Peptide Material Research Articles

Demonstration of circulating antibodies to neurophysin in patients treated with pitressin.

Commercially produced Pitressin (Parke Davis and Co.), used in the treatment of diabetes insipidus, is prepared from bovine and porcine posterior pituitary tissue and contains a variable amount of peptide material in addition to vasopressin. It would be expected to contain neurophysin (Acher & Fromageot, 1955) since this is the major protein constituent of the posterior pituitary. Neurophysin is considered to function as a binding protein for oxytocin and vasopressin, which are found in association with it, both in the hypothalamus and the posterior pituitary gland. It is known that nasal insufflation of 'pituitary snuff' (acetone-dried posterior pituitary tissue) can result in the production of antibodies to its protein constituents (Pepys, Jenkins, Lachman & Mahon, 1965; Mahon, Scott, Ansell, Manson & Fraser, 1967). However, when more purified preparations are used, hypersensitivity reactions are uncommon. In rare cases, antibodies to vasopressin itself have been present after treatment with Pitressin (Roth, Glick,

Incorporation of 32P into ribosomal RNA, transfer RNA and inositol hexaphosphate in germinating pea cotyledons

1. 1. RNA synthesis in germinating pea cotyledons has been studied by incorporation of radioactive phosphate. 2. 2. Sucrose density gradient centrifugation of RNA extracts showed that 28-S and 18-S rRNA are synthesized in an approximately linear fashion for up to 6 days. Radioactive label is also incorporated into tRNA. 3. 3. RNA extracts also contained highly labelled low molecular weight material which obscures the tRNA labelling region on sucrose gradients. The labelled material is soluble in 5 % trichloroacetic acid and can therefore be resolved from tRNA. 4. 4. The trichloroacetic acid-soluble material from RNA extracts was labelled very rapidly with 32P on germination and its labelling approximated to curves for water uptake up to 36 h. This was resolved by DEAE-cellulose chromatography and Sephadex G-25 chromatography into two major components, one of which was impure, being associated with carbohydrate and peptide material. In some preparations the partially purified 32P-labelled material co-chromatographed with ATP and in others with inorganic phosphate; it is likely that the latter case represented breakdown of ATP. The other rapidly labelled compound was identified as inositol hexaphosphate. 5. 5. 32P-labelled inositol hexaphosphate was found only in germinating cotyledons and was not detected in the developing embryo tissue.

The subcellular localization of a cardioexcitatory peptide in the pericardial organs of the crab, Cancer borealis

A cardioexcitatory peptide is associated with a sedimentable fraction of homogenized pericardial organs from Cancer borealis; this fraction contains numerous electron-dense granules of diameter ca 1500 Å. The association of the peptide with the fraction depends on the osmolarity of the granule suspension. The cardioexcitatory peptide exists in a “free” form, which can be released from a granule suspension by dilution with distilled water, and in “bound” form, which can be released by further treatment of the suspension with acetic acid. 5-hydroxytryptamine is probably associated with a sedimentable particle different from that containing the peptide material.

Characterization of amino acids and low-molecular-- weight peptides bound to cytoplasmic granules from the posterior pituitary.

A number of peptides and amino acids, representing 30–40% of the total acid-extractable, ninhydrin-positive material of the tissue, were associated with cytoplasmic granules (sedimenting at 3 000 000 g-min after preliminary removal of "nuclei and debris") isolated from bovine posterior pituitary glands. Acetic acid (0.2 N) extracts of a purified neurosecretory granule fraction showed only slight differences in the pattern of peptides and amino acids from extracts of the total cell particulate fraction. Gel filtration of extracts on Sephadex G-25 yielded three major fractions: fraction I consisting of peptide material of molecular weights > 4000, fraction II of molecular weights averaging about 3000, and fraction III of molecular weights < 2000. Fraction III was further resolved by anion-exchange chromatography into 12 subfractions. Vasopressin and oxytocin were contained in subfractions 2 and 3, respectively. Each of these subfractions was in turn chromatographed on a cation-exchange resin and resolved into a total for fraction III of 22 major components: lysine, arginine, phenylalanine, ammonia, and 18 peptides. Three of the peptides contained only aspartic and glutamic acids in the ratios 8:1, 5:1, and 4:1. The sequences of four dipeptides were ascertained. Another peptide was not retarded by Dowex 50 and yielded glutamic acid upon acid hydrolysis. Still another peptide yielded tyrosine plus an unknown ninhydrin-positive component after hydrolysis. The amino acid compositions were determined for nine other peptides containing three to nine residues. Additional peptides in fraction III were detected in lesser or trace amounts. Isolated granule fractions from both bovine posterior pituitary and rat liver were dialyzed against isotonic sucrose or distilled water. The rate of loss of ninhydrin-positive material from the sample dialyzed against water indicated that a large proportion of the "free" amino acids and peptides of these tissues were contained within intracellular organelles.

Rapid sequence analysis of small peptides

Small amounts of peptide material can be analyzed by a modified sequential degradation plus dansylation procedure. The method requires about 5 nanomoles per step, and is primarily designed for application to peptides of up to five or six residues. Alternatively it can be applied to the terminal sequence of larger peptides, but it is not generally applicable to proteins. Several cycles of degradation can be carried out in a typical working day, so that the entire sequence of a pentapeptide is established within a two-day period.

Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease.

1. Treatment of rat liver polysomes in a buffer containing 2.5mm-magnesium chloride with T(1) ribonuclease at a concentration of 330units/ml. of reaction medium at 37 degrees for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of (3)H-labelled polysomes (structural RNA labelled with orotic acid) with T(1) ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying (14)C-labelled nascent peptide chains with T(1) ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0.48mumole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying (14)C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0.5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying (14)C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0.3m-potassium hydroxide at 20 degrees for 24 hr., neutralized to pH7.6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent (14)C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [(3)H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20 degrees .

IDENTIFICATION OF PROTEIN-CONTAINING PAINT MEDIA BY QUANTITATIVE AMINO ACID ANALYSIS

In the present study the amino acid analyses of five protein-containing media were first ascertained. Then several specimens, both antique and modern, whose identity was unknown to the analyst, were assayed. The application of quantitative amino acid assay was found to be a useful and accurate means of determining the nature of protein media when sufficient peptide (protein) material was present.

Inhibition of the peptide bond synthesizing cycle by chloramphenicol.

To study the mechanism by which chloramphenicol inhibits bacterial protein synthesis, we examined the kinetics of the puromycin-induced release of peptides from transfer ribonucleic acid (tRNA) in the presence and in the absence of chloramphenicol. Washed Escherichia coli ribosomes with nascent peptides which had been radioactively labeled in vivo were used for this study. When such ribosomes were incubated in the presence of 10 mug of puromycin per ml, approximately one-fourth of the radioactive peptide material was rapidly released from tRNA. This rapid, puromycin-dependent reaction is assumed to be equivalent to the peptidyl transferase reaction. Chloramphenicol inhibited the extent of the puromycin-induced release of peptides by only 50%, demonstrating that some of the peptide chains which are present on active ribosomes react with puromycin, even in the presence of chloramphenicol. The addition of the supernatant fraction and guanosine triphosphate (GTP) increased the extent of the puromycin-induced release; this additional release was completely inhibited by chloramphenicol. Peptidyl chains on washed ribosomes prepared from chloramphenicol-inhibited cells were not released by puromycin in the presence of chloramphenicol and reacted slowly with puromycin in the absence of chloramphenicol. The release of peptidyl groups from these ribosomes became largely insensitive to chloramphenicol after preincubation of the ribosomes with GTP and the supernatant fraction. We conclude that chloramphenicol does not inhibit the peptidyl transferase reaction as measured by the puromycin-induced release of peptides from tRNA, but rather inhibits some step in the peptide synthesis cycle prior to this reaction.

Insulin biosynthesis in the rat: demonstration of two proinsulins.

Proinsulin, a single-chain precursor of insulin, has been detected in biosynthetic studies in vitro,(1-4) and in crystalline preparations of insulin from several species.(1, 5-7) It appears that proinsulin normally serves neither as a major storage nor secretory form of the hormone.(8) Its primary functions seems to be to facilitate the efficient formation of the disulfide bonds of insulin.(9)Although several species of fish have two insulins differing slightly in primary structure,(10-12) the rat is the only mammal where this is known to be the case.(13, 14) As the two rat insulins are nonallelic(14) and presumably coded by two distinct genes, a separate proinsulin would be expected to precede each insulin. We have demonstrated the biosynthesis of both insulins(15) and of a proinsulin corresponding to each in isolated islets of Langerhans, and have detected probable intermediates in the conversion of these proinsulins to insulin. Labeled peptide material corresponding to the free connecting segment of proinsulin (C-peptide) was detected in insulin-containing fractions isolated from incubated rat islets. It was noted that islets incubated in vitro secreted small amounts of newly synthesized proinsulin as well as the two insulins and essentially equivalent amounts of C-peptide. Increasing the glucose content of the medium selectively enhanced the incorporation of radioactivity into proinsulin and insulin and also seemed to increase the relative proportion of labeled proinsulin appearing in the medium.

Studies on the chemical constitution of cell-wall lipopolysaccharides from Neisseria perflava.

Lipopolysaccharides (LPS) were prepared from Neisseria perflava cells and separated into chloroform-soluble and chloroform-insoluble fractions in about equal proportions. The preparations were almost free from protein and contained only traces of peptide material. Both LPS fractions contained 3-deoxyoctulosonic acid (KDO), lipid A, glucose, rhamnose, heptose, glucosamine, galactosamine, ethanolamine, phosphate, and fatty acids. Both fractions had the same proportions of hexosamines and ethanolamine and the same fatty acid components (chiefly β-hydroxymyristic, β-faydroxylauric, octadecenoic, and palmitic acids), but differed markedly in their relative molar proportions of KDO to neutral sugars and of neutral sugars to total fatty acids. Equimolar proportions of hexosamine, ethanolamine, and phosphate in the lipid A moieties suggested that ethanolamine phosphate was linked to glucosamine, probably at the C-6 position. Fatty acids were bound in the LPS by both amide and ester linkages. Heptose units were linked linearly by 1 → 3 glycosidic bonds. About 25% of the glucose units were linked 1 → 3 in a chain; other glucose units were branched as were the rhamnose units. The proportions and arrangement of the heptose, KDO, and lipid A components suggest that the backbone part of the N. perflava lipopolysaccharide is very similar to that found in the Salmonella-Escherichia group.

The proteolytic activity of insulin.

The action of insulin on hemoglobin at pH 7.5 was studied. Four different methods were used to determine the degree of proteolysis. After mixtures of hemoglobin and insulin were incubated for 2 hours, very little amino acid or peptide material was liberated from the proteins. As many amino acids are liberated from hemoglobin when it is incubated alone under the same conditions. It is concluded that autolysis is responsible for the observed increases in non-protein amino acids and that insulin has no proteolytic activity under the conditions used in this study.

Biosynthesis of penicillin N and cephalosporin C. Antibiotic production and other features of the metabolism of Cephalosporium sp.

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp.

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.

Acid mucopolysaccharides of amyloid tissue

The acid mucopolysaccharides (MPS) were isolated from different organs from patients with primary and secondary amyloidosis, and the MPS composition of these tissues was compared with those isolated from control individuals. Although each organ contains different amounts and types of MPS, amyloid tissue in general shows an increase in total MPS, primarily due to heparitin sulfate. In the myocardium involved in primary (familial) amyloidosis (only one sample available for study), the increase in MPS is apparently due to hyaluronic acid, in contrast to liver, spleen, kidney, and heart tissue involved in secondary amyloidosis. A slight increase in chondroitin sulfate B was also noted in certain secondary amyloid tissues. The amino acid composition of peptide material bound to heparitin sulfate was determined, and no abnormality was observed from the similar MPS-protein complex isolated from control tissue.

Some characteristic of glycogen from a fly pupa (Calliphora erythrocephala meig.)

1.1. The blowfly pupa glycogen can be fractionated into four types of glucan by the use of phenol-water extraction, TCA- and La3+-precipitation and papain digestion. The content of the glucans have a rhythmic variation during the pupal development, but their rhythms are not quire synchronous.2.2. The glucans are to a varying degree associated with peptides and there are quantitative differences in the amino acid composition of the four glucans. The peptides are bound to an inner part of the glucan molecule and undergo concentration when the glucans decompose.3.3. To a varying degree in the different glucans the bound peptide material inhibits the branch-splitting by α-amulase and inhibits a mineral acid hydrolysis.

Amino acids liberated into the culture medium by pea seedling roots

The amino acid composition of the medium in which seedlings of peas had been grown aseptically with and without quartz sand were determined by ion-exchange chromatography. Besides considerable amounts of amino acids, peptide material was also present.

Fractionation of S-Carboxymethyl- κ-casein and characterization of the components

1. 1. A method is described for the fractionation of SCM-κ-casein ( S-carboxymethyl- κ-casein) into several fractions by chromatography on DEAE-cellulose in the presence of 20% dimethylformamide as dissociating agent. Essentially the same conditions have been used to fractionate reduced and alkylated whole casein into four main fractions corresponding to α s-, β, SCM- κ- and γ-caseins. 2. 2. Analysis of the fractions of SCM-κ-casein obtained has shown that the carbohydrate of SCM-κ-casein is concentrated in those components which migrate most rapidly during starch-urea gel electrophoresis. The phosphorus of SCM-κ-casein appears to be evenly distributed among the components. 3. 3. The isolation of a major fraction of SCM-κ-casein having virtually no carbohydrate but possessing full micelle-stabilizing ability and rennin sensitivity leads to the conclusion that the carbohydrate plays no significant role in these functions. 4. 4. Examination of the action of rennin (EC3·4·4·3) on a fraction containing no carbohydrate and on another fraction, rich in carbohydrate, leads to the conclusion that the previously observed heterogeneity, with respect to trichloroacetic acid solubility, of the peptide material released by rennin is mainly a result of the heterogeneity of the original κ-casein with respect to carbohydrate content. 5. 5. All the components of SCM-κ-casein give rise to the same two para-derivatives, a major and a minor one, and it is suggested that they arise as a result of the attack of rennin at two different sites in the κ-casein molecule.

The papain digestion of human serum α2-macroglobulin

Human α 2-macroglobulin has been digested with papain (EC 3.4.4.10) at pH values of 5.7 and below to give an immunologically detectable, low-molecular-weight component ( s 20, w approx. 3 S). The digestion process also results in the liberation of a considerable amount of peptide material. Digestion appears to proceed by a series of components of intermediate starchgel electrophoretic mobility, the final component moving in the post-albumin position. Gel diffusion studies reveal that the product is deficient in primate specific antigenic groupings, but carries the antigenic determinants responsible for its cross reactivity with the α 2-macroglobulin homologues of other mammalian species.

ISOLATION OF RIBONUCLEOTIDE-PEPTIDE COMPLEXES FROM PASTEURELLA MULTOCIDA.

SummaryRNA was extracted with 10% NaCl from acetone-dried cells of Past, mul-tocida and deproteinized by treatment with the chloroform-octanol mixture. After alkaline hydrolysis, CMP, AMP, UMP and GMP were separated by anion exchange chroma-tography and paper ionophoresis. The nu-cleotides were tightly bound to peptide material. Nucleotide-peptide complexes were also detected in the TCA-soluble extract from Past, multocida.

Reaction of histidine with the biuret reagent in the presence of oxygen

A simplified system is described which involves the reaction of the amino acid histidine with oxygen and the biuret reagent, yielding a biuret-positive color on standing or being heated. The reaction product appears to be a three-membered complex of histidine or a derivative of histidine, oxygen or possibly a derivative of oxygen (H 2O 2), and copper. The participation of cuprous copper in the complex is indicated by the facility with which a variety of reducing agents effect immediate biuret positivity at room temperature, and by the speed of the reaction in the presence of cuprous copper without the addition of any other reducing agent. In the absence of a prolonged incubation period or the application of heat, a reducing agent such as cysteine must be added to enable the reaction to proceed rapidly to biuret positivity at room temperature. It is concluded that, in the absence of added reducing agents, histidine itself may act as the reducing agent. The reaction differs in hue and color intensity from the immediate blue color-producing reaction obtained by mixing relatively large quantities of histidine with biuret reagent; it also differs in respect to other variables such as the necessity for O 2. A brief discussion of the present significance of this reaction, its use as a quantitative method for estimating histidine, and its potential for introducing gross error in the biuret determination of protein and peptide material is given. The reaction may hold some promise as a sensitive chemical dosimeter for ionizing radiation.