Structure-activity relationships of chromogranin A in cell adhesion. Identification of an adhesion site for fibroblasts and smooth muscle cells.

Previous studies showed that chromogranin A (CgA), a glycoprotein stored and co-released with various hormones by neuroendocrine cells and neurons, can modulate cell adhesion. We have investigated the structure-activity relationships of CgA using fibroblasts and coronary artery smooth muscle cells in adhesion assays. A recombinant CgA fragment 1-78 and a peptide 7-57 containing reduced and alkylated cysteines (Cys(17) and Cys(38)) induced cell adhesion after adsorption onto solid phases at 50-100 nm. Peptides lacking the disulfide loop region, including residues 47-68, 39-59, and 39-68, induced cell adhesion, either bound to solid phases at 200-400 nm or added to the liquid phase at 5-10 microm, whereas peptide 60-68 was inactive, suggesting that residues 47-57 are important for activity. The effect of CgA-(1-78) was blocked by anti-CgA antibodies against epitopes including residues Arg(53), His(54), and Leu(57). Substitutions of residues His(54), Gln(55), and Asn(56) with alanine decreased the cell adhesion activity of peptide 47-68. These results suggest that the region 47-57 (RILSILRHQNL) contains a cell adhesion site and that the disulfide bridge is not necessary for the proadhesive activity. The ability of soluble peptides to elicit proadhesive effects suggests an indirect mechanism. The high sequence conservation and accessibility to antibodies suggest that this region is important for the physiological role of CgA.

Combinatorial synthesis of highly selective cyclohexapeptides for separation of amino Acid enantiomers by capillary electrophoresis.

Cyclic peptide libraries dissolved in the electrolyte solution can be used as chiral selectors in capillary electrophoresis. In the present investigation, the resolution obtained in capillary electrophoresis for a set of dinitrophenyl-d,l-amino acids was the parameter used to screen for the most effective selectors contained in a mixture of thousands of components of a cyclic hexapeptide sublibrary with three randomized positions. The deconvolution procedure was simplified by fractionating the sublibrary components according to the hydrophobicity of the amino acids in the randomized positions through reversed-phase HPLC. By comparing the resolution obtained with the separated fractions, a set of hydrophobic amino acids was recognized as essential to achieve adequate enantioselectivity. The whole deconvolution process, which made it possible to select two highly selective cyclopeptides, required the synthesis and the evaluation of 15 sublibraries instead of the 54 syntheses required by a classical procedure of serial deconvolution.

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41.

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577-599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization.

Role of the exon 11 of the insulin receptor gene on insulin binding identified by anti-peptide antibodies.

The insulin receptor exists in two isoforms differing by the absence (HIR-A) or presence (HIR-B) of 12 amino acids in the C-terminus of the alpha-subunit as a consequence of alternative splicing of exon 11. It was shown that the two isoforms exhibit different binding affinities for insulin, thus suggesting that the sequence encoded by exon 11 may be important for insulin binding. To further investigate this issue, we generated polyclonal antibodies against C-terminal peptides of the two HIR alpha-subunit variants. Herein, we characterized two antibodies, PA-11 and PA-12, directed against the C-terminus or the N-terminus of the sequence encoded by exon 11, respectively, and one (PA-13) directed against a sequence in the carboxy-terminal region of the alpha-subunit which is common to HIR-A and HIR-B. Antibodies were characterized for their ability to immunoprecipitate the receptor and to inhibit [125I]insulin binding to both isoforms. We found that PA-13 immunoprecipitates both the HIR-A and the HIR-B, PA-12 immunoprecipitates exclusively the HIR-B, and PA-11 fails to precipitate both isoforms. Interestingly, PA-12 inhibits specifically insulin to the HIR-B, whereas other PAs fail to affect insulin binding to either isoforms. Furthermore, PA-12 linearises the Scatchard plot of binding data, and retards the dissociation rate of insulin, thus suggesting that antibody affects cooperative interactions among binding sites. We conclude that the sequence encoded by exon 11 may play a role in modulating the binding of insulin to the receptor and negative cooperativity.

The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways.

Two forms of cytochrome b5 are present in rat tissues, with a sequence identity of approximately 60% in the cytoplasmically exposed, tryptic fragments (Lederer, F., Ghrir, R., Guiard, B., Cortial, S., and Ito, A. (1983) Eur. J. Biochem. 132, 95-102). It has been suggested that the two isoforms have partially overlapping subcellular distributions, with each form localized to some extent on both endoplasmic reticulum and outer mitochondrial membranes (Ito, A. (1980) J. Biochem. (Tokyo) 87, 73-80). To investigate the degree of specificity of the localization of cytochrome b5 isoforms, we studied their subcellular distributions with antipeptide antibodies, one specific for microsomal cytochrome b5, one specific for outer membrane cytochrome b, and one against a sequence common to the two cytochromes. We first identified outer membrane Cyt b as a tightly bound, Triton X-114-extractable, 23-kDa polypeptide. We then analyzed biochemically characterized rat liver subcellular fractions by Western blotting and found that outer mitochondrial membrane cytochrome b was not present on endoplasmic reticulum membranes. Conversely, microsomal cytochrome b5 was present on outer mitochondrial membranes in extremely low concentration, at a level < 5% of that on endoplasmic reticulum membranes. Thus, the subcellular distribution of microsomal cytochrome b5 is more restricted than previously thought, suggesting that novel posttranslational targeting mechanisms direct it to the endoplasmic reticulum.

Anti-peptide specific antibodies for the characterization of different alpha subunits of alpha-bungarotoxin binding acetylcholine receptors present in chick optic lobe.

Chick optic lobe express alpha-Bungarotoxin receptors. We have recently purified these receptors which, when reconstituted in a lipid bilayer, behave as functional acetylcholine gated channels. In order to characterize this purified preparation, we raised polyclonal antibodies against peptides obtained from the putative cytoplasmic domain between the hydrophobic sequence M3 and M4 of two previously cloned alpha-Bungarotoxin receptor subunits, alpha 7 and alpha 8. Both antibodies recognized the receptors present in the membrane extract and in the purified preparation, although the amount of the alpha-Bungarotoxin receptors precipitated by the two antibodies was quantitatively different. In Western blots of both purified and membrane-bound receptors, these antibodies specifically reacted with an M(r) 57000-55000 band. A study was also undertaken to quantify the receptors containing these subunits in different chick brain areas; it was found that the number of these subunits, as well as their ratio, was similar in all the tested areas. Furthermore, the alpha-Bungarotoxin receptors were present in at least two subtypes, one containing only the alpha 7 subunit and the other both alpha 7 and alpha 8 subunits.

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin.

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.

Opioid peptides in micellar systems: conformational analysis by CD and by one-dimensional and two-dimensional 1H-NMR spectroscopy.

beta-Endorphin has been studied in SDS micelles by one- and two-dimensional nmr spectroscopy (1D and 2D nmr), and to explore the influence of peptide length and composition on the polypeptide structure, the investigation was extended to a number of fragments. The nmr results are compared with those obtained from CD experiments and discussed in terms of a secondary structure that involves the central region of beta-endorphin.

Bovine seminal plasma contains a low-molecular-weight factor that inhibits RNA synthesis.

A low-MW factor (800-1000 daltons) extracted from bovine seminal plasma (bSP) and partially purified by a five-step fractionation is very active in inhibiting RNA synthesis by E. coli RNA polymerase with calf thymus DNA as template (70% inhibition at factor:DNA ratio of about 1:100). The same factor also inhibits RNA synthesis in isolated liver nuclei but to a lesser extent. The bSP factor probably exerts its inhibitory activity on initiation rather than on the elongation processes. DNA heat denaturation experiments indicate that the factor stabilizes double-stranded DNA. The activity of bSP factor is almost destroyed by protease (pronase) digestion. Trypsin digestion is ineffective. Consequently, peptide integrity seems to be important for the biological activity. The factor is heat stable and does not contain nucleic acid components. Preliminary analysis indicates the presence of acidic amino acids with no basic or aromatic ones and that the active factor is not a product of histone or protamine degradation. When injected i.p. into 25-day-old female rats, the bSP factor has an inhibinlike activity.

A non-steroidal gametic factor linked to DNA modulates delta-aminolevulinic acid synthase inducibility acting on liver transcriptional and translational processes.

The effect of an acidic factor of low molecular weight (about 1,000 daltons), extracted from bovine spermatozoan DNA, on the inducibility of delta-aminolevulinic acid synthase by ethanol during aging in rat has been examined. The increased enzyme inducibility in 600-day old rats is supported by stimulation of transcriptional and translational processes; on the contrary, in 30-day old rats, the higher enzyme values induced by ethanol are significantly decreased after factor treatment. The active factor is strongly DNA-bound in the native spermatozoan DNA. This would imply a possible role in regulating gene expression in vivo.

Effect of a nonsteroidal gametic factor on senile cataract in the dog.

We have studied the clarifying activity of a purified peptide fraction of gametic origin in senile cataract of the dog. The active substance, administered intramuscularly, had significant and durable clarifying effects on the cortical area of the lens.

Activity of a purified nonsteroidal gametic factor on the inducibility of hepatic delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase in senescent rats.

The effect of a water soluble nonsteroidal factor extracted from the male gamete on the activity of certain liver inducible enzymes during aging has been examined. Three enzymes have been studied: delta-aminolevulinic acid synthetase, NADPH-oxidase and tyrosine aminotransferase whose inducibility by ethanol, phenobarbital and ACTH, respectively, show age dependent alterations. The results here reported show that this factor is able to restore the enzyme inducibility in the liver of aging (600-day-old) rats without affecting the response of young (40-day-old) rats. Since the enzyme inducibility is altered during aging, and in the majority of rat hepatomas this factor might enter, possibly, in the regulation of enzyme activity also of neoplastic cells.

Effect of the inhibin-like factor from bull spermatozoa on sexual maturation of the male rat.

The s.c. administration for 25 days of the inhibin-like factor extracted from bull spermatozoa to immature 20-day-old male rats reduced serum FSH levels by about 60% and serum LH and testosterone levels by about 50%. However, neither tubular nor interstitial modifications of the histological appearance of the testis were found and there were no changes in the activity of delta 5-3 beta-hydroxysteroid dehydrogenase. The assay described could be usefully employed for evaluating the activities of inhibin-like factors.