Interaction of F1-ATPase and its inhibitor peptide. Effect of dinitrophenol, nucleotides and anions

1. ATPase natural inhibitor interacted in a mixed non-competitive manner with compounds affecting hydrolytic activity. 2. Ka's for DNP, HCO3- and free ATP, and Ki's for SCN- and ADP became smaller as inhibitor peptide concentration increased, reflecting an increase in affinity of F1-ATPase for these compounds induced by the peptide. 3. Activators increased the peptide inhibitory effect, whereas inhibitors decreased it. 4. A two-step model for the peptide-enzyme interaction is suggested in which ATP hydrolysis is a key factor.     

Purification of human haptoglobin 1-1, 2-1, and 2-2 using monoclonal antibody affinity chromatography

Similar to blood type, human plasma haptoglobin (Hp) is classified as 3 phenotypes: Hp 1-1, 2-1, or 2-2. The structural and functional relationship between the phenotypes, however, has not been studied in detail due to the complicated and difficult isolation procedures. This report provides a simple protocol that can be used to purify each Hp phenotype. Plasma was first passed through an affinity column coupled with a high affinity Hp monoclonal antibody. The bound material was washed with a buffer containing 0.2M NaCl and 0.02 M phosphate, pH 7.4, eluted at pH 11, and collected in tubes containing 1M Tris-HCl, pH 6.8. The crude Hp fraction was then chromatographed on a HPLC Superose 12 column in 0.05 M ammonium bicarbonate at a flow rate of 0.5 ml/min. The homogeneity of purified Hp 1-1, 2-1, or 2-2 was greater than 95% as judged by SDS-polyacrylamide gel electrophoresis. Essentially, each Hp isolated was not contaminated with hemoglobin and apolipoprotein A-I as that reported from the other methods, and was able to bind hemoglobin. Neuraminidase treatment demonstrated that the purified Hp possessed a carbohydrate moiety, while Western blot analysis confirmed alpha and beta chains corresponding to each Hp 1-1, 2-1, and 2-2 phenotype. The procedures described here represent a significant improvement in current purification methods for the isolation of Hp phenotypes. Circular dichroic spectra showed that the alpha-helical content of Hp 1-1 (29%) was higher than that of Hp 2-1 (22%), and 2-2 (21%). The structural difference with respect to its clinical relevance is discussed.     

Fluorescent probe studies of haptoglobin type 2-1.

Haptoglobin is an alpha2 serum protein that forms an irreversible complex with hemoglobin. The combination between these two macromolecules resembles the binding of an antigen to its antibody except that the complex remains soluble. This investigation was undertaken to determine the nature of the hydrophobic sites on haptoglobin type 2-1. The interaction of 1-anilinonphthalene-8-sulfonate (ANS) with haptoglobin type 2-1 is characterized by a flourescence intensity in solutions containing ANS and haptoglobin as the pH is decreased from 9 to 4. The dissociation constant for the ANS interaction with haptoglobin 2-1 is 5.8 x 10--5 M at pH 7.0, 5.2 X 10--5 M at pH 5.0 AND 30.3 X 10--5 M at pH 4.0. Fmax shows no change in the pH range 6-9 but does show an increase at pH 4.0 when compared to the neutral region.     

The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Meiotic Gene Conversion Mutants in SACCHAROMYCES CEREVISIAE . I. Isolation and Characterization of pms1-1 and pms1-2

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.