Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Transaminase of brainched chain amino acids. X. High activity in stomach and pancreas.

The activity of branched chain amino acid transaminase (EC 2.6. 1.6) was found to be 8 to 10 times higher in rat stomach and pancreas than in heart and kidney, which were previously thought to be the tissues with the highest activity. For comparison, the activities of two other transaminases, aspartate transaminase (EC 2.6.1.1) and alanine transaminase (EC 2.6.1.2) in different parts of the digestive tract were measured. However, their activities were not especially high in the stomach and pancreas, and in the pancreas the activity of branched chain amino acid transaminase was higher than those of the two other transaminases. The isozyme of branched chain amino acid transaminase in the stomach and pancreas was identified as enzyme I by DEAE cellulose chromatography and immunochemistry. The rates of oxidation of [U-¹⁴C]-L-leucine by slices of stomach and pancreas were also higher than by slices of other tissues.     

Changes in the exercise-induced hormone response to branched chain amino acid administration.

It was the aim of the present experiment to detect possible effects of branched-chain amino acids (BCAA) on the endocrine response to 1 h of continuous running. Blood samples were collected from 14 long-distance runners (age 24-42 years) in two different trials performed at 1-week intervals. In both trials (E and P) blood samples were collected at the following times: 9 a.m. (basal values sample), 10.30 a.m. (sample 90), 11.30 a.m. (sample 150), 12.30 p.m. (sample 210); the athletes performed 1 h of running at a constant predetermined speed between samples 90 and 150. Following the basal sample a mixture containing BCAA (E trial), or not containing BCAA (P trial) was ingested. In both trials no hormone basal concentrations, except insulin, were changed before exercise. In P trial, following exercise (sample 150), human growth hormone (HGH), prolactin (PRL), adrenocorticotropic hormone (ACTH) and cortisol (C) increased, while testosterone (T) decreased. In sample 210, after 1 h of rest, while ACTH, PRL and HGH had recovered to basal concentrations, C remained elevated and T displayed a further decrease. In the E trial a similar pattern of change was observed in sample 150 for HGH, PRL, ACTH and C; in sample 210 HGH and PRL displayed significantly lower values than in the corresponding P trial samples. The T was not modified by the running exercise and increased during the recovery period. It is, therefore, suggested that BCAA administration before exercise affects the response of some anabolic hormones, mainly HGH and T.     

The amino acid sequence of rabbit J chain in secretory immunoglobulin A.

The primary structure of rabbit J chain, which occurs covalently bound to secretory IgA, was determined. J chain was isolated in its S-carboxymethylated form, in one step, by SDS/PAGE followed by electro-elution; 5 nmol of protein (approx. 75 micrograms), in all, was necessary for the determination of the complete sequence by the 'shot-gun' microsquencing technique; with the use of several site-specific endoproteinases, the various digests of S-carboxymethylated J chain were separated by micro-bore reverse-phase h.p.l.c. and the partial N-terminal sequences of all peptides were analysed. From the sequence alignment, gaps were filled by further extensive sequencing of the relevant overlapping fragments isolated from selected digests. Rabbit J chain comprises 136 amino acid residues, out of which eight are conserved cysteine residues, and is more closely similar to the human sequence (73.5% identify) than to the mouse sequence (68% identity). There is one unique glycosylation site at asparagine-48.     

Porcine pancreatic lipase. Sequence of the first 234 amino acids of the peptide chain.

The single polypeptide chain of about 460 amino acids of porcine pancreatic lipase (EC 3.1.1.3) has been fragmented into five peptides by cyanogen bromide cleavage [Rovery, M., Bianchetta, J. & Guidoni, A. (1973) Biochim. Biophys. Acta, 328, 391--395]. The sequence of the first three cyanogen bromide peptides (CNI, CNII, CNIII), including a total of 234 amino acids, was fully elucidated. Automatic or manual Edman degradation was performed on the different peptides. Fragmentations of the CN peptides were accomplished by digestions with trypsin (after citraconylation or 1,2-cyclohexanedione treatment), chymotrypsin and Staphylococcus aureus external protease. Hydrolysis of unreduced material by pepsin and thermolysin, performed in order to determine the S-S bridge positions, provided useful overlapping peptides. The glycan moiety of lipase is bound to Asn-166. The non-essential tyrosine specifically blocked by diisopropylphosphorofluoridate is Tyr-49 in a cluster of asparagine and glutamine residues. The existence of a highly hydrophobic sequence (206--217) at the C terminus of the CNII fragment is noteworthy.     

Branched chain amino acid aminotransferase isoenzymes of Pseudomonas cepacia

Pseudomonas cepacia grew rapidly using a mixture of all three branched chain amino acids as carbon source, but failed to use individual branched chain amino acids as sole carbon source. Extracts of bacteria grown on branched chain amino acids had between 2- and 3-fold higher levels of -ketoglutarate-dependent branched chain amino acid aminotransferase activity than extracts of glucose-grown bacteria. The increase in enzyme activity was due to the presence of a second aminotransferase not detected in extracts of glucose-grown bacteria. The enzyme, which presumably plays a role in branched chain amino acid degradation, had an apparent molecular weight (mol. wt.) of 75,000. The other aminotransferase was formed constitutively and apparently functions in synthesis of branched chain amino acids. It was more stable than the 75,000 mol.wt. enzyme, and was purified to homogeneity and found to be a 180,000 mol.wt. oligomer containing 6 subunits of approximately 30,000 mol.wt. Antiserum prepared against the purified enzyme inhibited its activity but failed to influence the activity of the 75,000 mol.wt. aminotransferase, suggesting that the two isoenzymes are encoded by different genes.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Characterization of tryptic peptides from porcine haptoglobin light chain and an amino acid sequence.

The tryptic peptides derived from porcine haptoglobin light chains have been separated and characterized by composition, chromatography, electrophoretic mobility, and partial sequencing. Depending on homology with the corresponding human polypeptide, the amino acid sequence of the chain is proposed. Twenty differences from the human chain are indicated in the total of 84 residues.     

Site-directed mutagenesis at amino terminus of recombinant ricin A chain

Successful immunotoxin therapy may depend upon reduction of the size of the components in order to decrease antigenicity and rate of clearance. In initial attempts to modify the A chain of ricin by deletion analyses within a prokaryotic expression system, coding sequences were modified by the insertion of unique restriction endonuclease sites and by the removal of 22 codons near the 5' terminus. The work presented here examines the expression, solubility, and activity of these mutant proteins and demonstrates that while amino acid residues may be altered in this region, the deletion of residues 19 through 40 yields an insoluble and inactive toxin molecule.     

The amino acid sequence of a human κ light chain

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the kappa type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other kappaI chains (repeated variants). Based on these repeated variants a subdivision of the kappaI subgroup is proposed.     

Variability in the amino terminus of myosin light chain 1

Three naturally occurring variants of myosin light chain 1, type I, II, and III from avian fast-twitch muscle, have been analyzed by reverse-phase HPLC peptide mapping and amino acid sequencing. Difference peptides were absent from accompanying digests of the related protein, myosin light chain 3, indicating that the heterogeneity was located in the N-terminal 50 residues unique to light chain 1. The type II variant possessed the previous published sequence for the protein [Nabeshima Y., Fujii-Kuriyama, Y., Muramatsu, M., & Ogata, K. (1984) Nature (London) 308, 333-338]. The type I variant, which migrates faster than the type II on SDS gene electrophoresis, contained a Pro----Ala substitution at residue 15, turning the Lys-Pro-(Ala)5(Pro-Ala)7 stretch in this region into Lys-Pro-(Ala)7(Pro-Ala)6. The type III variant, which migrates just faster than the type I, had an (Ala)2 deletion in the (Ala)5 run, yielding Lys-Pro-(Ala)3-(Pro-Ala)7. As indicated by the SDS gel migration rates, the type I and III variants are significantly shorter in length than the type II. The benign nature of the changes is consistent with a flexible arm function for the N-terminal region of light chain 1, with the structural changes in the variants occurring in the spacer region of the arm.(ABSTRACT TRUNCATED AT 250 WORDS)