A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs.

A novel glutathione S-transferase (GST) was purified from broccoli (Brassica oleracea var. italica). Partial amino-acid sequencing indicated that the protein shared significant homology with several different plant GSTs from maize, silene, Dianthus, Nicotiana and Triticum, but little homology to yeast (Issatchenkia) GST. One region of the polypeptide near the N-terminal also shared significant homology to a region of rat 5-5, rat 12-12 and human theta-GST (collectively referred to as the theta-GST-class) but little structural homology to the common mammalian cytosolic GSTs (alpha-, mu- or pi-classes). The broccoli GST was retained on a novel membrane based glutathione affinity matrix and displayed activity towards 1-chloro-2,4-dinitro-benzene (CDNB), a general GST substrate, as well as 4-nitrophenethyl bromide, a marker substrate for the theta-class of GSTs. The characteristics of the broccoli GST potentially define it as a member of the theta-class. This is consistent with the view that the theta-class may have arisen prior to the divergence of animals and plants while the mammalian mu-, pi- and alpha-classes evolved after the two kingdoms were established.

High resolution 2-D peptide mapping with subsequent analysis of peptides by microsequencing or lectin binding directly from PVDF membrane blots.

Peptide mapping is a technique that is frequently used to characterize proteins. Typically, the method involves the cleavage of proteins in solution or in a gel with the subsequent separation of the peptide fragments on a 1-D SDS PAGE gel. Electrophoretic peptide maps are often used to compare homologous proteins from related organisms to derive evolutionary relationships. Other applications of peptide mapping include immunoblotting studies of selected proteins. Two-dimensional peptide mapping, a less common technique, has traditionally involved a combination of thin layer or paper chromatography and electrophoresis. Amino acid sequencing of peptides that were separated using this method and then subsequently blotted to PVDF membrane was reported recently. However, the resolution achieved with these methods is far below that which can be achieved with conventional 2-D electrophoresis of proteins in polyacrylamide gels. This report describes an electrophoretic system for the high resolution 2-D separation of peptides in gels with subsequent blotting to a novel cationic PVDF membrane, Immobilon-CD, and microsequencing directly from the 2-D blot. In addition, the high resolution peptide maps can be further analyzed by techniques such as lectin probing to determine post-translational modifications.

Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.