Biomedical subjects

J Spiess

Publications and source records attributed to J Spiess.

At least 19 recordsLinked to original sources

PubMed1992

Isoproterenol enhances a calcium-independent potassium current in mouse anterior pituitary tumor cells.

1. The patch-clamp technique was used to study the action of the beta-adrenergic agonist (-)-isoproterenol in anterior pituitary tumor cells of the mouse. 2. (-)-Isoproterenol induced an inward-rectifying potassium conductance with half-maximal stimulation at a concentration of approximately 67 nM. The isomer (+)-isoproterenol was less effective in stimulating the current. 3. The effect of (-)-isoproterenol was abolished by cholera toxin treatment, indicating the involvement of a Gs protein, whereas pertussis toxin treatment did not exhibit a current reduction. 4. We blocked or stimulated phosphorylation pathways in cells to test the involvement of adenosine 3',5'-cyclic monophosphate (cAMP). It was concluded that the current stimulation probably was not exclusively mediated by cAMP. 5. Activation of calcium-dependent potassium channels by an isoproterenol-induced calcium influx into the cell could be excluded. 6. Therefore it is suggested that the observed activation of a potassium current by isoproterenol could be directly mediated by a Gs protein.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Isolation and characterization of hypothalamic growth-hormone releasing factor from common carp, Cyprinus carpio.

A growth hormone-releasing factor (GRF)-like peptide was isolated from the hypothalamus of common carp, Cyprinus carpio, by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against rat GRF, and multiple steps of HPLC using octadecyl columns. Based on Edman degradation and peptide mapping, this teleost GRF was established to be a 45-residue peptide with the following primary structure: His-Ala-Asp-Gly-Met-Phe-Asn-Lys-Ala-Tyr-Arg-Lys-Ala-Leu-Gly-Gln-Leu-Ser- Ala-Arg - Lys-Tyr-Leu-His-Thr-Leu-Met-Ala-Lys-Arg-Val-Gly-Gly-Gly-Ser-Met-Ile-Glu- Asp-Asp-Asn-Glu-Pro-Leu-Ser. Carp GRF is closely related structurally to peptides of the glucagon-secretin superfamily, and more particularly to mammalian vasoactive intestinal peptide (VIP) precursors and the N-terminal portion of mammalian GRFs. A synthetic replicate of this peptide is highly potent [50% effective dose (ED50) approximately 0.08 nM] in stimulating GH release from cultured goldfish pituitary glands and in elevating serum GH levels 30 min after injection (0.1 micrograms/g) in goldfish.

Amino Acid Sequence↗

PubMed1991

Primary structure and functional expression of a glutaminyl cyclase.

A glutaminyl cyclase (QC) that is probably involved in the biosynthesis of pyroglutamyl peptides such as gonadotropin-releasing hormone and thyrotropin-releasing hormone has been purified to homogeneity from bovine anterior pituitary. On the basis of N-terminal sequence analysis, a 2088-base-pair cDNA clone was isolated from a bovine anterior pituitary library. From the nucleotide sequence of this clone, the primary structure of a 330-residue protein and a preceding 31-residue prepropeptide sequence was deduced. By transfection of COS-7 monkey cells with a QC cDNA/pCDM8 vector construct, QC activity was expressed. Hybridization with mRNAs of various bovine tissues revealed expression of QC mainly in brain tissue.

Acyltransferases↗

PubMed1991

Improved method for identification of proteins using two-dimensional electrophoresis with immobilized pH gradient isoelectric focusing.

Recent advances in protein sequence analysis now permit the determination of partial N-terminal and internal primary structure from low picomole quantities of protein. The major remaining hurdles to sequence analysis of small amounts of protein are the identification, isolation, and handling of microgram and submicrogram quantities of protein. The technique of two-dimensional electrophoresis using immobilized pH gradient isoelectric focusing circumvents many of these problems. However, poor correlation between the first and second dimension have prevented use of this technique for the identification of some proteins which can only be assayed prior to the denaturing conditions used in the second dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis procedure. An improved method is presented which allows correlation of the native biological activity (first dimension) to a silver stained protein (second dimension) with a high degree of confidence.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1991

Primary structure and tissue distribution of anglerfish carboxypeptidase H.

Most peptide hormones are synthesized as part of larger precursor proteins which must be processed after translation to generate bioactive peptides. This usually involves cleavage of the precursor by an endopeptidase at sites marked by basic amino acids, followed by removal of N- or C-terminal basic residues by the action of an aminopeptidase or carboxypeptidase. These processing events have been observed in a variety of species, from yeast to mammals. As part of an effort to characterize prohormone processing enzymes in the anglerfish, Lophius americanus, we have cloned and sequenced a cDNA for the fish prohormone processing carboxypeptidase H (CPH). Polyadenylated RNA from anglerfish (AF) islet organs was used to construct a cDNA library in phage lambda gt11. The library was screened with a probe derived from the cDNA for rat CPH. A 2400 base pair AF cDNA clone was isolated. This cDNA encodes a polypeptide which is similar in size and composition to mammalian CPH. The sequence data indicate that the AF CPH precursor is a 454 amino acid polypeptide. The derived amino acid sequence of the putative fish CPH is 81% homologous to the rat and bovine CPH enzymes. Significantly, all of the amino acid residues thought to be important for metal ion and substrate binding, glycosylation, and catalytic activity of mammalian CPH are conserved in the fish enzyme. Northern hybridization using RNA from AF tissues indicates that a 2.5 kb fish CPH mRNA is expressed in brain, pituitary and islet organs, but not in other tissues which do not secrete peptide hormones.

Amino Acid Sequence↗

PubMed1991

The anglerfish somatostatin-28-generating propeptide converting enzyme is an aspartyl protease.

An enzyme that performs the conversion of anglerfish prosomatostatin-II (pro-SS-II) to anglerfish SS-28 has been identified using an improved two-dimensional electrophoresis procedure. The enzyme is a single chain 39 kDa polypeptide with its isoelectric point at pH 5.9. The converting enzyme has an acidic pH optimum, consistent with the lowered pH of the intracellular site of propeptide conversion. Secretory granule extracts were examined to determine the inhibitor sensitivity and pH optimum of the conversion of anglerfish pro-SS-II to anglerfish SS-28 in this organelle. Production of anglerfish SS-28 by secretory granules was maximal at pH 4.2 and was completely inhibited by the addition of pepstatin. Since pepstatin is a specific inhibitor of aspartyl proteases, these results indicate that the purified enzyme is a member of this enzyme family. This conclusion was supported by the data from partial amino acid sequence analysis. Because these results are consistent with the role of the purified enzyme in the in vivo production of anglerfish SS-28, the identified aspartyl protease has been termed the anglerfish SS-28-generating propeptide-converting enzyme.

Animals↗

PubMed1991

Identification of a somatostatin-14-generating propeptide converting enzyme as a member of the kex2/furin/PC family.

A propeptide converting enzyme capable of producing somatostatin-14 has been identified and partially characterized from anglerfish pancreatic islets. Results from N-terminal protein sequence analysis indicate that this enzyme is a member of the kex2/furin/PC family. This observation provides strong corroborative evidence that the kex2/furin/PC protease family is involved in propeptide conversion. Comparison of the obtained protein sequence with the cDNA sequence of mammalian PC2 also suggests that the active enzyme is derived from a precursor by cleavage at a site containing four consecutive basic amino acids.

Amino Acid Sequence↗

PubMed1990

Purification of prosomatostatin-converting enzymes.

The enzymes responsible for performing cleavage of propeptides at basic amino acids have proven difficult to characterize. Using the processing of anglerfish islet prosomatostatin (PSS) as a model system, we are pursuing the characterization of both a single basic amino acid-specific and a dibasic amino acid-specific converting enzyme. We describe here the model system and protein isolation methods that have allowed significant progress toward complete characterization of the somatostatin-generating propeptide converting enzymes (PCEs).

Animals↗

PubMed1990

Mechanism of action of somatostatin: an overview of receptor function and studies of the molecular characterization and purification of somatostatin receptor proteins.

To determine whether somatostatin receptor subtypes arise from molecular heterogeneity of the receptor protein, we have cross-linked the putative receptor in normal rat tissues and in AtT-20 and GH3 cells, both chemically with SS-14, SS-28 and Tyr3 SMS ligands, as well as by photoaffinity labeling with an azido derivative of Tyr3 SMS (EE 581). Three prominent somatostatin receptor proteins of 58-kDa, 32-kDa, and 27-kDa size have been identified. These proteins exhibit a tissue-specific distribution, ligand selectivity, and relative preference for SS-14 and SS-28 binding, and thus qualify as somatostatin receptor subtypes. Using EE 581 as a photoaffinity probe, the 58-kDa and 32-kDa proteins have been purified to homogeneity from brain and AtT-20 cells by successive SDS-PAGE. The 58-kDa form has been trypsinized and amino acid sequence data obtained from four tryptic fragments. With the help of synthetic oligonucleotides derived from these sequences, work is currently in progress to clone the 58-kDa protein to elucidate its complete sequence, its expression, and its functional relationship to the somatostatin receptor and its pharmacological subtypes.

Amino Acid Sequence↗

PubMed1989

Thyrotropin-releasing hormone (TRH) precursor processing. Characterization of mature TRH and non-TRH peptides synthesized by transfected mammalian cells.

Prepro-thyrotropin-releasing hormone (TRH) contains five TRH progenitor sequences and at least six other potential peptides (Lechan, R. M., Wu, P., Jackson, I. M. D., Wolf, H., Cooperman, S., Mandel, G., and Goodman, R. H. (1986a) Science 231, 159-161). Previous studies using radioimmunoassays developed against discrete regions of prepro-TRH have demonstrated that several of the potential peptides are present in rat brain and pancreas (Wu, P., Lechan, R. M., and Jackson, I. M. D. (1987) Endocrinology 121, 108-115; Wu, P. and Jackson, I. M. D. (1988a) Brain Res. 456, 22-28; Wu, P., and Jackson, I. M. D. (1988b) Regul. Pept. 22, 347-360). However, the low level of peptides present in intact tissues has made isolation of the peptides difficult. CA77 cells, a medullary thyroid carcinoma cell line, also express prepro-TRH and display processing similar to that found in tissues. However, peptide content in this tumor cell line is enhanced only 3-fold compared with normal tissues (Sevarino, K. A., Wu, P., Jackson, I. M. D., Roos, B. A., Mandel, G., and Goodman, R. H. (1988) J. Biol. Chem. 263, 620-623). To achieve higher levels of expression for facilitating peptide sequencing studies and to see if alternate processing of prepro-TRH could be detected in different cell types, we transfected into 3T3, GH4, AtT20, and RIN 5F cells a cDNA vector under control of the cytomegalovirus immediate-early promoter. 3T3 and GH4 cells failed to process prepro-TRH beyond cleavage of the signal sequence. Both AtT20 and RIN 5F cells efficiently cleaved the precursor at dibasic sites to generate mature TRH and the non-TRH peptides previously identified in vivo. Peptide content was up to 30 times greater than in hypothalamic extracts and 10 times greater than in CA77 cells. Secretion experiments with transfected AtT20 cells demonstrated that both mature TRH and the non-TRH peptides were secreted via a regulated secretory pathway similar to that utilized by endogenously synthesized peptides. We isolated several of the non-TRH peptides synthesized by transfected AtT20 cells and characterized these peptides by sequential Edman degradation. These studies identified the signal sequence cleavage site and determined that the non-TRH peptides are generated by cleavage at the dibasic sites flanking the five TRH progenitor sequences. Further, we determined that processing occurs at the Arg51-Arg52 site located in the amino-terminal portion of the precursor, the only dibasic site not flanking a TRH progenitor sequence.

Amino Acid Sequence↗

PubMed1988

Molecular characterization and chromosomal mapping of melanoma growth stimulatory activity, a growth factor structurally related to beta-thromboglobulin.

Melanoma growth stimulatory activity (MGSA) is a mitogenic polypeptide secreted by Hs294T human melanoma cells. Comparison of the N-terminal sequences of the 13 and 16 kd MGSA species with the cDNA sequence revealed that the mature form of human MGSA is maximally 73 amino acids long. Expression of the cDNA in mammalian cells results in the secretion of this peptide with mitogenic activity. MGSA is structurally related to the platelet-derived beta-thromboglobulin and to several other polypeptides. These factors may constitute a family of growth factors. MGSA mRNA was detected in a variety of cell types. The level of MGSA mRNA in melanoma cells is strongly elevated by treatment with MGSA. MGSA is the gene product of a recently detected gene gro. The gene was mapped to chromosome 4 (region q13----q21). This same region also contains genes for two of the structurally related factors, for c-kit, a receptor for an as yet unidentified ligand, and for 'piebald trait', an inherited skin pigmentation disorder.

Amino Acid Sequence↗

PubMed1988

Amino acid sequence of the BSC-1 cell growth inhibitor (polyergin) deduced from the nucleotide sequence of the cDNA.

The complete amino acid sequence of the BSC-1 cell growth inhibitor, including its precursor polypeptide, is reported. The sequence was deduced from the nucleotide sequence of the cDNA. The N-terminal amino acid sequence of the mature bioactive BSC-1 cell growth inhibitor is identical with the N-terminal sequences of the factors that have been called type beta 2 transforming growth factor and cartilage-inducing factor B, suggesting that these are identical. The complete amino acid sequence of the mature BSC-1 cell growth inhibitor differs from that of human type beta transforming growth factor in 32 of the 112 amino acids. Polyergin is proposed as the name for the BSC-1 cell growth inhibitor.

Amino Acid Sequence↗

PubMed1988

Characterization of neurophysin in the anterior pituitary gland of sheep.

Portions of sheep anterior pituitary lobe tissue were extracted under acid conditions and assayed for the two neurophysins (oN-III and oN-I/II) by radioimmunoassay. In all tissues examined, oN-III and oN-I/II immunoreactivities were detected. Using a combination of isoelectric focusing and polyacrylamide gel electrophoresis, the neurophysins of the anterior pituitary gland behaved like oN-III and oN-I/II. oN-III of the anterior pituitary was purified by affinity chromatography and high-performance liquid chromatography. This corticotroph oN possessed an amino acid analysis similar to that of oN-III and an N-terminal amino acid sequence, including residues 1-24, identical to that of authentic oN-III. These findings support the work of others who have identified neurohypophysial hormones in the anterior pituitary gland.

Amino Acid Sequence↗

PubMed1987

Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides.

Extracts from bovine pituitary were found to contain an activity catalyzing the conversion of glutaminyl peptides such as [Gln1]gonadotropin-releasing hormone, [Gln1, Gly4]thyrotropin-releasing hormone (H-Gln-His-Pro-Gly-OH), and H-Gln-Tyr-Ala-OH to the respective pyroglutamyl peptides. The TRH precursor fragment H-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-OH and the D-glutaminyl stereoisomer of H-Gln-Tyr-Ala-OH did not react under the same conditions. The conversion products were identified by Edman degradation, amino acid analysis, and reversed-phase HPLC. That this activity was exhibited by an enzyme, glutaminyl cyclase, was concluded from the protein character of the activity (revealed by its abolition with trypsin and heat), the Michaelis-Menten relationship between substrate concentration and conversion rate, and the substrate specificity. It was determined that glutaminyl cyclase had a molecular weight of 43,000-50,000, a pH optimum at pH 8, and Km and Vmax values in the range of 60-130 microM and 390-690 pmol/microgram per hr, respectively. Glutaminyl cyclase was not observed to require ATP and could be inhibited with 1.0 M ammonium chloride, which increased the Km and decreased the Vmax value. The subcellular distribution of glutaminyl cyclase corresponded to the one of peptidylglycine alpha-amidating monooxygenase believed to catalyze C-terminal amidations during posttranslational precursor processing. It was also observed that the formation of pyroglutamyl from glutaminyl peptides occurred nonenzymatically; however, the enzymatic reaction carried out with crude extract was found to be approximately 70 times faster than the nonenzymatic reaction enhanced by phosphate. It is speculated that glutaminyl cyclase may participate in the posttranslational processing of hormonal precursors to pyroglutamyl peptides.

Acyltransferases↗

PubMed1986

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding.

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1986

Isolation and sequence analysis of oxytocin from the sheep corpus luteum.

Studies have revealed that the corpus luteum (CL) of the sheep releases oxytocin (OT) -like immunoreactivity under normal and physiological conditions. We have now purified and completely characterized the OT-like species from ovine CL and established by Edman degradation and comparative reverse-phase HPLC its identity with hypothalamic oxytocin. On the basis of radioimmunoassay, the characterized oxytocin was the only peptide possessing OT-like immunoreactivity. This study represents the first identification by sequence analysis of oxytocin outside the central nervous system.

Amino Acid Sequence↗

PubMed1986

Inhibin A-subunit cDNAs from porcine ovary and human placenta.

Inhibin, a gonadal protein that preferentially suppresses the secretion of pituitary follicle-stimulating hormone, has been isolated from porcine follicular fluid and characterized as a 32-kDa protein composed of 18-kDa and 14-kDa subunits. In the present work, oligonucleotide probes predicted from amino-terminal inhibin amino acid sequences have been used to isolate, from a porcine ovarian lambda gt11 cDNA library, clones encoding the 18-kDa subunit, or A chain, of inhibin. DNA sequence analysis showed that the inhibin A chain is initially synthesized as a larger precursor protein and is predicted to be a glycopeptide. Inhibin A-chain mRNA is present specifically in the gonads, and its synthesis can be induced by treatment of the animal with gonadotropins. The porcine probe was used to isolate a human inhibin A-subunit cDNA from a placental cDNA library. The human precursor is highly homologous to its porcine counterpart and is predicted to generate an 18-kDa glycosylated inhibin A subunit.

Amino Acid Sequence↗

PubMed1986

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue.

Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96-residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS-14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS-14 from aPSS-I.

Amino Acid Sequence↗