Peptide Material Research Articles

The role of iodination in the formation of hormone-rich peptides from thyroglobulin.

Reduced thyroglobulins from several animal species contain hormone-rich iodopeptides of 20,000-26,000 and 15,000-18,000 daltons. The present study has investigated the role of iodination in their production. Experimental approaches have included: iodination in vitro of thyroglobulin from rabbit thyroid slices incubated with [3H]leucine and subsequent analysis of 3H distribution by gel electrophoresis; iodination in vitro of low iodine thyroglobulin from a human goiter, followed by isolation of the major iodopeptides and quantitation of their peptide content; and injection of iodine-deficient rats with Na125I and assessment of the distribution of isotope among the iodopeptides at successive time intervals. From these experiments the following general pattern has emerged: 1) at low levels of iodine (less than 5 atoms/molecule of thyroglobulin) in vitro or at short time intervals after iodine administration in vivo (less than 4 h), the principal iodinated component of reduced thyroglobulin is a approximately 230,000-dalton peptide; 2) with moderate increases in iodine (5-40 atoms/molecule) or longer time intervals after administration (greater than or equal to 4 h) there is less of the 230,000-dalton iodopeptide, and an iodothyronine-rich approximately 20,000- to 26,000-dalton iodopeptide appears; 3) at higher levels of iodine (greater than 40 atoms/molecule) the amount of approximately 230,000-dalton iodopeptide decreases further, the amount of 20,000- to 26,000-dalton iodopeptides may decrease, and an iodothyronine-rich 15,000- to 18,000-dalton peptide appears. Progressive iodination gives the same changes in distribution of peptide material among these iodopeptides as it does in iodine distribution, and the changes seen with iodination in vitro are similar to those occurring over time in vivo. We conclude that during the process of iodination discrete peptide bonds of thyroglobulin are cleaved to produce the hormone-rich iodopeptides. This is probably a normal part of thyroglobulin maturation in vivo and may be a necessary event preceding hormone formation.

Peptide factors as pharmaceuticals: criteria for application.

AbstractThe increasing interest in the pharmaceutical use of peptide factors in human medicine presents formidable challenges for peptide chemistry. Fully reproducible multistage syntheses with a definite assessment of the degree of purity represent the crucial premise for the introduction of peptide factors as pharmaceuticals. Extensive studies of the stability of peptidic material on storage allows identification of the most suitable form of administration. Nevertheless, the high clearance rate of peptides as pharmaceuticals presents new challenges for improvement by structural modification of resistance to enzyme degradation without creating new problems related to metabolites.

Structural and dipolar properties of the voltage-dependent pore former alamethicin in octanol/dioxane

Dielectric constant and loss of the membrane-active peptide alamethicin in octanol/dioxane mixtures have been measured at frequencies between 5 kHz and 50 MHz. On the basis of a rotational mechanism of dipolar orientation, the observed dispersion provides information regarding size, shape, and dipole moment of the structural entities which the solute may assume in media of diverse lipophilicity. Particularly detailed results are obtained in a pure octanol solvent where an apparent molecular weight of alamethicin could be determined. It turns out that in this quite lipophilic medium most of the peptide material exists as a monomer particle that has approximate length and diameter of 35 and 13 A, respectively. It carries a dipole moment of approximately 75 Debye units (directed nearly parallel to the long axis). At our concentrations of a few milligrams per milliliters, appreciable formation of dimers by head-to-tail linkage is indicated. When the octanol content is reduced by adding greater amounts of dioxane, larger particles are encountered. This is accompanied by a decrease of the effective polarity. The inherent increase of hydrophilicity in the dioxane-enriched solvent apparently favors another monomer conformation that has a low dipole moment and easily aggregates to some kind of micelle.

A peptide-containing fraction of plasma from schizophrenic patients which binds to opiate receptors and induces hyper-reactivity in rats

A serum fraction from schizophrenic patients has been investigated for its effects on opiate receptor sites and on behaviour in rats. Serum from schizophrenic patients was ultrafiltered and fractionated on DEAE-Sephadex A-25. The concentration of peptide material eluting under 0.1 M HCl (fraction I) was further purified on Sephadex-G10 and four major peaks were identified (fractions II to V). When injected intracerebroventricularly in rats, fraction II produced a characteristic behavioural syndrome, which included hyperactivity associated with hyperemotionality. The effects were long lasting, bouts of hyperemotionality accompanied by analgesia were recorded over a two-week period. The same fraction from control non-patients produced a transient and much reduced, but qualitatively similar response. There was evidence that fraction III was also active. An in vitro opiate receptor binding test showed that fraction II from schizophrenic patients inhibited [ 3H]naloxone binding.

Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens.

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases ('TIMP') and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.

31] Retinyl peptide isolation and characterization

Publisher Summary This chapter discusses the retinyl peptide isolation and characterization. Retinal may be reduced to its protein binding site in rhodopsin by the use of strong reducing agents such as sodium borohydride, sodium cyanoborohydride, or boranedimethylamine. The resulting retinylopsin is digested in detergent solution with proteolytic enzymes to produce retinyl peptides. Peptides from the digest that are soluble in ethanol are chromatographed on a silica gel column using alkaline chloroform/methanol. Rod outer segment membranes may be extracted in digitonin and the retinal reduced to rhodopsin by illuminating in the presence of sodium borohydride. The use of [ 3 H] retinal offers a convenient method for detection and quantification of the retinyl peptide during its formation and purification. The purified [ 3 H] retinal rhodopsin is dissolved in 67 mM sodium phosphate (pH 7.0) containing 50 mM octyl glucoside. The retinyl peptide is visualized by UV illumination and scraped from the plate. Peptide material is eluted by extracting with three 5-ml portions of solvent II.

A non-calcitonin secretory peptide derived from preprocalcitonin.

The terminal hexadecapeptide sequence of preprocalcitonin (termed carboxyl-adjacent peptide, CAP, for its position relative to calcitonin) has been synthesized to establish a radioimmunoassay for an investigation of cross-reacting peptides in extracts of normal and neoplastic calcitonin-producing tissues. Gel filtration chromatography of a rat thyroid extract revealed a major peak of immunoreactive peptide of approximately the same molecular weight as synthetic CAP. A minor peak of higher molecular weight immunoreactive material was also observed. The isoelectric point of both the thyroid peptide and synthetic material was about 5.2. Immunoreactive CAP and calcitonin were found in approximately equimolar amounts in normal thyroid and in anaplastic and well differentiated rat medullary thyroid carcinomas. Immunoreactive CAP was not detected in any tissue which did not contain calcitonin. Monolayer cultures of a rat medullary thyroid carcinoma contained and secreted equimolar amounts of immunoreactive CAP and calcitonin under basal (1 mM Ca/sup 2 +/) and stimulated (4 mM Ca/sup 2 +/ and 1 ..mu..M glucagon) conditions. These data indicate that the thyroidal peptide is probably the hexadecapeptide CAP which is derived from the same translation product as calcitonin. The similarity between the processing of precursors to the amidated peptide melanocyte-stimulating hormone and the corticotropin-like intermediate lobemore » peptide and processing of calcitonin and CAP suggests that the sequence Gly-Lys-Lys-Arg is one amidation codon in mammalian systems.« less

Induction of extrajunctional acetylcholine sensitivity of rat EDL muscle by peptidic component of peripheral nerve.

The induction of extrajunctional ACh-sensitivity was studied electrophysiologically 4 days after implantation of segments of the sciatic nerve, segments of dorsal and ventral spinal roots and silastic plates, containing some components of peripheral nerve onto surface fibres of innervated rat extensor digitorum longus muscle. It has been found that extrajunctional ACh-sensitivity can be induced by a piece of rat sciatic nerve and by peptidic material of about 8,000 and 10,000 mol. wt. No sensitivity was induced by segments of ventral and dorsal roots, by a piece of rabbit sciatic nerve and by rat sciatic nerve sectioned 7 days prior to implantation. The active nerve fractions lost its inducing potency after pepsin pre-treatment and irradiation with UV-light. Peripheral nerve probably contains some specific factor, peptidic in nature, which is able to induce the ACh-sensitivity of muscle fibre membrane.

Uptake and Metabolism of Hydroxyproline in Endosperm Cells of Lolium multiflorum (Ryegrass)

Hydroxyproline is rapidly removed from the medium by suspension-cultured endosperm cells of ryegrass (Lolium multiflorum) in a process which exhibits Michaelis-Menten kinetics and is dependent on temperature and metabolic energy. Following uptake by the cells, hydroxyproline is rapidly converted to proline by a route which appears to be more direct than the pathways of hydroxyproline metabolism in mammalian and bacterial systems and which results in substantial conservation of the pyrrolidine ring. Four hours after providing the cells with 5.5 nM [3H]hydroxyproline, approximately 50% of the radioactivity is recovered as intracellular imino acids, intracellular protein and extracellular peptide material. The radioactivity is associated predominantly with proline and hydroxyproline residues. In common with other plant and animal systems, hydroxyproline is not incorporated directly into protein. However, radioactivity originating in extracellular hydroxyproline is detected eventually in peptide linkage, where it appears first as peptidyl proline. In the extracellular fraction which contains arabinogalactan-protein, peptidyl proline is hydroxylated to form peptidyl hydroxyproline and, after 4 h, approximately 18% of the original 3H label is found in this fraction.

Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain.

The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [3H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes.

Evidence for presence of an internal thiolester bond in third component of human complement.

Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.

Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content.

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.

The chemistry of the collagen cross-links. Purification and characterization of cross-linked polymeric peptide material from mature collagen containing unknown amino acids.

A polymeric form of the alpha 1-chain C-terminal peptide alpha 1 CB6 (poly-alpha 1 CB6) was purified from CNBr digests of insoluble bovine tendon type-I-collagen by gel filtration and ion-exchage chromatography. The purified material had a molecular weight of 1.5 x 10(6)-5 x 10(6) on gel filtration and an amino acid content virtually identical with that of monomeric peptide alpha 1 CB6. The material could be adsorbed on affinity gels containing immobilized anti-(alpha 1 CB6-peptide non-helical region) antibodies and was an inhibitor of haemagglutination by the same antibodies of alpha 1 CB6-peptide-coated sheep erythrocytes. Periodate treatment of the material had no effect. Alkali hydrolysates were shown to contain two unknown amino acids, which were purified by gel filtration and ion-exchange chromatography in volatile buffers and are believed to be components of the mature cross-link of collagen.

Analysis of the most tightly bound proteins in eukaryotic DNA

DNA isolated by procedures generally considered to be most efficient for purifying DNA still contains detectable peptide components. The characteristics of this material and the stability of its linkage to DNA were investigated: DNA released from [ 35S]methionine-labelled cells by SDS in the presence of proteases contains a significant amount of 35S label which is not removed by additional treatment with proteases and phenol and which cosediments and cobands together with DNA on alkaline gradients. Furthermore, some peptide material which is copurified with native DNA and which remains complexed with DNA after alkali treatment can be labelled with 125I and analyzed on SDS-polyacrylamide-gels. The amino acid analysis of hydrolysates of purified DNA gives a rough estimate of the amount of the peptide material which is copurified with DNA. The results indicate that distinct proteins between 54 000 and 68 000 daltons in size are not removed from DNA by phenol, proteases, alkali or by any combination of these treatments. They can only be isolated by degradation of DNA. This extreme stability of the DNA-protein linkage indicates that these proteins are not merely contaminants which are difficult to eliminate but are rather covalently or otherwise bound (alkali-stable) to DNA. The size of these proteins and the stability of their linkage to DNA suggests that they are related to the class of non-histone proteins which are thought to be involved in chromatin structure e.g. by keeping DNA in a supercoiled state. Other possible functions are discussed.

Peptides as requirement for immunotherapy of the guinea-pig line-10 tumor with endotoxins

The transplantable line-10 hepatocellular carcinoma of guinea-pigs has been used as a model for the study of immunotherapy of malignant tumors. Cure rates of up to 100% have been obtained with ReGl-CM from 0 antigen-deficient (Re) mutant strains of Enterobacteriaceae, provided they were combined with mycobacterial trehalose dimycolate (cord factor, P3). Whereas highly endotoxic LPS extracts from all wild-type strains so far tested have failed to cause tumor regression, acid hydrolysis of such LPS samples led to residual fractions (RESI) that cross-reacted serologically with ReGl-CM samples (Chang and Nowotny, 1975) and provided cure rates up to 100%. RESI from Serratia marcescens was essentially nonpyrogenic and 100 times less lethal for chick embryos than potent endotoxins. Antigenic material associated with endotoxic extracts appears to be cryptic or sterically hindered from being effective in wild-type LPS but is exposed in ReGl and RESI samples. Reduction of the aminoacid content of ReGl-CM by microparticulate silica gel chromatography or by treatment with Triton X-100 significantly lowered the ability to bring about tumor regression without affecting endotoxicity. Antitumor activity could be restored by the addition of synthetic N-acetyl-muramyl-l-seryl-d-isoglutamine (MDP) or a nontoxic lipoid side fraction recovered during the isolation of ReGl-CM, which contained a small amount of peptidic substances. It is concluded that the addition of peptidic material, which may act as an adjuvant, to endotoxins is required to make them useful for immunotherapy of the weakly immunogenic line-10 tumor. Chemical procedures known to ‘detoxify’ endotoxins while retaining adjuvanticity, such as succinylation and phthalylation, resulted in complete loss of endotoxicity and tumor-regressive potency of ReGl-CM. Transesterification with sodium methoxide led to a water-soluble phase, which cured 50% of tumor-bearing animals even though lethality and pyrogenicity were reduced by 100 times and 50 times, respectively. Thus there was no direct correlation between endotoxic potency and tumor-regressive activity. In addition, our findings indicate that a low level of toxicity may be required to obtain optimal levels of tumoricidal action.

Identification of rapidly labeled, small molecular weight hydroxyproline-containing peptides in developing mouse limbs in vitro and in vivo

Limbs from embryonic mice labeled with radioactive proline either in vitro or in vivo readily synthesized hydroxyproline-containing peptides, the majority of which were of a molecular size less than intact collagen α-chains. The hydroxyproline isomers detected in these peptides included trans-3-hydroxyproline, trans-4-hydroxyproline, and cis-4-hydroxyproline. The abundance of small peptide material containing these hydroxyproline isomers is unusual in that as much as 85% is present in such forms at 10 days gestation when the limb is initially forming and 35% at 14 days when the limb is fully developed. The identification of small molecular weight hydroxyproline-containing peptides in limbs removed from embryos labeled in vivo indicate they are not organ culture artifacts.

Isolation and partial characterization of immunologically reactive fractions from chitinase digested cell wall of Trichophyton mentagrophytes

Cell wall preparations of Trichophyton mentagrophytes were digested with chitinase following which various fractions were isolated by ultrafiltration and Sephadex gel filtration. All fractions isolated contained both polysaccharide and peptide material. A correlation was seen between those fractions capable of eliciting immediate and delayed skin reactions in sensitized guinea pigs and those capable of stimulating the in vitro proliferation of lymphocytes taken from sensitized guinea pigs. These immunologically active fractions also developed precipitin lines with antiserum taken from sensitized animals. A low molecular weight fraction was found to be completely reactive immunologically (UM2(a)), and appeared to have a molecular weight in the range of 2,000--4,000 as assessed by ultrafiltration and gel filtration studies.

Formation and properties of a thrombin generated factor IX inhibitor.

Abstract The digestion of purified bovine Factor IX with purified thrombin at various concentrations removed peptide material from only the COOH-terminal end of the single chain molecule. The peptide was obtained in two forms, differing in carbohydrate concentration. Limited digestion with thrombin reduced the molecular mass of Factor IX by about 8,000 daltons. This smaller molecule, called Factor IXβ, was purified and found to be an inhibitor when added to a suitable mixture of purified Factor Xa and purified prethrombin 1 in 25% sodium citrate solution. It was also an inhibitor of thrombin formation in a system consisting of purified prothrombin complex, phospholipid, purified Factor V and calcium ions. With the ultracentrifuge, Factor IXβ sedimented as a single component and S o 20,w = 3.68 − 0.064X as compared with S o 20,w = 3.8 − 0.024X for Factor IX. Factor IXβ developed classical Factor IXa activity in the presence of Factor XIa and calcium ions.

Detection and kinetic behavior of preproinsulin in pancreatic islets.

Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.

Demonstration of biological activity of brain gastrin-like peptidic material in the human: its relationship with the COOH-terminal octapeptide of cholecystokinin.

The previously described peptide material that reacts with antibodies to gastrin and is found in the central nervous system of various vertebrates is present in only the 100,000 X g pellet of postmortem human cerebral cortical grey matter. This immunoreactive material, extractable in boiling water, is biologically active on rat pancreatic preparations. On the basis of size, charge, immunological specificity, and patterns of biological activity, most of this material is closely related to the COOH-terminal octapeptide of cholecystokinin in its complete, sulfated biologically active form.