Character Of Amino Acids Research Articles

Bioenergetic and structural consequences of allotopic expression of subunit 8 of yeast mitochondrial ATP synthase. The hydrophobic character of residues 23 and 24 is essential for maximal activity and structural stability of the enzyme complex.

Subunit 8 (Y8), a mitochondrially encoded subunit of the F0 sector of the F1F0-ATP synthase is essential for oxidative phosphorylation. We have previously introduced the technique of allotopic expression to study the structure/function of Y8, whereby an artificial Y8 gene is expressed in the nucleus of cells lacking a functional mitochondrial Y8, thus generating assembly of a functional F1F0-ATPase complex. In this paper we show that when a gene encoding an essentially unmodified version of Y8 is allotopically expressed, ATP synthesis and hydrolysis rates, as well as efficiency of oxidative phosphorylation, were similar to those of the parental wild-type strain in which Y8 is naturally expressed in mitochondria. We then tested the requirement for the hydrophobicity of the central domain (residues 14-32), which possibly represents a transmembrane stem, by introducing adjacent negative charges at different positions of Y8. One of the variants thus generated, which carries the double substitution Leu23-->Asp, Leu24-->Asp, when expressed in a strain lacking endogenous Y8, gave rise to cells which grew very slowly by oxidative phosphorylation. Measurement of bioenergetic parameters showed two major defects in these cells relative to control cells allotopically expressing unmodified Y8. First, the activity of the F1F0-ATP synthase was significantly decreased. ATP synthesis and state 3 of respiration were reduced by approximately 30-40%. ATP hydrolysis was reduced by approximately 30% and was almost insensitive to the F0 inhibitor oligomycin. Second, the physical coupling between the two sectors of the enzyme, as well as the stability of the F1 sector itself, were affected as shown by decreased recovery of F0 sector [8, 9, b, oligomycin sensitivity-conferring protein (OSCP), d, h and f] and F1 sector (alpha, gamma, delta) subunits in immunoprecipitates of ATP synthase. This study indicates that Y8 not only performs an important role in the structure of the mitochondrial complex but also in its activity. We conclude that the hydrophobic character of amino acids 23 and 24 in the middle of the putative transmembrane stem of Y8 is essential for coupling proton transport through F0 to ATP synthesis on F1.

Structural Studies of a UDP-glucuronosyltransferase gene from the plaice ( Pleuronectes platessa)

A partial length cDNA coding for the putative PAH-inducible phenol-conjugating UDP-glucururonosyltransferases (UGT) isoform of plaice was used to isolate overlapping clones from a plaice genomic library. Sequence analysis revealed the presence of a complete gene spanning 4.1 kb for a plaice UGT which showed a strong conservation in exon structure, amino acid character and amino acid sequence with mammalian UGT1 family genes, although additional alternative upstream exon 1s were not identified in the present study. Southern blot analysis revealed a low copy number for the gene and some degree of structural polymorphism in gene structure between individuals. This was also reflected in the finding that there were significant variations between the nucleotide sequences of the plaice gene and the cDNA previously isolated from a different individual fish. Future studies will investigate the possibility that there may be phenotypic variations which could lead to alterations in susceptibility to pollutant toxicity.

Phylogenetic Information Content of Five Nuclear Gene Sequences in Animals: Initial Assessment of Character Sets from Concordance and Divergence Studies

The phylogenetic information content of five nuclear genes encoding amino acid sequences was assessed on the basis of their character support of well-established phy? logenies and their ability to recover these phylogenies using parsimony methods. The five genes encode dopa decarboxylase, phosphoenolpyruvate carboxykinase, RNA polymerase ll (largest subunit), elongation factor-2, and elongation factor-la. The analyses employ the unambiguously alignable portions of all available animal sequences for these genes, representing a range of divergence times from about 10 to >550 million years ago. Separate analyses, including character mappings on the test phylogenies and, when different, on the most-parsimonious trees, were performed with nucleotides separated by codon position and with amino acids. Pairwise sequence divergences were calculated for all comparisons and are related to character information content. Amino acid and most nucleotide character sets recovered the test phylogenies, although with varying degrees of support, homoplasy, and resolution, confirming that these genes contain substantial phylogenetic information. An estimate is provided of the time span over which each character set in each gene will prove phylogenetically most informative. These genes should prove widely useful for systematics. (Nuclear genes; animals; character sets; sequence diver? gences; concordance studies.)

Some aspects of the organization and evolution of the genetic code

In this paper, I define a measure of the relative position of each amino acid in the genetic code by means of a 21-dimensional vector describing its potential for mutation, in a single step, to each of the other amino acids, or to a chain termination codon. This measure allows us to make a systematic investigation of the type and number of the physicochemical properties of the amino acids that were involved in evolution. The polar character and size of amino acids are identified in this analysis as properties that played a leading role in the evolutionary history of the genetic code. The application of cluster analysis and discriminant analysis reveals the characteristics of the structural organization of the genetic code. Finally, I suggest the existence of a relationship between the molecular weight of the amino acids and the number of synonymous codons.

A biochemical study of the toxicity of S-dichlorovinyl- l-cysteine

A study has been made of the biochemical effects of S-dichlorovinyl- l-cysteine (DCVC) and the corresponding mercaptopropionic acid (DCVMP) and cysteamine (DCVCA) derivatives in the rat and guinea-pig. In rat-kidney slice experiments using glucose as substrate DCVC and DCVMP inhibited respiratory activity at a concentration of the order of 10 −3M. DCVCA was without effect. The inhibitory action of DCVC was also demonstrated on guinea-pig kidney slices. Loss of respiratory activity was accompanied by increased levels of pyruvic and lactic acids in the incubation medium. With liver and kidney mitochondria from both species moderate or substantial inhibition of both unstimulated and dinitrophenol (DNP)-stimulated respiration occurred appropriate to the concentration of DCVC in the medium. Similar results were obtained with DCVMP in both types of rat mitochondria but DCVCA, however, exercised no effect on rat-kidney mitochondrial respiration. Intraperitoneal doses of 50 mg DCVC/kg impaired DNP-stimulated respiration of rat-kidney mitochondria which persisted for over 48 hr and could not be reversed by simple washing techniques. With 15 mg DCVMP/kg administered intraperitoneally the effect on DNP-stimulated respiration was both less marked and less persistent. There was no evidence to suggest that DCVC or DCVMP acts as an alkylating agent. The incorporation of 14C-labelled amino acids into protein of the pancreas, but not of the liver or kidney, was inhibited by intraperitoneal administration of DCVC. DCVMP also inhibited amino acid uptake into pancreatic and in addition kidney protein. These and other observations seem to indicate that the amino acid character of DCVC is not important with regard to its toxicity which is possibly mediated at the mitochondrial level.