The human Hb (mu) class glutathione S-transferases are encoded by a dispersed gene family

The human glutathione S-transferases are products of a gene superfamily which consists of at least four gene families. The various glutathione S-transferase genes are located on different human chromosomes, and new gene(s) are still being added to the gene superfamily. We have characterized a cDNA in pGTH4 encoding human glutathione S-transferase subunit 4 (GST mu) and mapped its gene (or a homologous family member) on chromosome 1 at p31 by in situ hybridization. Genomic Southern analysis with the 3' noncoding region of the cDNA revealed at least four human DNA fragments with highly homologous sequences. Using a panel of DNAs from mouse-human somatic cell hybrids in genomic DNA hybridization we show that the Hb (or B) genes of human glutathione S-transferases are on three separate chromosomes: 1, 6, and 13. Therefore, the glutathione S-transferase B gene family, which encodes the Hb (mu) class subunits, is a dispersed gene family. The GST mu (psi) gene, whose expression is polymorphic in the human population, is probably located on chromosome 13. We propose that the GST mu (psi) gene was created by a transposition or recombination event during evolution. The null phenotype may have resulted from a lack of DNA transposition just as much as from the deletion of an inserted gene.     

Enhancement of the catalytic properties of human carbonic anhydrase III by site-directed mutagenesis

Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.     

Binding of myotoxin a to sarcoplasmic reticulum Ca(2+)-ATPase: a structural study.

The interaction of myotoxin alpha with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca(2+)-ATPase, the major SR protein responsible for calcium loading. Ca(2+)-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca(2+)-ATPase was established by isolating chemically cross-linked myotoxin a-Ca(2+)-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with 125I-myotoxin a showed that the C-terminal fragment competed better against 125I-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with 125I-myotoxin a, while a second peptide (residues 13-28) did not.     

Mutual regulation between mitochondrial ATPase and respiratory chain activities

Compounds made from the reaction of fluorescamine with simple primary amines and with mycosamine-containing macrolide antibiotics (e.g., amphotericin B) are used to investigate possible interactions between ATPase and respiration enzymes in rat liver mitochondria. The following observations have been made. (1) The acyclic form of the benzyl amine-fluorescamine compound stimulates the ATPase-linked inorganic phosphate formation, and this stimulation is not affected by rotenone, antimycin A, and potassium cyanide. In contrast, the respiratory inhibitors are able to prevent the stimulation of ATPase activity that is caused by conventional uncouplers e.g., 2,4-dinitrophenol. (2) The acyclic form of the amphotericin B-fluorescamine compound has no effect on ATPase-linked inorganic phosphate formation rate. However, in the presence of the antibiotic-fluorescamine compounds, the respiratory inhibitors are no longer able to prevent the uncoupler-stimulated ATPase activity. (3) The amine-fluorescamine modifiers have no effect on rotenone-sensitive NADH-cytochrome c reductase, on succinate-cytochrome c reductase, and on cytochrome oxidase in submitochondrial particles. (4) The amine-fluorescamine modifiers decrease the rate of the ATP-driven NAD⁺ reduction by succinate in submitochondrial particles. (5) The amine-fluorescamine modifiers inhibit the stimulation of respiration that is caused by conventional uncouplers, although the modifiers have no effect on the kinetics of the proton influx induced by uncouplers. The data are consistent with the hypothesis that the ATPase-linked and respiration-linked proton pumps may interact directly with each other, and this step establishes the mutual regulation between ATPase and respiratory activities.     

Taxonomic relations between archaebacteria including 6 novel genera examined by cross hybridization of DNAs and 16S rRNAs

DNAs from 16 species of archaebacteria including 6 novel isolates were hybridized with 16S rRNAs from 7 species representing different orders or groups of the urkingdom of archaebacteria. The yields, normalized for the number of genes per microgram of DNA, and the temperature stabilities of all hybrids were determined and related to each other. A taxonomic tree constructed from such fractional stability data reveals the same major divisions as that derived from comparative cataloging of 16S rRNA sequences. The extreme halophiles appear however as a distinct order besides the three known divisions of methanogens. The methanogens, the halophiles and Thermoplasma form one of two clearly recognizable branches of the archaebacterial urkingdom. The order represented by Sulfolobus and the related novel order Thermoproteales form the other branch. Three novel genera, Thermoproteus, Desulfurococcus and the "stiff filaments" represent three families of this order. The extremely thermophilic methanogen Methanothermus fervidus belongs to the Methanobacteriales. SN1, a methanogen from Italy, appears as another species of the genus Methanococcus. Another novel methanogen, M3, represents a genus or family of the order Methanomicrobiales.     

Chemical and functional homology of myotoxin a from prairie rattlesnake venom and crotamine from South American rattlesnake venom

Myonecrosis is a serious result of rattlesnake bite and constitutes a persistent clinical problem. In the current study we have isolated crotamine from the venom of Crotalus durissus terrificus to test its ability to cause structural damage to skeletal muscle, and to make direct chemical comparisons with Myotoxin a, a myotoxic polypeptide we recently isolated from prairie rattlesnake (Crotalus viridis viridis) venom. Disc gel electrophoresis, isoelectric focusing, circular dichroic spectroscopy, and amino acid analysis, all indicated a high degree of chemical similarity. Light microscope histology revealed that crotamine caused vacuolizationof skeletal muscle fibers, qualitatively the same as the vacuolization caused by Myotoxin a. The ability of these two basic snake venom polypeptides to cause structural damage to skeletal muscle fibers has significant implications toward more complete understanding of the cause of snake venom-induced myonecrosis.     

Nucleotide recognition seouence at the cleavage site of Haemophilus aegyptius II (Hae II) restriction endonuclease

We have determined the nucleotide sequence recognized by the restriction endonuclease Hae II from Haemophilus aegyptius which cleaves the simian virus 40 (SV40) DNA at a single specific site. By using terminal radioactive labeling of the cleavage site at both the 5′ and 3′-ends we have deduced the recognition sequence,

with elements of a two-fold rotational symmetry. The endonuclease produces staggered ends with protruding 3′-terminated single-strands, four nucleotides in length. In plasmid RSF 2124 DNA, which contains multiple Hae II cleavage sites, it was observed that the 5th nucleotide from the 3′ terminus is either a pdA or a pdG, indicating alternating recognition sequences.