Immunoreactivity of a 70 kD protein purified from Mycobacterium bovis Bacillus Calmette-Guerin by monoclonal antibody affinity chromatography.

The protein antigens from Mycobacterium bovis (BCG), M. tuberculosis, and M. leprae share a number of common determinants. We have used a murine mAb (L7) recognizing such a determinant on a protein of Mr 70,000 to purify this antigen from M. bovis sonicate by affinity chromatography. Enrichment of the protein in column eluates was confirmed by immunoblotting and in antigen inhibition assays. After radiolabelling with 125I, the protein could be immunoprecipitated with human lepromatous leprosy sera. Stimulation of peripheral blood mononuclear cells from BCG-vaccinated and naturally mantoux-positive individuals induced proliferation and IFN-gamma secretion, while intradermal injection of purified antigen into the same subjects resulted in a delayed-type hypersensitivity reaction. Thus, the 70,000 molecule carried epitopes capable of reacting with B cells, and eliciting a potentially protective T cell response. The first 15 N-terminal residues were sequenced using a gas-phase sequenator.

Potent effects of human galanin in man: growth hormone secretion and vagal blockade.

Human galanin (hGAL) is a 30-amino acid neurohormone that has recently been shown to differ significantly from porcine and rat GAL. We investigated the endocrine and cardiovascular effects of hGAL in eight male volunteers. On three separate occasions, each received a 90-min infusion of saline, low dose (33 pmol/kg.min) and high (132 pmol/kg.min) dose hGAL, combined with an iv glucose bolus (to assess effects on insulin and GH release). hGAL was undetectable, 1.4 +/- 0.2 nmol/L, and 3.7 +/- 0.5 nmol/L during control, low dose, and high dose studies, respectively. The half-life of hGAL was 3.5 +/- 0.5 min. GH levels rose significantly in both studies (vs. control) and were not suppressed by hyperglycemia [low dose area under the curve (AUC), 1827 +/- 348 micrograms/min.L (P < 0.05); peak, 19.5 +/- 5.3 micrograms/L; high dose AUC, 1896 +/- 401 micrograms/min.L (P < 0.005); peak, 28.0 +/- 7.5 micrograms/L]. PRL levels rose significantly with the high dose study only (AUC, 12.8 +/- 1.1 micrograms/min.L; P < 0.01). FSH, LH, and catecholamine levels were unchanged. Glucose-stimulated insulin release was not inhibited. There was a dose-dependent increase in pulse rate and a profound decrease in sinus arrhythmia, but no change in blood pressure. These cardiovascular effects have not been reported with studies in humans using GAL of other species. We conclude that hGAL may play an important role in man in modulating GH secretion and cardiac vagal tone, but not insulin release.

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apo VLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences.

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellenin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.

Major acute phase alpha 1-protein of the rat is homologous to bovine kininogen and contains the sequence for bradykinin: its synthesis is regulated at the mRNA level.

The complete amino acid sequence of major acute phase alpha 1-protein of the rat (MAP) was derived from the nucleotide sequence of cloned cDNA. Amino acid analysis and partial sequencing supported the predicted sequence. The amino acid compositions of MAP and rat low-Mr kininogen are identical within experimental variation. The sequence is homologous (60%) to that of bovine low-Mr kininogen and both proteins carry the sequence for the vasoactive nonapeptide bradykinin in their C-terminal region. The rate of synthesis of MAP and the levels of MAP mRNA change coordinately during the acute phase response to inflammation.

Chemical techniques employed for the primary structural analysis of proteins and peptides.

This chapter summarises modern microchemical approaches to the purification, identification and primary structure analysis of peptides and proteins. Discussion of high-sensitivity purification methods is restricted to two-dimensional polyacrylamide gel electrophoresis (2-DE) and microbore/capillary column reversed-phase high-performance liquid chromatography (RP-HPLC). Associated techniques are discussed, particularly with respect to analysis of the products with current automated amino acid sequencers and mass spectrometers.

Amino acid analysis using phenylisothiocyanate prederivatization: elimination of the drying steps.

Results of experiments on the procedure for amino acid analysis via analysis of the phenylthiocarbamyl amino acids are reported. It was found that yields of some amino acids varied in the presence of salt and with changes in the vacuum drying steps. An improved procedure is described which includes a standard addition of salt to the hydrolysate before drying it; the redrying step is omitted and the post derivatization drying is replaced by a simple addition of heptane to the reaction mixture.

Formation of proline thiohydantoin with ammonium thiocyanate: progress towards a viable C-terminal amino-acid-sequencing procedure.

Pure amino acid thiohydantoins are required as reference standards for development of C-terminal-sequencing procedures based on thiohydantoin formation of the C-terminal amino acids of peptides and proteins. Proline thiohydantoin was prepared using a straightforward method involving reaction of acetylproline with ammonium thiocyanate. It was characterized by UV spectrophotometry, mass spectrometry and back-hydrolysis to the free amino acid. These data establish unequivocally that the thiocyanate procedure is applicable to proline as well as to the other common amino acids. This work also validates earlier claims that proline thiohydantoin can be prepared by reaction with thiocyanic acid.

The protein sequenator: a new extraction principle and a metal reaction cup.

A simpler and more efficient extraction procedure is proposed for solvent extraction of the Edman degradation procedure in the protein sequenator. The method is less likely than the conventional one to cause wash-out of peptides or proteins from the reaction cup while the very small solvent volumes required substantially decrease the running costs of the instrument. It has been used successfully (98% repetitive yield) with a metal reaction cup in the sequenator.

Primary structure of apovitellenin I from hen egg yolk and its comparison with emu apovitellenin I.

The amino acid sequence of apovitellenin I from hen egg yolk has been determined using both automatic and manual procedures; it comprises 82 residues. Hen apovitellenin shows considerable homology with emu apovitellenin I which contains 84 residues. Besides two deletions in the sequence, the hen protein differs in 28 positions from the emu protein; 26 of these positions may have arisen from single base changes. The changes are largely conservative ones, which suggest that the structure and function have been preserved despite extensive mutation.

Amino acid sequence of cyanogen bromide fragment CN2 from Hong Kong influenza haemagglutinin heavy chain.

The amino acid sequence of cyanogen bromide peptide CN2 from the heavy chain (HA1) of the haemagglutinin of the Hong Kong variant A/Memphis/102/72 has been obtained by direct, automated sequence analysis on the whole fragment and by manual dansyl-Edman degradation of tryptic, peptic and chymotryptic peptides. It was found to contain 92 amino acid residues, including a large, insoluble, tryptic core peptide (residues 62-87). It did not contain any half-cystine residues or carbohydrate. The determination of its structure was complicated by the presence of an Asn-Ile bond at positions 48-49 which was readily cleaved by both trypsin and chymotrypsin.

Amino acid sequences of alpha-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment.

1. The helical fragments obtained by partial chymotryptic digestion of S-carboxymethylkeratine-A, the low-sulphur fraction from wool, were fractionated into type-I and type-II helical segments in aqueous urea under conditions limiting carbamoylation. 2. The amino acid sequence of a 109-residue type-II segment was completed by using the sequenator. 3. When the data were incorporated into a helical model of 3.6 residues per turn the hydrophobic residues generated a band aligned at a slight angle to the helical axis. This result is in accord with the postulated coiled-coil structure of the crystalline regions of alpha-keratin.