Radioactive Lysine Research Articles

My Early Days with Ari Helenius: Detergents and Viruses.

My Early Days with Ari Helenius: Detergents and Viruses.

Tissue distribution of lysine metabolism in commercial turkeys

The essential amino acid lysine is typically the first or second limiting amino acid in diets fed to growing turkeys. However, there is little information about how and where lysine is degraded. Therefore, an investigation of the tissue distribution of lysine catabolism was performed using liver, kidney, enterocytes, breast, thigh, heart and lung of four and eight week old turkeys. The tissues were assayed for the initial enzyme activity of the primary pathway of lysine oxidation, lysine ketoglutarate reductase (LKR). Additionally, lysine oxidation (LOX) was determined based on the recovery of 14CO2 from radioactive lysine. All tissues assayed had measureable LKR activity except lung. Liver had the highest activity (5.2 mmol/min*g liver). Intestine, kidney and liver oxidized lysine (P<0.05) and liver had the highest lysine oxidation (15.9 μmol/min*g tissue). A significant week * tissue interaction and week effect were detected. A positive correlation (P<0.05) was observed between LKR activity and LOX. In conclusion, it is clear that extra hepatic tissues contribute to whole body lysine metabolism. (Support: HATCH WVA 470, Virginia Poultry Grower Cooperative)

A comparison of the proteins of the amoebal and plasmodial phases of the slime mould, Physarum polycephalum.

1. The proteins of amoebae and plasmodia of strain CL of Physarum polycephalum have been compared by two-dimensional gel electrophoresis. Both forms of the organism were labelled by growth on formalin-killed bacteria labelled with [35S]sulphate, [3H]lysine or [14C]lysine. Plasmodia were also labelled from radioactive lysine in the medium. 2. Of 306 relatively abundant proteins examined, 26% were phase-specific, that is they were found only in amoebae or in plasmodia. About a quarter of these apparent differences in gene expression may be due to minor changes in charge and/or size. 3. Amongst the 74% of the proteins present in both amoebae and plasmodia, there are substantial differences in differential rates of synthesis and these have been measured for a representative set of proteins by a double-label procedure.

Cellular Retinaldehyde-binding Protein Ligand Interactions: GLN−210 AND LYS−221 ARE IN THE RETINOID BINDING POCKET

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Effect of feeding clinoptilolite (zeolite) on the performance of three strains of laying hens

1. One hundred and twenty 16‐week‐old single combed pullets of three strains were fed on a diet containing 135 g protein/kg with or without 50 g clinoptilolite/kg in a trial with 20 hens per treatment. Sterile river sand replaced clinoptilolite in the control diet in order to keep the diets isoenergetic. The hens were individually caged in a naturally ventilated laying house and fed one of the two diets for ten 28‐d periods. 2. Significant dietary effects of feeding clinoptilolite were observed with number of eggs laid per hen, shell thickness, efficiency of food utilisation and droppings moisture content. No significant dietary effects between treatments were observed with body weight, age at first egg, egg weight, Haugh units, food intake/hen and rate of amino acid absorption of radioactive lysine and methionine into the bloodstream. Significant differences between strains were observed with regard to all parameters except food intake/hen. There were no significant strain × clinoptilolite interactions.

Multiple Forms of tRNALys3in HIV-1

tRNALys3is the primer for HIV-1 reverse transcriptase (RT) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNALys3, multiple forms of tRNALys3are detected in the virus released into the cell culture media. These tRNALys3isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analysis using a tRNALys3-specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNALys3to be the major abundance form of tRNALys3in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNALys3species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3′ end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNALys3with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNALys3. These radioactive tRNALys3species retained their relative mobilities when rechromatograped on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNALys3. These results indicate that the major tRNALys3species present in the virus is also the major tRNALys3isoacceptor used as the primer for reverse transcription.

The immediate precursor of the nitrogen-containing ring of rapamycin is free pipecolic acid

Studies were carried out to determine the precursor of the nitrogen-containing ring of rapamycin. The addition of l-lysine and d,l-pipecolate to the medium of Streptomyces hygroscopicus did not affect the specific production of rapamycin. In a basal medium containing no l-lysine except for that in the yeast extract component, l- 14C-lysine and d,l- 3H-pipecolate were incorporated to a high degree into rapamycin, d,l- 3H-pipecolate being incorporated at a higher efficiency. Unlabeled pipecolate reduced the incorporation of radioactive lysine much more than unlabeled lysine decreased the incorporation of radioactive pipecolate. These results indicate that pipecolate is the more direct precursor of rapamycin, lysine being converted to pipecolate and free pipecolate being incorporated directly into rapamycin. Rapamycin samples enriched with 14C-lysine and 3H-pipecolate were hydrolyzed and analyzed to provide evidence of specific incorporation of these precursors into the pipecolate moiety .

Studies on the Extractive Components of Ascidians-V. Biosynthesis of Halocynine in the Ascidian Halocynthia roretzi.

In order to know whether halocynine, a novel betaine discovered by us in the muscle of the ascidian Halocynthia roretzi, is endogenous or not, the incorporation of radioactivity into it was examined by injecting L-(U-14C)lysine into the muscle of ascidian specimens. Halocynine isolated from the muscle extract by ion-exchange column chromatography and cellulose thin layer chro-matography was found to be radioactive in the specimen which had been kept in aerated sea water for 10 days after the administration of radioactive lysine. However, the isolated halocynine exhibited only a very weak activity and no activity in the specimens kept for 90 and 60h, respec-tively. These results unequivocally indicate that the ascidian is capable of synthesizing halocynine, although the biosynthetic rate is very slow under the conditions employed.

Biochemical changes and cytotoxicity associated with the degradation of polymeric glutaraldehyde derived crosslinks

The reversibility of glutaraldehyde crosslinks has been suggested as a reason for failure of long-term bioprosthetic implants. The stability of such crosslinks was investigated in tendons and model compounds. Small but cytotoxic levels of glutaraldehyde were still released from crosslinked tendons even after these tendons were extensively rinsed for up to 6 months. The toxic effect was evidenced by the death of fibroblasts surrounding a midsection piece of rinsed crosslinked tendon, while the end section pieces did not show toxic effects. The formation and stability of glutaraldehyde modified [14C]-L-lysine derivatives were investigated. The polymerization of glutaraldehyde with amino compounds was initially fast but continued to proceed slowly for months. Degradation of high-molecular-weight soluble polymers was detected by gel filtration chromatography. Low-molecular-weight soluble materials were also released from insoluble products which were formed when high concentrations of glutaraldehyde and radioactive lysine were reacted. These chemical and biological studies suggest that local cytotoxicity of glutaraldehyde crosslinked bioprostheses may be due to unstable glutaraldehyde polymers that persist in the interstices of crosslinked tissues.

Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.

N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.

Mechanism of pneumococcal cell wall degradation in vitro and in vivo

We compared the products of autolytic amidase-catalyzed wall degradation in vivo (in penicillin-induced lysis) and in vitro. Pneumococci labeled in their cell wall stem peptides by radioactive lysine were treated with penicillin, and the nature of wall degradation products released to the medium during lysis of the bacteria was determined. At early times of lysis (20% loss of wall label), virtually all the radioactive peptides released (greater than 94%) were of high molecular size and were still attached to glycan and teichoic acid. At times of more extensive bacterial lysis (56%), progressively larger and larger fractions of the released peptides became free, i.e., detached from glycan and teichoic acid. Analysis of the nondegraded residual wall material by high-resolution high-pressure liquid chromatography revealed that this in vivo-triggered autolysis did not involve selective hydrolysis of some of the chemically distinct stem peptides. Parallel in vitro experiments yielded completely different results. Purified pneumococcal cell walls labeled with radioactive lysine were treated in vitro with low concentrations of pure amidase, and the nature of wall degradation products released during limited hydrolysis and after more extensive degradation was determined. In sharp contrast to the in vivo experiments, the main products of in vitro hydrolysis were free peptides. After a short treatment with amidase (resulting in a 20% loss of label), the material released was enriched for the monomeric stem peptides. At all times of hydrolysis (including the time of extensive degradation), only a relatively small fraction of the released wall peptides was covalently attached to glycan and teichoic acid components (17% as compared with 40% in the intact cell wall). We propose that the in vivo-triggered amidase activity first attacks the amide bonds in some strategically located (or unprotected) stem peptides that hold large segments of cell wall material together. The observations indicate that the in vivo activity of the pneumococcal autolysin is under topographic constraints.

Beta-2-microglobulin of rat major histocompatibility complex class I antigens.

Detection of the beta 2-microglobulin (beta 2m) component of the rat MHC class I antigens has been difficult. In the present report, we have addressed this issue by a systematic study of rat class I antigens from red blood cells or from lymphocytes that were freshly isolated or cultured in the presence of autologous or heterologous sera and surface-labeled with 125I or intrinsically labeled with radioactive amino acids. First, specific radioiodination of rat beta 2m in association with the antigen heavy chain on red blood cells or lymphocytes is minimal, resulting in its poor identification by SDS-PAGE. Second, labeling with radioactive methionine or lysine gives a more intense beta 2m band with respect to the heavy chain than labeling with arginine or tyrosine. Third, the beta 2m component shows a large increase in intensity compared to the heavy chain when the antigens are isolated from lymphocytes that are cultured in the presence of fetal bovine serum prior to 125I-labeling. This increase is due to exchange of endogenous rat beta 2m with bovine beta 2m and to a much higher level of radioiodination of the latter. Fourth, rat red blood cells and lymphocytes contain free surface beta 2m molecules in addition to those associated with the antigen heavy chains. The free molecules show a much higher level of radioiodination than those associated with the heavy chains, and there is little exchange between the antigen-bound and the free beta 2m after radioiodination.(ABSTRACT TRUNCATED AT 250 WORDS)

Biosynthesis of collagen crosslinks: in vivo labelling of neonatal skin, tendon, and bone in rats.

Collagen crosslinks in neonatal rats were labelled in vivo by a single intraperitoneal injection of 200 microCi of [14C]lysine. Rats were killed at times ranging from 30 minutes to 10 weeks after injection. Whole skin, tendon, and bone were analyzed, after reduction and hydrolysis, for collagen crosslink content by HPLC. Crosslinks and amino acids were visualized by their incorporation of radioactivity from [14C]lysine and also fluorometrically by post-column derivatization with o-phthalaldehyde. The incorporation of 14C from labelled lysine into the principal difunctional reducible crosslinks, N6.6'-dehydro-5,5'-dihydroxylysinonorleucine and N6.6'-dehydro-5-hydroxylysinonorleucine, increased most rapidly between 4 and 12 hours after injection, results similar to those observed by others studying crosslink biosynthesis in vitro. Incorporation of 14C into the tetrafunctional crosslink histidinohydroxymerodesmosine proceeded more slowly than it did for the difunctional crosslinks. Values for the amount of radioactivity incorporated into the various crosslinks reached an apparent constant value between 3 and 5 days after injection for all three tissues studied. These values remained approximately constant for the duration of the experiment except for HHMD in tendon, which showed an increase in incorporated radioactivity at 8 and 10 weeks after injection. Direct chemical quantification of these same crosslinks by determination of the fluorescence of their o-phthalaldehyde adducts was also performed. We conclude that in vivo labelling of collagen crosslinks can be studied, at least in rapidly growing neonates, after a single injection of radioactive lysine. The results of such studies support previous suggestions by others about the rate of formation of difunctional crosslinks based upon studies using in vitro systems. Our results further suggest that formation of the tetrafunctional reducible crosslink histidinohydroxymerodesmosine proceeds relatively rapidly in vivo. Finally, we conclude that such labelled crosslinks are apparently quite stable after biosynthesis, suggesting the possibility of studies of the metabolic fate of collagen crosslinks over appreciable fractions of the lifetime of a rat.

Rapid exchange of histones H2A and H2B in sea urchin embryo chromatin

Chromatin from sea urchin blastula and gastrula was partially digested with micrococcal nuclease and separated electrophoretically in two dimensions into various nucleosome fractions and their component histones. After labelling of the embryos with radioactive lysine the various isohistones of the H2A and H2B group in all nucleosome fractions had incorporated approximately twice as much the radioactivity as the histones H2 and H4. The results are interpreted in terms of a labile nucleosome structure during transcription.

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli ( speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with M r s corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.

Stimulation of retinal capillary pericyte protein and collagen synthesis in culture by high-glucose concentration.

The influence of glucose concentration on cell multiplication and protein synthesis was studied in synchronized, long-term cultures of bovine retinal microvessel pericytes. The cell multiplication rate and the mitotic rate were reduced in media containing 20 mM glucose to 57% and 54%, respectively, of that obtained in media containing 5 mM glucose. Elevated glucose, however, did not change the DNA content of individual cells. Protein and collagen synthesis were measured by the incorporation of radioactive proline and lysine, or the posttranslational production of hydroxyproline and hydroxylysine, respectively. High glucose stimulated protein and collagen synthesis per cell 2.2 +/- 0.10 (SD) and 2.1 +/- 0.06 times, respectively. Aspirin (0.5 mM), an inhibitor of nonenzymatic glycosylation, did not alter the effect of elevated glucose concentration on protein and collagen synthesis.

Biodegradation of fatty acylamino acids byfusarium culmorum

Abstract Biodegradation of the fatty acylamino acids by Fusarium culmorum, measured in terms of the release of radioactive aspartate and lysine, occurred maximally at pH 6.5 and pH 7.0, respectively in 10 day cultures. Thirty‐six percent and twenty‐four percent of the total radioactivity recovered were in released aspartate and lysine, respectivley at 30°C. Twenty degrees (C) was the minimum temperature for biodegradation of these compounds by F. culmorum. Greater degradation was observed at 15°C and 30°C. The data suggest the activity of hydrolytic isoenzymes, with optima at different pH's and temperatures, operating in the biodegradation process.

The role of lysine-rich proteins in the acute steroidogenic response of rat adrenal cells to ACTH

The possibility that a lysine-rich protein was involved in the acute stimulation of steroidogenesis by ACTH was investigated using [ 3H] labelled lysine and isolated adrenal cells. The results demonstrated that cycloheximide inhibited steroidogenesis in a dose-dependent, rapid fashion and inhibited the incorporation of radioactive lysine into protein. However cells incubated in a lysine-free medium showed the same response to ACTH as cells incubated in a lysine-containing medium. It was also demonstrated that ACTH had no effect on the incorporation of tritiated lysine into the protein or small peptide fractions. These observations suggest that a rapidly synthesised, lysine-rich protein is not involved in the acute response to ACTH.

Intermolecular reducible cross-links in rat glomerular basement membrane.

Glomerular basement membrane (GBM) was purified from adult rats and treated with tritiated borohydride for analysis of the reducible cross-links. After acid hydrolysis, samples were subjected, with and without prior gel filtration on Biogel P-2, to a chromatographic system standardized with known cross-links. The major peak of radioactivity co-eluted with authentic di-hydroxylysinonorleucine (di-OHLNL) standard. Derivation of this cross-link from lysine was corroborated by demonstrating its presence in GBM purified after incubation of isolated glomeruli with [14c]-lysine and after in vivo injection of [14C]-lysine. GBM labeled in vitro with radioactive lysine also contained peaks co-eluting with hydroxylysinonorleucine and lysinonorleucine standards, as well as at unidentified positions in the chromatogram. The findings indicate that rat GBM contains lysine-derived cross-links, of which di-OHLNL is the major reduced form.

The synthesis and deposition of the prolamin storage proteins (secalins) of rye

The synthesis and deposition of the endosperm storage proteins of rye, usually termed secalins, has been studied. The rate of accumulation of secalin in developing rye grain was at a maximum between 3 and 5 weeks after anthesis. Some changes in the proportions of the four major groups of secalin polypeptides were observed during maturation, notably an increase in γ-secalins of Mr 75k and a decrease in ω-secalins. In-vitro translation of mRNA fractions prepared from 4-week-old endosperms showed that secalin polypeptides were synthesised on membrane-bound polysomes. The secalin products were identified by their mobilities on SDS-PAGE and their relative incorporation of radioactive lysine, glycine, proline, leucine and methionine. Protein bodies prepared by sucrose density ultracentrifugation contained reduced amounts of γ-secalins of Mr 40k and ω-secalins compared with the total secalin fraction, but these components were present in the expected amounts when 1.0 M NaCl was added to the buffers. Treatment of the protein bodies with proteinase-k resulted in the digestion of their contents regardless of the presence of NaCl, indicating that the surrounding membrane was incomplete. It was concluded that the NaCl reduced the loss of secalins from the protein bodies by decreasing secalin solubility rather than by affecting the integrity of the protein body membrane. The results reported for the synthesis and deposition of secalins are consistent with the results of previous studies on the prolamins of wheat and barley.