Raymond J. Paxton

Induction of a human carbonyl reductase gene located on chromosome 21

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.

Characterization and partial amino acid sequence of human plasma glutathione peroxidase

Human plasma glutathione peroxidase was purified to homogeneity and partially sequenced. Overlapping peptide fragments from three endopeptidase digests permitted the determination of one sequence of 32 contiguous amino acids and one sequence of 23 contiguous amino acids. Five additional unique peptide sequences without obvious overlaps were obtained. The sequence of 32 amino acid residues aligns with positions 82-113 of human cytosolic glutathione peroxidase with nine mismatches without gaps or insertions. The sequence of 23 amino acid residues aligns with positions 157-178 with six mismatches and an insertion of one residue. Three additional peptide sequences with no obvious sequence homology to glutathione peroxidase can be aligned based on the sequence of a cDNA clone encoding plasma glutathione peroxidase that was isolated from a human placental library. The plasma enzyme is a homotetramer composed of 21-kDa subunits which cannot reduce phospholipid hydroperoxides. These results indicate that the plasma glutathione peroxidase is distinct from both the classical cytosolic enzyme and the monomeric phospholipid hydroperoxide glutathione peroxidase. Only a negligible amount of glutathione peroxidase activity was detected in bile, indicating that the liver exports plasma glutathione peroxidase exclusively to the circulation.

Highlights of protein structural analysis

This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.

Cysteine and Tryptophan Amino Acid Analysis of ABRF92-AAA

Publisher Summary This chapter presents a study involving the cysteine and tryptophan amino acid analysis of ABRF92-AAA. The 1992 ABRF amino acid analysis test sample was bovine pancreatic chymotrypsin, and was chosen because of its relatively high Cys and Trp content. The Sigma preparation was dissolved in 0.1% trifluoroacetic acid, dialyzed against the same solvent to remove salts, the concentration determined by amino acid analysis and 56 μ g (2.2 nmol) aliquots of chymotrypsin dried in small plastic tubes using a Savant Speed Vac. The sample was sent as an unknown to 231 ABRF facility directors for analysis using amino acid analysis methods of their choice. Each participating laboratory was asked to report pmol of amino acids found using their standard analysis method, and to quantify Cys and Trp using additional methods and analyses. The standard analysis results formed the major part of most facilities composite data, and absolute yield of protein and average error was calculated from these data. In a few instances, the standard data set was patently inferior to that obtained by the Cys or Trp method, and the better data set was used to calculate yield of protein and average error.

The hydrolysis process and the quality of amino acid analysis: ABRF-94AAA collaborative trial

Publisher Summary Amino acid analysis remains an indispensable tool in a variety of biological research and development fields, for example, the biochemical study of proteins, quality control in biotechnology and nutrition, and in clinical analyses. This chapter discusses primarily on discriminating between hydrolysis and all other aspects of amino acid analysis as potential sources of error. Two samples are distributed, one pre-hydrolyzed and one to be hydrolyzed, and their absolute yields are determined with very similar accuracy and standard deviations. The average errors in determining individual amino acids, however, are significantly different for the two samples, with smaller errors in the pre-hydrolyzed sample. However, direct cystine analysis of the pre-hydrolysate is as successful as that of the protein sample, suggesting that chromatography and derivatization, and not hydrolysis are the most important factors in successful analysis of cystine by this simple methodology. Thus, it is clear that excellent analyses can be and are done by any of the available technologies, with proper concern for the many pitfalls peculiar to each method.

Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences

Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.

Collaborative Trial Analyses of ABRF-91AAA

Publisher Summary The ABRF studies to date have examined the recovery, accuracy, and precision of amino acid analysis using either synthetic peptides, purified proteins, or a protein hydrolysate. These studies concentrate on the sensitivity of analysis with hydrolyse and analyses being performed in the 0.1–5 μg range. The accuracy of analyses has generally been in the 80–90% range. This chapter discusses an attempt at investigating the quality of analyses under conditions where laboratories might feel more comfortable and where ample supply of sample was available. Three general aspects were explored. The first two were a realistic assessment of the accuracy of absolute quantitation and of the accuracy of relative yields of residues obtainable at present. The third aspect was the analysis of the problem amino acids tryptophan and cystine/cysteine.

Amino Acid Analysis of Phospho-Peptides: Abrf-93AAA

Publisher Summary This chapter describes the amino-acid analysis of phospho-peptides. The 1993 ABRF amino-acid analysis test sample (ABRF-93AAA) was a 30 residue synthetic peptide with four intended phosphorylation sites. The chapter presents average peptide yields obtained by the participating facilities. With all 49 sites included, the overall average peptide yield was 11.1 ± 23.8 nmol. When 10 sites were eliminated from both extremes, this value narrowed considerably to 7.0 ± 0.7 nmol. The average peptide yields from these central 29 sites were very similar and within 8% of the amount distributed. The chapter discusses the average percentage error obtained by each participating laboratory in the analysis of common amino acids in ABRF-93AAA. The post-column techniques were more accurate and had a narrower error range. Eighty-two percent of the sites with errors greater than the overall average error were pre-column sites. The lowest error was similar for both techniques and obtained using a pre-column method.

Structural Analysis of Carcinoembryonic Antigen (CEA) and a Related Tumor-Associated Antigen (TEX)

Carinoembryonic antigen (CEA), first described by Gold and Freedman(1), is one of the most widely investigated human tumor- associated antigens. CEA was originally detected in colonic adenocarcinoma and fetal gut, but has since been detected in other malignant and non-malignant tissues. The chemistry, biochemistry, immunology, tissue distribution and clinical aspects of CEA were recently reviewed(2). Using anti-CEA sera, immunologically crossreacting antigens have been detected in several tissues as described in Table 1. The crossreacting antigens share at least one antigenic determinant (epitope) with CEA and for the most part share epitopes with each other.

Protein Sequence Analysis of Highly-Glycosylated Proteins: Carcinoembryonic Antigen (CEA) as a Model System

Post-translationally modified proteins, especially glycoproteins, offer unique challenges to the protein chemist. In general, glycoproteins are resistant to proteolytic enzymes, and those peptides that are generated are difficult to separate (1,2). Secondly, the identification of glycosylation sites in these proteins is usually accomplished by indirect methods (1–4). When a glycopeptide is sequenced, a phenylthiohydantoin is usually not observed for the glycosylated residue. If the amino acid composition for the glycopeptide agrees with the observed sequence and suggests asparagine (in the case of N-glycosylation) or serine or threonine (in the case of O-glycosylation), then the blank cycle is assumed to be the glycosylated residue. This subtractive method relies on an accurate amino acid composition and a complete sequence for the peptide, excepting the blank cycle.