Coupling of DTPA to proteins: a critical analysis of the cyclic dianhydride method in the case of insulin modification.

The reaction between the cyclic dianhydride of diethylenetriaminepentaacetic acid (DTPA), a bifunctional reagent, and proteins under various conditions was studied using porcine insulin as a model protein. The reaction was compared with that between citraconic anhydride, a monofunctional reagent, and insulin. Products were characterized chromatographically and electrophoretically before and after deesterification by hydroxylamine. A DTPA-conjugated product was further characterized by proteolytic fragmentation. The reaction with citraconic anhydride yielded the expected number of products exclusively acylated on amino groups. In contrast, the reaction with the cyclic dianhydride of DTPA under all conditions examined yielded a much higher number of products than expected. Among the products formed were O-acylated ones and products of intermolecular cross-linking. It is concluded that the use of the cyclic dianhydride of DTPA does not allow the reliable preparation of proteins or other macromolecules conjugated with a high number of DTPA molecules in which each molecule of DTPA is linked to one amino group of the macromolecule through a single amide bond.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

On the reaction of methyleneaminoacetonitrile in aqueous media

Methyleneaminoacetonitrile was found to give a number of products under mild hydrolytic conditions. The products identified by ion-exchange chromatography include iminodiacetic acid, iminodiacetonitrile, N-(cyanomethyl)glycine, N-(carbamoylmethyl) glycine, and N-(cyanomethyl)glycine amide along with glycine, aminoacetonitrile, and glycine amide. Compounds of biological significance such as peptides and hydroxy amino acids were not found. The results were well consistent with those obtained for aminoacetonitrile under similar conditions.     

Mechanisms of amino acid polycondensation on silica and alumina surfaces

Chemisorption products of bifunctional amino acid vapours on the surface of silica and alumina have been studied by the method of infrared spectroscopy. On the basis of the analysis of spectral data it is supposed that heterogeneous polycondensation of amino acids with formation of peptides proceeds under these conditions. The supposition was confirmed by the study of products of interaction of amino acid vapours with silica and alumina by the method of fast atom bombardment mass-spectrometry. It is established that in contrast to alumina the condensation of amino acids into linear peptides on silica surface proceeds only at presence of at least small amounts of water. The most probable mechanisms of extending of peptide chains are proposed on the basis of obtained experimental data.     

Biomphalaria glabrata: respiration, calcium and end products of carbohydrate metabolism

1. Lactic acid and succinic acid (end products of anaerobiosis) also occur under aerobic conditions in the haemolymph and excretion products of Biomphalaria glabrata. This phenomenon has been investigated in more detail. 2. Experiments on oxygen uptake, and analyses of organic acid, amino acid and calcium were carried out under various aerating conditions, various temperatures and in various water qualities. 3. No differences were found in the concentrations of the organic acids and calcium in the haemolymph under different aerating conditions. 4. Neither snail-conditioned water, nor artificial crowding effects played a role in the initiation of anaerobic respiration. 5. A low exposure temperature (4 degrees C) initiated anaerobic respiration in spite of the aeration.     

The degradation of glycoproteins with lithium borohydride. Isolation and analysis of O-glycopeptides with reduced C-terminal amino acid residue

By the example of fetuin and a blood-group-specific mucin from porcine stomach, we showed that, under conditions of reductive degradation of glycoproteins with LiBH4-LiOH in 70% aqueous tert-butyl alcohol, the reduction and cleavage of amide bonds occur much faster than the simultaneous beta-elimination of carbohydrate chains O-linked with Ser and Thr residues of the peptide chain. The major degradation products containing the O-linked glycans are the O-glycosylated derivatives of 2-aminopropane-1,3-diol and 2-aminobutane-1,3-diol (the products of reduction of glycosylated Ser and Thr) and the glycopeptides containing 2-4 amino acid residues with reduced C-terminal amino acid. Seventeen homogeneous O-glycopeptides were isolated from the fetuin degradation products by ion-exchange and reversed-phase HPLC. Their structures were determined by MALDI-TOF mass spectrometry and by analyses for amino acids, amino alcohols, and carbohydrates. The application of the reaction for characterization of O-glycans and localization of O-glycosylation sites in O- and N,O-glycoproteins is discussed.     

Aspects of the Drought Tolerance in Creosotebush (Larrea divaricata)

In order to understand better the physiological adaption of creosotebush (Larrea divaricata Cav.) to drought conditions, its carbohydrate and nitrogen metabolism after a 7-day desiccation period under controlled conditions were studied. Although fructose was not significantly altered in the leaves of desiccated plants, as compared to those maintained under normal moisture conditions, both glucose and sucrose were significantly reduced. Total amino acids more than doubled under moisture stress, the increase being predominantly due to proline, phenylalanine, and glutamic acid. Significant increases also occurred in alanine, arginine, histidine, isoleucine, and valine. Increases or decreases in other amino acids were not significant. These stress-induced changes in certain amino acids are considered in relationship to protein hydrolysis, to accumulation of nitrogen degradation products translocated from the roots, and to the possible function of specific amino acids (e.g., proline) in NH₃⁺ storage.     

Effects of environmental conditions on microbial proteolysis in a pork myofibril model system

P.M. KENNEALLY, N.G. FRANSEN, H. GRAU, E.E. O'NEILL & E.K. ARENDT.1999.A number of bacterial strains used for meat fermentations were screened for proteolytic activity. A strain of Micrococcus which was found to be proteolytic was evaluated for the effects of environmental conditions on its proteolytic activity against pork myofibrillar proteins using response surface methodology. Three strains of micrococci were also tested for the ability to produce free amino acids from pork myofibrils. Analysis of the effects of environmental conditions showed that proteolytic activity would be minimal under conditions normally found in fermented sausages, thereby suggesting that proteolysis in these products is largely due to endogenous meat enzymes. The three strains of micrococci were shown to produce free amino acids from pork myofibrils, thereby demonstrating the presence of peptidase activity in these strains.     

Étude du mécanisme d'établissement des liaisons glutaraldéhyde-protéines

Commercial aqueous 25 per cent glutaraldehyde solutions contain no stable derivative of this aldehyde, but compounds of variable molecular weight which easily revert to glutaraldehyde. The effect of pH on the reaction of glutaraldehyde with amino acids and on the stability of the products under acid conditions, shows the importance of the structure modification of the dialdehyde which occurs when pH increases, and even leads to precipitation in highly alkaline solutions. This precipitate results from aldol condensation of glutaraldehyde molecules. It contains aldehyd groups conjugated with ethylenic double bonds. Such a structure reacts with amino groups to give an imino bond, stabilized by resonance with the ethylenic bond, and does not undergo Michael-type addition reactions.Therefore, glutaraldehyde does not react with proteins under its free form, but as an unsaturated polymer, which gives imino bonds stabilized by conjugation.     

Polypeptide modification and cross-linking by oxidized 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.     

Analysis of folding intermediates from the main toxin of Naja naja samarensis containing two blocked cysteines

The formation of folding intermediates blocked by iodoacetate from the main toxin of Naja naja samarensis was monitored by FPLC and the populations were identified by amino acid analyses. We have determined conditions allowing for the highest yield in the populations with two blocked cysteines. The free cysteines remaining under these conditions were labeled with iodo[14C]acetate and localized by peptide mapping in one of the products isolated by ion-exchange chromatography (NT3III). We have also investigated the effects on the native-like characteristics of such molecules of an incubation at equilibrium with a mixture of cysteine and cystine. We find that many different molecular populations are present during the folding process and that disulfide exchanges allow for the reconstitution of native-like products having open disulfides even under strongly denaturing conditions.     

Cleavage of Mengovirus Polyproteins In Vivo

The synthesis of mengovirus-specific proteins in vivo was studied by labeling the viral proteins with radioactive amino acids under conditions in which host protein synthesis was almost completely inhibited. Pulse-chase experiments enabled the kinetic analysis of the cleavages of certain viral protein precursors and the formation of others. The pattern of cleavages of mengovirus precursor polypeptides is similar to that of encephalomyocarditis virus. The major difference between the two viruses seems to be in the molar concentration in which the various primary products are produced. The molar ratio of the A protein, which is the precursor of the capsid proteins, to that of the primary products F and C, is approximately 1.5 to 2.0: 1: 1. Possible explanations for the unequal appearance of the structural and nonstructural proteins are discussed.     

Role of conserved amino acids in the catalytic activity of Escherichia coli primase

The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate-size products. From these data we discuss the significant step of the 2-nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability.     

In vitro nutritional requirements and metabolic products of pathogenic and nonpathogenic strains of Cryptobia salmositica: essential carbohydrates and amino acids

Pathogenic and nonpathogenic strains of Cryptobia salmositica cultured in minimum essential medium (MEM) with several monosaccharides, disaccharides and amino acids were observed for differences in multiplication and motility. Metabolic end products (i.e. alanine, aspartate, carbon dioxide, lactate and pyruvate) were measured for logarithmically growing cells under aerobic conditions. The pathogenic strain of C. salmositica multiplied more readily in MEM supplemented with D(-)ribose, D(+)xylose, D(+)galactose, D(+)glucose, D(+)mannose and D(-)fructose. However, there were no significant differences in multiplication when the strains were cultured with the monosaccharide D(-)arabinose. The nonpathogenic strain multiplied significantly better than the pathogenic strain in the presence of the disaccharides alpha-lactose, maltose and sucrose. It also multiplied more readily when the amino acids L-glutamine and D(-)proline were added to MEM. The end products of carbohydrate catabolism under aerobic conditions were alanine, aspartate, carbon dioxide, lactate and pyruvate.     

Effect of different cell culture conditions on the polypeptide integrity and N-glycosylation of a recombinant model glycoprotein

The effect of different short-term controlled cell culture conditions on the product quality of a genetically engineered human interleukin-2 N-glycosylation variant protein expressed from a baby hamster kidney cell line (BHK-21) has been investigated. A perfused 2-L stirred tank reactor was used. Products purified from the culture supernatant of cells grown under experimentally initiated nutrient limitations (glucose, amino acids, pO(2)) were characterized by their HPLC-elution profile, SDS-PAGE and western blotting, amino acid sequencing as well as for their N-linked carbohydrates, using "HPAEC-PAD fingerprinting" and methylation analysis. The glycoprotein products secreted from cells under the different culture conditions (kept for 24 h, after an adaption time period of 48 h) showed an almost identical oligosaccharide pattern. By contrast, short-term changes of the culture condition led to considerable differences in the ratio of glycosylated to unglycosylated protein forms. Significant amounts of NH(2)-terminally truncated polypeptide forms were observed. They lacked proponderantly the first two amino acids; however, under certain culture conditions forms lacking up to eight NH(2)-terminal amino acids were detected. (c) 1995 John Wiley & Sons, Inc.     

Simulation of cell growth,substrate consumption and product formation with recombinant Escherichia coli

Summary The growth of recombinant Escherichia coli, consumption of different amino acids and glucose as well as fusion protein formation are simulated by a structured mathematical model and compared with measurements carried out in a stirred tank reactor under well-controlled conditions. The model parameters were identified based on the variation in cell concentration, particular amino acids, and glucose as well as intracellular products during cultivation. The dependence of the model parameters on cultivation time and culture conditions (temperature, medium composition, dissolved oxygen concentration) are discussed.Offprint requests to: K. Schügerl     

Enzymatic hydrolysis of keratin-containing stock for obtaining protein hydrolysates

Optimization of the process of enzymatic hydrolysis of keratin-containing stock aimed at obtaining hydrolysates of high biological value has been performed. The increasing of the stock/water weight ratio, the amount of the alkaline protease preparation from Acremonium chrysogenium added and the temperature of the reaction mixture resulted in an increase in the yield and antioxidant capacity of hydrolysis products. The molecular masses of soluble products obtained under optimal hydrolysis conditions ranged from 3.55 to 3.60 kDa. High antioxidant capacity, 100% bioavailability and a well-balanced amino acid composition was characteristic of the hydrolysis products.     

Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge,a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. ¹³C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using ³H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions.