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PubMed1984

Isolation and characterization of the two giant secretory proteins in salivary gland of Chironomus tentans.

The two giant secretory proteins, sp-Ia and sp-Ib, in salivary-gland cells of the larva of the fly Chironomus tentans, were isolated by preparative gel electrophoresis and characterized chemically. Their amino acid compositions are dominated by polar amino acids, with about 30% of basic amino acid residues. Crossed immunoelectrophoresis of sp-Ia and sp-Ib provided evidence that they share antigenic determinants. They also have major methionine-containing tryptic peptides in common. CNBr cleavage of sp-Ib gives a small number of low-Mr fragments, indicating that this protein has a repetitive structure.

Amino Acids↗
PubMed1994

cDNA cloning and amino acid sequence of human mitochondrial delta 3 delta 2-enoyl-CoA isomerase: comparison of the human enzyme with its rat counterpart, mitochondrial short-chain isomerase.

We report the isolation of a cDNA encoding a mature human monofunctional delta 3 delta 2-enoyl-CoA isomerase and the determination of its nucleotide sequence. The purified uncleaved protein, as well as several internal tryptic and CNBr fragments, were subjected to N-terminal peptide sequencing. The deduced amino acid sequence of the mature protein consists of 260 amino acids with a predicted M(r) of 28735. The human mitochondrial isomerase exhibits a 74% (78%) sequence identity with the corresponding rat counterpart at amino acid (nucleotide) level(s). Many basic amino acid residues in rat isomerase have been changed to acidic or neutral residues in human enzyme, explaining the differences observed between these proteins.

Amino Acid Sequence↗
PubMed1994

Nuclear accumulation of interferon gamma.

Examination of the interferon gamma (IFN-gamma) amino acid sequence revealed two conserved basic amino acid clusters similar to the prototype nuclear localization signal. We followed the fate of cell surface receptor-bound IFN-gamma in murine leukemia L1210 cells. A time- and temperature-dependent accumulation of murine IFN-gamma in the cell nucleus could be demonstrated by autoradiography and indirect immunofluorescence after the rapid isolation of nuclei. Human IFN-gamma was also internalized and translocated to the nucleus of murine L1210 cells transfected with and expressing the human IFN-gamma receptor, but it appeared to be retained by the nucleus only transiently. IFN-gamma molecules chemically crosslinked to their cell surface receptor remain capable of being translocated to the nucleus even as part of a receptor-ligand complex. Thus, the bipartite nuclear localization signal sequence appears to be functional and suggests that nuclear targeting could participate in IFN-gamma signal transduction.

Animals↗
PubMed1995

Characterization of the mitotic specific phosphorylation site of histone H1. Absence of a consensus sequence for the p34cdc2/cyclin B kinase.

32P-Labeled histone H1 was isolated from synchronized Chinese hamster (line CHO) cells, subjected to tryptic digestion, and fractionated into 15 phosphopeptides by high performance liquid chromatography. These phosphopeptides were grouped into five classes having different cell cycle phosphorylation kinetics: 1) peptides reaching a maximum phosphorylation rate in S and then declining in G2 and M, 2) peptides reaching a maximum phosphorylation rate in G2 and then remaining constant or declining in M, 3) peptides with increasing phosphorylation throughout S and G2 and reaching a maximum in M, 4) one peptide that was phosphorylated only in M, and 5) peptides that had low levels of phosphorylation that remained constant throughout the cell cycle. Amino acid analysis and sequencing demonstrated that the mitotic specific H1 phosphopeptide was the 16-amino acid, N-terminal, tryptic peptide Ac-SETAPAAPAAAPPAEK of the H1-1 class. This peptide, which is phosphorylated on both the Ser and Thr, does not contain the consensus sequence (S/T)PXZ (where X is any amino acid and Z is a basic amino acid). This sequence is thought to be required by the p34cdc2/cyclin B kinase that has maximum phosphorylating activity in mitosis. These data indicate that this kinase either does not have an obligatory requirement for the consensus sequence in vivo as generally believed or that it is not the enzyme responsible for the mitotic specific H1 phosphorylation.

Amino Acid Sequence↗
PubMed1996

Identification of a region of Bacillus subtilis Ffh, a homologue of mammalian SRP54 protein, that is essential for binding to small cytoplasmic RNA.

Bacillus subtilis Ffh and scRNA are homologues of mammalian SRP54 and SRP RNA, respectively, which are components of the eukaryotic signal recognition particle (SRP). Ffh (446 amino acids) interacts with scRNA to form a stable complex in vivo. Here, we identified an RNA-binding domain of Ffh. The results obtained using a series of deletion mutants show that amino acid positions 364 to 432 in the C-terminal region of Ffh correlates with its ability to bind RNA. The amino acid sequence of this region is well conserved among members of the SRP54 protein family. This sequence contains two hydrophobic regions (h2, 364 to 391, and h3, 416 to 435), separated by the positively charged amino acid motif, 398RRKRIAKGSG407. Among the basic amino acid residues in this region, Arg-401 was essential for binding to scRNA, but Arg-399 and Lys-400 were not. The co-existence of Arg-398 and Lys-404 was necessary for the same affinity as wild type Ffh. The two glycine residues of the 405GSG407 were also essential. MH23 peptide (91 amino acids) encompassing from 356 to 446, consisting of h2-RRKRIAKGSG-h3, bound scRNA with the same affinity as wild type Ffh, whereas a 24-amino acid synthetic peptide 392DIINASRRKRIAKGSGTSVQEVNR415 did not. The region containing two hydrophobic segments separated by the positively charged motif is the minimal requirement of Ffh for RNA binding.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2. Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2.

The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.B., and Datta, K. (1991) Eur. J. Cell Biol. 56, 58-67). Here, we report the isolation of a partial cDNA clone from a lambda gt11 cDNA expression library of human skin fibroblast by immuno-screening with HA-binding protein antiserum. The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone. Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A.R., Mayeda, A., Kozak, D., and Binns, G. (1991) Cell 66, 383-394). Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins. As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography. The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein. The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e. two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid. All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid-binding protein family, the "hyaladherins."

Amino Acid Sequence↗
PubMed1981

Induction of lysine imbalance in rats: relation to competition for lysine transport into the brain in vitro.

The ability of amino acids inhibitory of lysine transport into brain slices to induce lysine imbalance was determined by feeding wheat gluten or casein diets with additions of such amino acids. Lysine transport was only moderately inhibited by amino acids; the most effective were basic amino acids or mixtures of indispensable (IAA) or branched chain amino acid (BCAA). Only mild depressions in growth and food intake occurred during a 10-day period when male, 60--65 g rats of the Sprague-Dawley strain were fed lysine-limiting, 18% wheat gluten diets with additions of these amino acids. The effects were prevented by added lysine. Rats allowed a choice between the lysine-imbalanced or non-protein diets selected the imbalanced, wheat gluten diets (in severe imbalances rats will choose the non-protein diet). Growth depression, prevented by added lysine, occurred in rats fed a 6% casein diet supplemented with IAA; individual amino acids were ineffective. Growth depressions also occurred when rats were fed a basal diet containing 6% case in + 5% of an equimolar mixture of nine IAA and supplemented with arginine or more IAA; BCAA were less effective. Additional lysine completely prevented the growth depressions, but growth of rats fed the diets containing arginine and BCAA was greater than that of those fed the extra IAA. It is difficult to induce a severe lysine imbalance; this is consistent with the failure of amino acids to cause under our conditions strong inhibition of lysine transport into brain.

Amino Acids↗
PubMed1977

Purification and characterization of active component and active fragment of colicin E3.

1. Two components of colicin E3, namely proteins A and B, were prepared by means of an improved method. 2. Protein A thus obtained was more than a thousand times as active as native colicin E3 when they were assayed in terms of activity for ribosome inactivation. 3. Protein A was reconstituted to colicin E3 simply by mixing with protein B. 4. Trypsin digestion of colicin E3 yielded two fragments, T1 and T2, probably by cleaving one specific bond of the A moiety of colicin E3. 5. T2 was a complex of T2A and B proteins. T2A showed an activity equivalent to that of protein A when assayed in the in vitro system, and its activity was neutralized by protein B. Thus T2A was assigned as an active fragment of protein A. 6. T2A has a characteristic amino acid composition rich in the basic amino acid, lysine. 7. The structure and function of the colicin E3 molecule is discussed based on the results obtained with its components as well as with fragments of the components.

Amino Acids↗
PubMed1992

Structure of the genes encoding the rod-core linker polypeptides of Mastigocladus laminosus phycobilisomes and functional aspects of the phycobiliprotein/linker-polypeptide interactions.

The 3' portion of the cpc operon in Mastigocladus laminosus encloses the genes 5'-cpcF-cpcG1-cpcG2-cpcG3 3'. The three cpcG genes encode different phycocyanin-associated rod-core linker polypeptides of the phycobilisomes with predicted 279, 247 and 254 amino acids in length. The gene products CpcG show a high similarity at their N-terminal domains (190 amino acids) and an overall identity of 47-53% to one another. Each of the three CpcG polypeptides is highly related to one of the four CpcG gene products of Anabaena sp. PCC 7120 (66-81% identity). It is suggested that these pairs of rod-core linker polypeptides mediate the same specific type of phycocyanin----allophycocyanin interaction in the similar phycobilisomes of M. laminosus and Anabaena sp. PCC 7120. The similarity of the CpcG1, CpcG2 and CpcG3 polypeptides to the single CpcG rod-core linker polypeptide of Synechococcus sp. PCC 7002 (36-41% identity) is lower. The rod-core linker polypeptides are more distantly related to the rod linker polypeptides associated with phycocyanin or phycoerythrin. However, six conserved domains were identified within the N-terminal 190 amino acids of these linker proteins, which bear similar amino acid sequences, including highly conserved basic amino acids. A similar amino acid sequence but with conserved acidic amino acids can be found in the beta subunits of phycocyanin, phycoerythrin and phycoerythrocyanin, which is protruding into the central cavity of the phycobiliprotein hexamers. It is suggested that these domains are sites of phycobiliprotein-hexamer/rod and rod-core linker interactions.

Amino Acid Sequence↗
PubMed1983

Evidence that pituitary cation-sensitive neutral endopeptidase is a multicatalytic protease complex.

Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.

Animals↗
PubMed1997

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.

Acetobacter↗
PubMed1983

Effects of carrier ampholyte contamination on the biological and biochemical properties of streptococcal pyrogenic exotoxin type C.

Three streptococcal pyrogenic exotoxins (SPEs), designated as SPE A, B, and C, have been purified and characterized. Routine purification of the SPEs includes the technique of isoelectric focusing. An earlier study showed that the removal of commercial carrier ampholytes (Ampholines) from SPE was difficult. The physiochemical properties of SPE C were previously reported; however, the SPE C preparation used in those experiments was contaminated with Ampholines. As an alternative to Ampholines, we used simple buffers to generate the isoelectric focusing pH gradient and used this SPE C in a comparative study to evaluate the effects of Ampholine contamination on the biological and biochemical properties of this toxin. We found that Ampholine contamination overestimates protein concentration; consequently, the biological activity of SPE C was actually greater than reported. The most serious effect of Ampholines in SPE C was on amino acid analysis. The presence of Ampholines causes an apparent increase in neutral amino acids and a decrease in basic amino acids.

Amino Acids↗
PubMed1992

LSA: a new liver-specific antigen in the rat. I. Purification and characterization.

A liver-specific antigen (LSA) was purified to homogeneity from rat liver by conventional methods of protein chemistry. By consecutive 100,000 g centrifugation, ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-200, ion-exchange chromatography on CM-cellulose and affinity chromatography on concanavalin-Sepharose, it has been possible to isolate a preparation that migrated as a single band on SDS-PAGE. This preparation gave a complete identity pattern with the original crude rat liver extract when tested by double immunodiffusion. This antigen has a molecular weight of 72.5 kD with an electrophoretic mobility in the region of alpha 2-globulins. The LSA proved to be thermolabile since exposure to 55 degrees C completely destroyed the antigen. Exposure of the LSA to different pH ranging from 4 to 10 had no detrimental effect on its antigenic activity. The amino acid composition of the LSA revealed that the acidic amino acids out-number the basic amino acids, with glutamic acid being the most abundant of them. Failure of beta-mercaptoethanol to split the LSA molecule suggests the absence of sulfhydryl groups related to its antigenic activity. Subcellular fractionation of rat liver revealed most of the antigenic activity in the 100,000 g supernate, i.e. the soluble cytoplasmic fraction of the liver (cytosol). By contrast, the LSA was absent from isolated Kupffer cells from rat liver. The absence of any carbohydrate or lipid from the purified preparation of this antigen, in conjunction with the destructive effects of trypsin suggest that the LSA is a protein or a moiety closely associated with proteins.

Amino Acids↗
PubMed2002

[Comparison of sequences of the hemagglutinin gene and phylogenetical analysis of H9 subtype avian influenza viruses isolated from some regions in China].

In order to explore the genetic mutaions of the hemagglutinin(HA) gene and the law of molecular epidemiology of H9 subtype avian influenza viruses in China, 23 H9 subtype avian influenza viruses(AIVs) were isolated from 12 provinces of China in recent years. Their nucleotide sequences of cDNA of HA gene were determined by RT-PCR and sequencing. Their nucleotide and putaive amino acid sequences homology was compared. The results showed that their nucleotide sequence homology was from 94.1% to 100% and that amino acid sequence homology was 95.4% to 100%. The sequences of the HA gene of these isolates were analyzed and compared with that of another 8 isolates from reference. The similedty indicated that HK170499 isolated from Hong Kong was close to the 2 isolates of Japan. And of the 31 isolates with complete HA gene sequences there were 5 isolates, HA gene of which were loss of one potelltial glycosylation site, which were CKGS199, CKTJ196, CKT296, CKSH300 and CKBJ197. Then 1098 nucleotide regions (bases 55 to 1,152) of HA gene of 23 isolates in this study were analyzed phylogenetically and compared with sequences from 31 H9 subtype viruses available in the GenBank database. Although considerable variation at the cleavage sites of the different viruses was observed, giving 10 different amino acid motits, none had multiple basic amino acids that correlate with highly pathogenic avian influenza (HPAI) isolates. Examination of amino acid sequences involved in repeptor binding site(RBS) revealed that the amino acid residue at position 191 characteristically distributed in the 54 isolates, that is, this amino acid residue of the isolates of mainland China and several Hong Kong strains was Asn(N) and that of the others was His(H). And the 141 143 amino acid residues, involved in forming the potential glycosylation sites, had the similary characteristic distribution with the 191aa position. The isolates with Asn-191. excluding CKBJ197, had NVS in the position 141aa143aa, meanwhile those with His-191 had NVT. Twenty-six mainland China isolates was genetically in Eurasian lineage but did not show distinctly geographical and temporal relationship. It is concluded that in recent years H9N2 subtype AIV, circulating in chicken flocks of mainland China, may have a common origini. These findings provides importan basis for establishment of scientifically preventive measurements to control H9 subtype avian influenza.

Amino Acid Sequence↗
PubMed1990

The 5'-terminal sequence of the hepatitis C virus genome.

The 5'-terminal sequence of the genome of hepatitis C virus (HCV) was determined for two distinct HCV strains in human and chimpanzee carriers. It had a 5'-noncoding region of at least 324 nucleotides, well preserved by the two strains with a high homology (99.1%), followed by 1348 nucleotides that continued to the documented sequence of prototype HCV spanning 7310 nucleotides (European Patent Application #88310922.5). Based on these results, HCV is considered to possess an uninterrupted open reading frame encoding at least 2886 amino acid residues. Two structural genes were postulated on the 5'-terminal sequence of the HCV genome. One gene in the upstream region, highly conserved by the two strains at the amino acid level and rich in basic amino acids such as arginine, appeared to encode the viral capsid protein. The other gene in the downstream region was divergent between the two strains at both nucleotide and amino acid levels. It coded for nine potential N-glycosylation sites, and was considered to encode the viral envelope protein. Disclosure of the 5'-terminal sequence of the HCV genome would facilitate its taxonomic classification, and contribute toward immunological diagnosis of infection and development of vaccines.

Amino Acid Sequence↗
PubMed1980

Purification and partial characterization of a preneoplastic antigen in liver carcinogenesis.

A preneoplastic antigen has been isolated from the microsomal fraction of the hyperplastic nodules of rat liver. During the purification, the presence of preneoplastic antigen was monitored by microcomplement fixation test and double immunodiffusion plate following reaction with antiserum specific for this antigen. The antigen was purified by seven steps with a purification factor of 167 and 11% yield. It migrated as a single protein band, both in nondenaturing and denaturing polyacrylamide gel electrophoresis; showed a single sharp peak in an isoelectric focusing column at pH 4.75; and had a single serine amino-terminal amino acid. In the absence of detergent during purification, this protein tends to form molecular aggregates. It appears to be a glycoprotein based on the positive periodic acid-Schiff stain and absorption by concanavalin A:Sepharose affinity column chromatography. Amino acid analysis indicated that the protein is acidic, having acidic amino acids (aspartate and glutamate) and basic amino acids (lysine, arginine, and histidine) in a ratio of 6.8. An estimate of the molecular weight by gel filtration gave a value of 145,000 and a calculated Stokes radius of 41.5 A. Electrophoresis under denaturing conditions on sodium dodecyl sulfate-polyacrylamide gel indicated that this protein is a dimer consisting of two subunits of equal molecular weight (M.W. 74,000). Circular dichroism spectra indicated alpha-helix contents at 298 and 222 nm of 25.5 and 28.3%, respectively. A similar protein is present in the endoplasmic reticulum of normal rat liver, but in a much lower concentration.

Amino Acids↗
PubMed2005

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

ADAM Proteins↗
PubMed1990

Rat pro-atrial natriuretic factor expression and post-translational processing in mouse corticotropic pituitary tumor cells.

Atrial natriuretic factor (ANF) is stored within atrial myocyte secretory granules as pro-ANF (ANF-(1-126] and is proteolytically processed co-secretionally C-terminal to a single basic amino acid to form ANF-(1-98) and the bioactive product ANF-(99-126). Pro-ANF is also expressed in certain non-cardiac neuroendocrine cell types (e.g. brain, adrenal). Although the relatively low levels of the peptide in these cell types have precluded detailed processing and secretion studies using cultured cells, some work with tissue extracts suggests that pro-ANF is pre-secretionally processed between or C-terminal to Arg101-Arg102 in such cells. In order to assess whether cultured non-cardiac endocrine cells process pro-ANF pre- or co-secretionally, and to establish whether both paired and single basic amino acids can serve as cleavage sites, transfection studies were carried out using the adrenocorticotropic hormone (ACTH)-producing pituitary tumor cell line AtT-20/D-16v. These cells normally cleave pro-ACTH/endorphin pre-secretionally at selected, but not all, pairs of basic amino acids to a variety of product peptides. A prepro-ANF expression plasmid was constructed and transfected into the AtT-20 cells. The resulting ANF/AtT-20 cell clone selected for this study expressed ACTH at levels similar to the untransfected wild type cells and secreted immunoreactive ANF-related material at a rate of approximately 1 fmol/min/10(5) cells, which was about 10% the rate of ACTH secretion. The rates of secretion of both ANF and ACTH could be increased 3-5-fold with a variety of known AtT-20 cell secretagogues including phorbol esters and the beta-adrenergic agonist, isoproterenol, thus indicating that both peptides were routed through regulated secretory pathways. Utilizing a combination of specific antisera directed against various regions of pro-ANF, size exclusion and reversed phase high performance liquid chromatography, and peptide mapping, it was shown that the ANF/AtT-20 cells contained and secreted the bioactive peptide ANF-(103-126) and -(1-97). These results indicate that the ANF/AtT-20 cells specifically cleave pro-ANF pre-secretionally at the same single basic site used by cardiac tissue; this single basic cleavage is apparently followed by removal of Arg98 by carboxypeptidase H. It is also apparent that the cells can cleave at the sole paired basic site in pro-ANF, which is the probable cleavage site used by neurons and some other endocrine cells that express low levels of the prohormone.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenocorticotropic Hormone↗