Horse Liver Alcohol Dehydrogenase

Abstract

1. The ethanol-active isoenzyme of horse liver alcohol dehydrogenase has been carboxymethylated with iodo[14C]acetate and different samples of the labelled protein treated with trypsin (both with and without previous maleylation of the substrate), chymotrypsin, pepsin or cyanogen bromide. In each case most of the resultant, peptides have been purified and characterized.2. Data are given for those peptides originating from an N-terminal region comprising about 4001, of the protein chain and the amino acid sequence of the first 140 residues is deduced.3. It is established that over this region the two chains of the enzyme have an identical sequence. The N-terminus is an acetylated serine residue and the "active site" cysteine residue occupies position no. 46. One oft he two tryptophans of the chain, but none of the four tyrosines, is in the sequence given. The region around position 100 is rich in cysteine residues. Some serine residues were found to have reacted during maleylation of the protein. 4.The amino acid sequence is compared to the previously known sequences of glyceraldehyde 3-phosphate dehydrogenase from two species. Similarities strongly suggest a true homology, a t least of an N-terminal region, and support theories of a common ancestral origin for these enzymes. The reactive cysteine residues are, however, a t widely different positions in the primary structures.

Isolation and characterization of bovine vasoactive intestinal peptide (VIP)

“…Digestion of bovine VIP (134 pg) with TPCKtreated trypsin (5 pg) in 0.1 M ammonium bicarbonate (50 ~1) was performed at 37°C for 4 h. Fragments were separated by high-voltage paper electrophoresis at pH 6.5 [8], and eluted with water. Total compositions were determined on a Beckman 121 M amino acid analyser, after hydrolysis at 110°C for 24 h in evacuated tubes with 6 M HCI containing 0.5% phenol.…”

“…Total compositions were determined on a Beckman 121 M amino acid analyser, after hydrolysis at 110°C for 24 h in evacuated tubes with 6 M HCI containing 0.5% phenol. N-terminal amino acids were analyzed by the dansyl method and manual sequence analysis was performed with the dansyl-Edman method [8,9]. Liquid phase sequencer degradation was carried out in a Beckman 890 C instrument, using the 0.1 M quadrol peptide program and double identifications of residues liberated [lo].…”

Rat liver alcohol dehydrogenase. Characterisation of alkylated cysteine residues in the carboxymethylated protein

“…A chymotryptic digest of 1'" C] carboxymethylated rat liver alcohol dehydrogenase ( 100 mg with 5 X 10 7 cpm) was separated into six fractions [4] by chromatography in 0.1 M NH4 HC03 on Sephadex G-50 (column dimensions: 2.5 X 100 cm; recovery of radioactivity: over 95%). Purification of l4 C-peptides in each fraction yielded a total of 12 different major radioactive peptides.…”

The tryptic peptides of rabbit muscle triose phosphate isomerase

“…The solvent pyridine-acetic acid-waterethanol (9:16:1000:30, by vol.) (Jornvall, 1970) was useful for the identification of Dns-histidine.…”