 from FM3A cells.

The requirements of cofactor DNA for DNA-dependent ATPases B and C3 were analyzed in detail. ATPase B and C3 required the presence of a polynucleotide for their activities. Among the DNAs tested, ATPase B showed a preference for poly(dT) as its cofactor. The other deoxyhomopolymers, except poly(dG) and heat-denatured DNA also were effective. The alternating polydeoxyribonucleotide, poly[d(A-T)] had an efficiency 23% that of heat-denatured DNA. Unlike ATPase B, ATPase C3 showed almost no activity with deoxyhomopolymers. The most effective cofactor for ATPase C3 so far tested is poly[d(A-T)]. Relatively high activity was obtained with heat-denatured DNA. The high activity of ATPase B with poly(dT) was reduced by the addition of poly(dA). The addition of noncomplementary homopolymers did not affect enzyme activity. ATPase C3 activity in the presence of 10 microM poly(dT) increased gradually with concentrations of poly(dA) up to 20 microM, after which it decreased. Almost no increase in activity was observed when noncomplementary homopolymers were added. The relatively high activity of ATPase C3 with heat-denatured DNA was suggested by its high sensitivity to ethidium bromide to be due to the double-stranded region in the heat-denatured DNA formed by self-annealing.

Multiple deoxyribonucleic acid dependent adenosinetriphosphatases in FM3A cells. Characterization of an adenosinetriphosphatase that prefers poly [d(A-T)] as cofactor.

Four chromatographically distinct DNA-dependent ATPases, B, C1, C2, and C3, have been partially purified from mouse FM3A cell extracts. These ATPases are distinguished from each other by their physical and enzymological properties. DNA-dependent ATPases B, C1, C2, and C3 have sedimentation coefficients in 250 mM KCl of 5.5, 5.3, 7.3, and 3.4 S, respectively. ATPases B, C2, and C3 hydrolyze dATP as efficiently as ATP, whereas C1 does not. ATPase B hydrolyzes other ribonucleoside triphosphates with relatively high efficiency as compared to the other three enzymes. ATPase C3 prefers poly[d(A-T)] to poly(dT) as cofactor, whereas the other three enzymes prefer poly(dT) to poly[d(A-T)]. Among the four ATPases, ATPase C3 has been highly purified and characterized in detail. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction of ATPase C3 showed two major bands corresponding to molecular weights of 66 000 and 63 000. The Km values of the enzyme for ATP and dATP are 0.53 and 0.86 mM, respectively. As cofactor, poly[d(A-T)] is the most effective among the DNAs tested. Heat-denatured DNA and native DNA are also effective but used with less efficiency. Almost no or very little activity has been detected with ribohomopolymers and oligonucleotides. The activity attained with poly(dT) and poly(dA) is 11 and 6% of that with heat-denatured DNA, respectively. When both polymers were added at a molar ratio 1 to 1, very high activity was obtained with these polymers. On the other hand, little activity was observed by the combination of noncomplementary homopolymers such as poly(dT) and poly(dG).