The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

The effect of age on enolase turnover in the free-living nematode, Turbatrix aceti.

The rates of synthesis and degradation of enolase and total soluble proteins slow with age in the free-living nematode, Turbatrix aceti. The half-lives are 73 and 58 h for soluble protein and enolase, respectively, in young organisms (5 days old). The respective figures are 163 and 161 h for old organisms (22–30 days old). Similar slowing of protein turnover occurs when the organisms are aged by a repeated screening procedure which avoids the use of fluorodeoxyuridine, an inhibitor of DNA synthesis normally added to aging cultures to obtain synchrony. The results support the idea that slowed protein turnover may be responsible for the formation of altered enzymes in old organisms.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.     

Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.     

Monoclonal antibodies to leucine enkephalin

Monoclonal antibodies to leucine enkephalin have been produced after fusion of mouse myeloma cells with spleen cells from hyper-immune mice. Hybrid clones 2D1 and SL1 were characterised using radioimmunoassay and an enzyme-linked immunosorbent assay. The antibody 2D1 was of low affinity and showed a maximum sensitivity of 0.1ng. The antibody binds equally well to the sulphated leucine enkephalin and to methionine enkephalin. It does not cross-react with dynorphin, methionine enkephalin-arg-phe or oxidised methionine enkephalin. The hybrid clone SL1 appears to be specific for leucine enkephalin. Preliminary immunocytochemical studies have shown that both antibodies bind specifically to leucine enkephalin in defined areas of the central nervous system.     

Changes in blood gas tensions and functional residual capacity in chronic asthmatics treated with disodium cromoglycate

Twenty chronic asthmatic subjects were treated with disodium cromoglycate in open trial. Although in the group as a whole spirometric findings improved, some patients showed a significant increase in arterial oxygen tension or a significant fall in functional residual capacity without any appreciable changes in spirometry. These changes may partly explain the clinical efficacy of this drug.