Ninhydrin-positive Spots Research Articles

Antimicrobial activity of marine sponge Clathria indica (Dendy, 1889)

Sponges are sessile filter feeders that have developed efficient defense mechanisms against foreign invaders such as viruses, bacteria or eukaryotic organisms. Antimicrobial peptides are known as major components of the innate immune defense system in marine invertebrates. The aim of the present work was to study the antimicrobial properties of the Indian sponge Clathria indica with special reference to the identification of antimicrobial peptides. Crude methanolic extract and its chloroform, n-butanol and aqueous fractions were tested against 16 human pathogens which include eleven bacteria with four of them being multidrug resistant and five pathogenic fungi. All fractions showed effective antibacterial activity against common and multidrug-resistant Salmonella typhi and antifungal activity against C. albicans and C. neoformans. However, they were ineffective against Escherichia coli, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus. Chloroform fraction being the most potent among the fractions tested on chemical investigation was indicative of the presence of peptides as evidenced by ninhydrin positive spots on TLC and presence of peptide bonds by NMR. Its ESI-MS showed presence of several peptides in the range of m/z 850 to 980. Structure of three peptides has been tentatively assigned by ESI-MS/MS or tandem mass analysis, on the basis of the amino acid sequence established. The results clearly show that the sponge C. indica represent an interesting source of marine invertebrates-derived antimicrobial peptides in the development of new strategies to treat various infectious diseases.

Distribution of a Diphytanyl Ether Analog of Phosphatidylserine and an Ethanolamine-containing Tetraether Lipid in Methanogenic bacteria

Summary Aminolipids were found to be widely distributed in methanogens. Some of them were major components of the polar lipids. The distribution of two aminolipids, a diether analog of phosphatidylserine and a phosphoethanolamine derivative of dibiphytanyl diglycerol tetraether, was studied using TLC. In addition to the simple comparison of TLC patterns, the introduction of radiolabeled internal standards greatly improved the reliability of TLC analysis of lipids. A ninhydrin-positive spot which cochromatographed with the 32 P-labelled diether analog of phosphatidylserine occurred as a major constituent in the total lipid in Methanobacteriaceae , but was abserit in Methanomicrobiaceae and Methanosarcinaceae . Using the same method, the ethanolamine-containing tetraether phospholipid was found only in the genera Methanobacterium and Methanosarcina . A highly polar phosphoglycolipid was found only in Methanobacteriaceae . An aminolipid which migrated on TLC between phosphatidylserine and phosphatidylethanolamine was confined to Methanomicrobiaceae . It is suggested that the occurrence of these polar lipids be used for the grouping of methanogens at the family level.

Simulation of Bacterial Fermentation of Milk and Possible Acylation of Its Proteins by Acidogen Hydrolysis

Gradual, controlled, and homogenous acidification of milk was achieved by addition to milk of acidogens: propionic anhydride, lactide, glucono delta lactone, ethyl butyrate with its esterase, and tributyrin with its lipase. Only glucono delta lactone gave satisfactory delicate creamy and jelly-textured yogurt-like product within a relatively short time. The gellification time was shortened significantly by preacidification of the milk by a mild buffer. Glucono delta lactone released smaller amounts of acid in the presence of proteins than in their absence. It caused the appearance of a new ninhydrin positive spot on the chromatogram of its slightly basic mixture with L-lysine. Therefore, I assumed that glucono delta lactone reacts with milk proteins and also may attack digestive tract tissue in addition to its spontaneous hydrolysis in aqueous medium.

Characterization of the host-specific phytotoxin victorin by high-pressure liquid chromatography

Summary The host-specific phytotoxin victorin was purified from culture filtrates of Bipolaris victoriae by solvent extraction, alumina adsorption chromatography and reverse phase high-pressure liquid chromatography (HPLC). Victorin activity was associated with an HPLC fraction containing a peak of absorption at 254 nm; by several criteria this ultra-violet (UV)-absorbing peak was identified as victorin. Victorin purified by HPLC ran as a single ninhydrin-positive spot in several thin-layer chromatography (TLC) systems. Purified victorin inhibited susceptible oat ( Avena sativa ) root growth half-maximally at 0.1 ng/ml, yet did not significantly inhibit resistant oat root growth at 50 g/ml.

Copper-binding Components in the Water-soluble Low Molecular Weight Fraction of Soybeans

The water-soluble fraction of soybeans was extracted with 0.01M Tris-HCl buffer (pH 7.4). The low molecular weight fraction (F-II, MW. F-IIC>F-IIA).All three Cu complexes in F-II were eluted together with nitrogen-containing materials, which were all ninhydrin-positive, but they were not eluted with sugars or phosphorus compounds.Amino acid analyses of the three Cu complexes in F-II were carried out. The amino acid composition of F-IIA was similar to that of F-IIB, but differed from that of F-IIC. Approximately 35% of the amino acid residues in F-IIC were found to be arginine.Both F-IIA and F-IIB showed ninhydrin-positive spots on a paper chromatogram. The Rf values of the spots from F-IIA and. F-IIB were different, and both were less than those of free amino acids. F-IIC showed several ninhydrin-positive peaks on a Sephadex G-10 chromatogram. Cu in F-IIC was eluted with ninhydrin-positive materials having an Rf value similar to that of arginine.

Difference in physical and chemical properties between muscle-type and spleen-type isoenzymes of pyruvate kinase in rat

Muscle(M)-type and spleen(S)-type isoenzymes of pyruvate kinase were prepared to a homogeneous purity from skeletal muscles and the tumor (Rhodamine sarcoma) of rats, respectively. The respective types were composed of four of the identical subunit of approximately 60,000 daltons. Their amino acid compositions and peptide maps resembled each other, although there were some definite differences. The total number of dicarboxylic and basic amino acids were higher and lower with M-type than with S-type, respectively, in accordance with the difference in isoelectric point. Of the ninhydrin-positive spots on the peptide maps of their tryptic digests, 61 spots were common for both types, whereas 4 spots were specific to M-type and 5 spots to S-type.

Prostaglandin-generating factor of anaphylaxis. Identification and isolation.

Anaphylaxis of human lung in vitro is accompanied by prostaglandin (PG) formation. To search for factors capable of generating PG, supernatants prepared by reversed anaphylaxis of human lung tissue were added to guinea pig lung and PG formation examined. The PG-generating activity fractionated between Mr = 500 and 5000 by ultramembrane filtration and was designated prostaglandin-generating factor of anaphylaxis or PGF-A. Partially purified PGF-A was soluble in 80% ethanol and insoluble in chloroform or ethyl acetate. Paper chromatography of anti-IgE-treated human lung preparations revealed a ninhydrin-positive spot at RF 0.27 to 0.41 with PG-generating activity. When subjected to gel filtration on Sephadex G-50 or G-25, PGF-A demonstrated peak PG-generating capacity at Mr = 1200 to 1800. Peak fractions were pooled and chromatographed on CM52 cellulose with PGF-A activity eluting at 0.16 to 0.3 M NH4COOH. The pooled peak of PGF-A activity from Sephadex G-25 filtration and CM52 chromatography eluted as a single peak at 0.024 M NH4HCO3 on DE52. Application of the peak off DE52 to a paper chromatogram revealed a single ninhydrin-positive spot with an RF 0.27 to 0.41 with the capacity to generate PGs. Amino acid analysis of three purifications revealed the following: Glu/Asp/Gly/Ser/Thr (6:3:2:1:1). Based on these analyses, a Mr = 1450 was calculated for the PGF-A. Therefore, PGF-A represents a novel mediator of lung anaphylaxis which may be partly responsible for PG generation accompanying allergic reactions.

The isolation of a low molecular weight serum factor which enhances the viability of SV3T3 cells.

A serum factor which enhances the growth/viability of SV40 transformed 3T3 mouse fibroblasts, but not untransformed 3T3 cells in tissue culture has been partially purified from calf serum. The purification, which involves acidification to pH 2, chromatography on Sephadex G-100 at pH 2 followed by either Sephadex G-25 or BioGel P-2, results in material exhibiting two ninhydrin positive spots on thin layer chromatography and five to six dansylatable bands on SDS gels. This substance, termed Peak III or Serum Factor III, has been found in every serum examined thus far (including calf, rat, mouse, goat, lamb, rabbit, pig, horse, and chicken) and appears to be a low molecular weight, heat-insensitive molecule. Attempts to characterize it by chemical analyses and specific enzymatic inactivation have been either negative or inconclusive. Its biological mode of action is presently obscure. While it does not effect DNA synthesis, it does greatly increase the viability of SV3T3 cultures growing in low serum (0.15-0.30% calf serum) medium and, when added to "stationary phase" cells, restores healthy proliferation. This and evidence reported by others suggest that Peak III may affect SV3T3 cells in either overcoming a potentially lethal block in G1 or in passage through G2/M.

An inhibitor of opiate receptor binding from human erythrocytes identified as a glutathione-copper complex

A “peptide-like” inhibitor of opiate receptor binding and of N-methyltransferase previously purified by us from rabbit brain was also found in human red blood cells. Boiled extracts of erythrocytes were fractionated on Sephadex followed by chromatography of the active fractions on silica gel layers. Both, a migrating ninhydrin-positive spot and a naturally blue substance which did not migrate from the origin coincided with the active fractions. The blue substance was identified as copper and the ninhydrin-positive material was identified as oxidized glutathione. While glutathione per se has no effect, copper and other transition metals are potent inhibitors of opiate receptor binding. A mixture of glutathione and copper plus serum albumin in proportions simulating erythrocyte extracts gave results identical to the latter. Several other laboratories have extracted, from various tissues and body fluids, “opiate-like peptides” which are distinct from the β-LPH derived endorphins. In view of our findings it is possible that metal bound to glutathione or to other peptide ligand may be a complicating factor in some of these studies.

Biochemical and physiological studies of certain ticks (Ixodoidea). Excretion during ixodid feeding.

Excreta from females of 9 ixodid species in the genera Hyalomma, Dermacentor , and Haemaphysalis , and from nymphal Dermacentor andersoni , contained 1.4–9.3% guanine, 2.9–15.0% “2nd purine,” 0.19–1.5% hematin, and 5.0–68.0% protein by weight. Polyacrylamide gel electrophoresis of excreta showed only hemoglobin and albumin. The quantity of protein in D. andersoni excreta differed when the ticks fed on different hosts. Thin-layer chromatography showed 1–9 ninhydrin-positive spots for each tick species; these were identified as arginine, serine, alanine, histidine, valine, glutamate, aspartate, cystcinc, glycine, and phenylalanine.

Primary sequence of the β-chain of badger haemoglobin

Badger ( Meles meles) haemoglobin was purified by paper electrophoresis and converted into globin. Chain separation was carried out on a CM-cellulose column in the presence of 8 M urea. The β-chain was aminoethylated, purified by gel filtration and submitted to tryptic digestion. A fingerprint obtained with the enzymic digests showed 17 distinct ninhydrin-positive spots from which 20 pure peptides were isolated by further electrochromatographic separations. These peptides were sequenced using Dansyl-Edman and Ptc-Edman degradation techniques. The presence of amide residues was confirmed after aminopeptidase M hydrolysis. Taking human haemoglobin β-chain as a model, the covalent structure could be completely resolved without the help of any further overlapping technique. The following substitutions were noted (badger/human, position): Ala/Pro 5, Ser/Ala 13, Tyr/Phe 41, Asp/Glu 43, Ser/Ala 70, Glu/Asp 73, Lys/Ala 76, Asn/His 77, Lys/Thr 87, Lys/Arg 104 and Gln/Pro 125. A comparison with other haemoglobin β-chains already sequenced shows a greater similarity with dog haemoglobin, the only example of β-chain of known structure in the order of Carnivores.

The rôle of ninhydrin-positive substances in osmoregulatton in the western rock lobster, Panuliris longipes (Milne Edwards)

Free leucine, valine, proline, tyrosine, alanine, taurine, glutamic acid, and glycine are present in both blood and muscle of Panulirus longipes (Milne Edw.). Arginine is present in muscle only. Five unidentified ninhydrin-positive spots appeared in thin-layer chromatograms of blood, and four in chromatograms of muscle. Non-protein, ninhydrin-positive substances (NPS) in muscle do not appear to function in the moulting process. Concentrations averaged 278 mM/kg wet weight of muscle and NPS were the most abundant of organic osmotically active substances (total ions were 325 mM/kg). Trimethylamine oxide and glycine betaine together amounted to a further 121 mM. With lowered salinity, NPS concentrations in muscle were sharply reduced within 24 h, but with increased salinity, concentrations rose slowly over 7 days suggesting that NPS are produced by intracellular processes. When rock lobsters were fully adapted to salinities ranging from 25 to 45‰ concentrations of muscle NPS were linearly related to external salinity. When the salinity was lowered the blood was initially flooded with NPS, concentrations reaching a maximum at 12 h, and returning to normal after 72 h. Gastric fluid concentrations also rose, and evidence indicates that a large part of the NPS lost from muscle is excreted into the external water via the gastric fluid. At a salinity of 30‰, 56 % of the NPS lost from muscle appears in the external water, the remainder being excreted as ammonia; at 27‰, 82 % of NPS was excreted into the water, the remainder as ammonia. It is concluded that a changing external salinity would cause a continual loss of NPS from the body of a decapod crustacean. Although wasteful of nitrogenous compounds, such a process is of high survival value in permitting osmotic adjustment in the absence of more efficient mechanisms, such as those present in teleost fishes.

On Metabolism of Bromvalerylurea. I. Detection of 2-(2-Amino-2-carboxy-ethylthio)-3-methyl-butyrylurea from Rabbit Urine

It is well known that 3-methylbutyrylurea is one of the metabolites of α-bromoisovalerylurea (bromvalerylurea), and the present experiment was carried out in order to find other metabolites of this hypnotic. After the administration of bromovalelrylurea to a rabbit, the urine was collected daily and passed through an anion-exchange resin column. As a result of thin-layer chromatography of the fractions eluted with dil acetic acid, a new Ninhydrin-positive spot was detected much more clearly in the first 24-hr urine than in any other cxcreted urine. From the first 24-hr urine, this substance was obtained as a pure crystalline compound. Mass spectra, infrared spectra, and other chemical analyses suggested that this Ninhydrin-positive spot is a cysteine derivative of bromvalerylurea. Finally, this compound was determined as 2-(2-amino-2-carboxyetylthio)-3-methylbutyrylurea by comparison with a synthetic compound.

A method for determining 2-aminoethane-phosphonic acid in rumen contents

The work described is an attempt to develop a reliable method for determining AEP when mixed with related substances from micro-organisms in the rumen. Hydrochloric acid hydrolysates of rumen contents, rumen bacteria, rumen ciliate protozoa and clarified rumen contents were applied to a column (10 × 1 cm) of Dowex 50–X8, eluted with 0·6 N hydrochloric acid and 2·4-ml fractions collected. Inorganic phosphorus was separated in column fractions 2–10 and AEP appeared in fractions 11–23.When fractions containing AEP were spotted on to Whatman No. 1 filter-paper strips the developed chromatograms showed six ninhydrin-positive spots in addition to that of AEP, which was the slowest acid to migrate (RF= 0·3). The substance giving spot No. 4, another phosphonic acid (RF= 0·59), was present in ciliate protozoa and ciliate-free fractions of rumen contents, whereas AEP was confined to protozoa. A highly significant correlation was found between the AEP concentrations and protozoal counts in samples of rumen contents collected at different intervals. It is therefore suggested that the concentration of AEP can be used as a marker of the protozoal growth in the rumen.

Occurrence of .DELTA.-aminovaleric acid in the culture medium of rumen ciliate protozoa.

An unknown ninhydrin positive spot on the paper chromatogram of the endogenous metabolites of mixed rumen ciliates was isolated by using ‘Dowex’ 50-X8 column and silica gel column and identified as δ-aminovaleric acid by the data of elementary analysis, specific optical rotation, mass spectrum, paper chromatogram and infrared spectrum.

Free amino acids in a cave beetleDarlingtonea kentuckensisValentine (Coleoptera: Carabidae)

Free amino acids of Darlingtonea kentuckensis were investigated by two-dimensional, thin-layer chromatography on Silica Gel G. Thirteen amino acids which could be identifìed (alanine, glutamic acid, glycine, histidine, isoleucine and/or leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and seven unidentified ninhydrin-positive spots were found. Beta-Alanine, alpha-amino-butyric acid, arginine, aspartic acid, cystine, hydroxyproline and methionine were not detected. No difference was observed in the free amino acids with respect to sex of beetle, time after feeding and method of sample preparation.

Studies on colistin nonapeptide derivatives. III. Chemical synthesis and characterization of n-fattyacyl mono-aminoacyl derivatives of colistin nonapeptide.

In order to examine the antimicrobial activity of n-fattyacyl mono-aminoacyl derivatives of colistin nonapeptide (CNP), twenty-one derivatives were synthesized and characterized on their chemical, physicochemical and antimicrobial properties. Five α-amino acids, glycine, l-alanine, dl-aminobutyric acid, dl-norvaline and dl-norleucine, and two ω-amino acids, β-alanine and γ-aminobutyric acid, were first acylated with each of three kinds of acid chloride, n-octanoyl, n-decanoyl and n-dodecanoyl chloride, and then esterified with p-nitrophenol prior to the coupling of those n-fattyacyl amino acids to the terminal threonine of CNP. The coupling reaction was carried out in an aqueous solvent buffered at pH 5.0 without any protection of the γ-amino groups of CNP, as reported previously.All of the derivatives obtained in hydrochloride salts were hygroscopic white amorphous powder showing decomposing points at 180~230°C. Each purified preparation exhibited a ninhydrin positive spot on a paper chromatogram, and ea...

Abnormal Pattern on High-voltage Paper Electrophoresis (HVPE) During Paracetamol Medication

An extraneous ninhydrin-positive spot on high-voltage paper electrophoresis (HVPE) was observed in urine samples collected from children receiving paracetamol medication. Interference by this derivative has not previously been reported. This extraneous spot could simulate an abnormal peptiduria.

Fidelity of translation of satellite tobacco necrosis virus ribonucleic acid in a cell-free Escherichia coli system.

Biochemistry @ Copyright 1973 by the American Chemical Society Volume 12, Number 2 January 16,1973 Fidelity of Translation of Satellite Tobacco Necrosis Virus Ribonucleic Acid in a Cell-Free Escherichia coli Systemt R. Rice* and H. Fraenkel-Conrat The polypeptides synthesized in an E. coli cell-free system under the direction of the small RNA of the satellite tobacco necrosis virus were compared with the authentic coat protein of this virus. Many of the tryptic peptides obtained from the two types of proteins moved identically upon high- resolution cation exchange column chromatography, but distinct qualitative and quantitative differences between the two peptide patterns were also evident. Gel electrophoresis in 9 M urea at pH 4.0 showed that the biosynthesized material was not homogeneous in regard to charge; some of it showed a ABSTRACT: E scherichia coli cell-free systems synthesize recognizable proteins when programmed with messengers from the small RNA bacteriophages (Stavis and August, 1970). These extracts generally respond better to exogenous messenger RNA, particularly in quantitative respects, than do incorporation systems from plant and animal sources. The genetic code, possibly including initiation (Stewart et at., 1971 ; Housman et a/., 1970; Marcus et a/., 1970) and termination (Goldstein et al., 1970) codons, seems to be identical in the animal, plant, and bacterial kingdoms. Hence, E. coli extracts have been used with animal and plant viral RNA messengers to investi- gate translation in these heterologous systems and to study the viral gene products in vitro. The most direct test to establish whether accurate translation of a viral messenger occurs has been to ascertain whether the viral coat protein is synthesized in vitro since other gene prod- ucts have not been as readily identifiable. Some studies have indicated considerable similarity between translation products and viral coat protein. For example, with the RNA from the t From the Department of Molecular Biology and Virus Laboratory, University of California, Berkeley, California 94720. Receired August 18, 1972. This investigation was supported by U. S. Public Health Service Grant No. G M 01389 from the National Institute of General Medical Sciences, and Grant No. GB 6209 from the National Science Foundation. R . Rice was a recipient of a National Science Foundation Graduate Fellowship. * Address correspondence to : Institute of Marine Resources, Uni- versity of California, Davis, Calif. 95616. mobility similar to that of the authentic coat protein. Gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of a wide range of molecular weight species in the biosynthesized protein ; the largest, representing about 5 % of the total, coincided with authentic virus coat pro- tein. It is concluded that much of the information on satellite tobacco necrosis virus RNA is translated into proper amino acid sequences in the E. coli system, but that only very little complete coat protein, and that probably carrying a terminal formyl-methionyl group, is synthesized. satellite of tobacco necrosis virus (STNV)' (Clark et a/., 1965) and the similarly small top a component of alfalfa mosaic virus (van Ravenswaay Classen et a/,, 1967), two-dimensional peptide maps have been reported as showing nearly complete coincidence of radioactive spots from the translation products and ninhydrin-positive spots from the coat proteins. In con- trast, earlier studies with TMV-RNA had indicated no clear evidence for coat-related material being synthesized in citro (Aach et a/., 1964; Schwartz, 1967). Studies with brome mosaic virus RNA (Stubbs and Kaesberg, 1967) and poliomyelitis virus RNA (Rekosh et al., 1970) gave somewhat ambiguous results and the recent claim that avian myeloblastosis virus RNA directs the synthesis of viral antigens (Siegert et a/., 1972) must await substantiation. In view of these widely varying results, it was our intention to reinvestigate one of the reports indicating positive results. We chose to characterize the translation products of STNV- RNA using more discriminating techniques which have be- come available since the original report appeared. The RNA of STNV is a convenient messenger for this kind of work in that it is small (0.4 X lo6) and can code maximally for only one small protein in addition to the viral coat protein (Roy et at., 1969). Since the completion of this work, an interesting study of the translation of STNV-RNA in E. coli and wheat embryo 1 Abbreviations used are: STNV, satellite tobacco necrosis virus; TMV, tobacco mosaic virus; TPCK, ~-(l-tosylamido-2-phenyl)ethyl chloromethyl ketone. BIOCHEMISTRY V O , L . 1 2 , NO.

Chemical Studies on the Subunit Structure of Rabbit Muscle Phosphoglucose Isomerase: IDENTIFICATION OF N-ACETYLATED NH2-TERMINAL ALANINE AND COOH-TERMINAL GLUTAMINE

Abstract Tryptic hydrolysates of native, denatured, or chemically modified phosphoglucose isomerase from rabbit skeletal muscle were subjected to standard peptide mapping techniques. Maps prepared from hydrolysates of either native, guanidine hydrochloride-denatured, or carboxymethylated enzyme were found to have only two-thirds to three-fourths of the number of ninhydrin-positive spots that would be expected (a) on the basis of the amino acid composition (82 lysyl and 42 arginyl residues per enzyme molecule of 132,000 mol. wt.) and (b) if it is assumed that the enzyme is composed of 2 identical subunits (physical studies have shown it to consist of 2 subunits of equal size). The lower-than-expected number of peptides obtained under these conditions was shown to be the result of incomplete hydrolysis, due to partial resistance of the enzyme to tryptic attack. However, tryptic peptide maps prepared from fully carbamylated phosphoglucose isomerase (all lysyl residues modified) yielded 20 to 22 unique peptides, the theoretical number for 2 identical subunits. The technique of peptide mapping after carbamylation of all of the lysyl residues proved to have numerous experimental advantages over mapping of the unmodified protein, suggesting this procedure as the method of choice for any protein composed of relatively large polypeptide chains. Standard methods of NH2-terminal analysis gave only negative results, leaving the possibility that the NH2 termini might be blocked. This was confirmed by gas chromatography which showed phosphoglucose isomerase to contain two acetyl groups per enzyme molecule. Application of gas chromatography and amino acid analysis after extensive proteolytic hydrolysis of the enzyme, followed by cation exchange chromatography of the products, yielded two equivalents of acetylalanine per enzyme molecule, i.e. one each at the NH2 terminus of each subunit. Treatment of the enzyme with carboxypeptidase A and hydrazinolysis followed by dinitrophenylation established the COOH-terminal end of each of the subunits to be -isoleucyl-glutamine. The results in their entirety indicate that rabbit muscle phosphoglucose isomerase is composed of 2 identical subunits.