Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase.

Escherichia coli isocitrate lyase was inactivated by iodacetate in a pseudo-first-order process. Complete inactivation was associated with the incorporation of only one carboxymethyl group per enzyme subunit. The substrate and products of the enzyme protected against inactivation, suggesting that the reactive group may be located at the active site. Isolation and sequencing of a carboxymethylated peptide showed that the modified residue was a cysteine, in the sequence Cys-Gly-His-Met-Gly-Gly-Lys. The reactivity of isocitrate lyase to iodoacetate declined with pH, following a titration curve for a group of pKa 7.1. The Km of the enzyme for isocritrate declined over the same pH range.     

Multielement analysis of biological samples by inductively coupled plasma-mass spectrometry

Interest in the biological behavior of a growing number of elements, along with increasing recognition of the importance of interactions among them, demands a versatile and reliable technique for multielement analysis of biological samples. Significant improvements over the sensitivity achieved with conventional inductively coupled plasma (ICP) optical emission spectrometries have been realized with the introduction of quadrupole mass spectrometry (MS) for detection of ions in the plasma. The hybrid technique of ICP-MS promises to be a method of rapid multielement analysis, at detection limits that approach or surpass those of other technologies. However, the application of ICP-MS to analyses of biological interest is truly in its infancy. Here we report the use of ICP-MS for the determination of more than 30 elements of biological interest in a tissue and a biological fluid (rat liver and serum, respectively). Experimental values of the elements serve as a basis for discussion of analytical protocols, performance criteria, and certain problems peculiar to ICP-MS.     

The glue protein of ribbed mussels (Geukensia demissa): a natural adhesive with some features of collagen

Summary The Atlantic ribbed musselGeukensia (Modiolus)demissa attaches itself to the roots of cord grass and other hard objects in tidal salt marshes by spinning adhesive byssal threads. The precursor of a protein apparently present in the adhesive plaques of the threads was isolated in quantity from the foot of the mussel. The protein has an apparent molecular weight of 130000, a pI of 8.1, and contains a high proportion of Gly, Glu/Gln, Lys and 3,4-dihydroxyphenyl-l-alanine (DOPA). Sequence of tryptic peptides suggests a pattern of repeated motifs, such as: Gly-DOPA-Lys, and X-Gly-DOPA-Y-Z-Gly-DOPA/Tyr-Lys, where X is Thr or Ala in octapeptides and Gln-Thr in nonapeptides. Y is variable, but more often than not hydrophobic; and Z is frequently Pro or 4-trans-hydroxyproline (Hyp). The presence of Pro-Gly and Hyp-Gly sequences of -hydroxylysine in the protein is reminiscent of typical collagens; however, the protein is not labile to clostridial collagenase, nor does collagen cross-react with antibodies raised against the mussel protein. Unlike typical collagens, Gly probably occurs only at every 4th or 5th residue in this unusual mussel protein.Abbreviations Anti-Gdfp anti-G. demissa foot protein - Dopa 3,4-dihydroxyphenylalanine - CTAB cetyltrimethylammonium bromide - HPLC high performance liquid chromatography - PAGE polyacrylamide gel electrophoresis Supported by grants from the National Science Foundation (DMB 8500301) and the Office of Naval Research (N00014-86K-0717)     

Substitution of an invariant nucleotide at the base of the highly conserved ''530-loop'' of 15S rRNA causes suppression of yeast mitochondrial ochre mutations.

We have determined the nucleotide sequence alteration in the 15S rRNA gene of a Saccharomyces cerevisiae strain carrying the previously described mitochondrial ochre suppressor, MSUI. The suppressor contains an A residue at position 633 of the yeast mitochondrial sequence, in place of the wild-type G. This position, located in the highly conserved region forming the stem of the '530-loop', corresponds to G517 of the Escherichia coli 16S rRNA and is occupied by G in all other known small rRNA sequences. This finding strongly supports the previous conclusions of others that the 530-loop region plays an important role in enhancing translational accuracy.     

Fluorescence lifetime studies with staphylococcal nuclease and its site-directed mutant. Test of the hypothesis that proline isomerism is the basis for nonexponential decays.

Using frequency domain methods, the fluorescence decay of Trp-140 in staphylococcal nuclease and its site-directed mutant (Pro-117----Gly) has been examined. Based on nuclear magnetic resonance (NMR) studies (Evans, P. A., C. M. Dobson, R. A. Kautz, G. Hatfull, and R. O. Fox. 1987. Nature [Lond.]. 329:266-268), it is believed that nuclease exists in two macroscopic, native conformations and that the slow interconversion of these conformations is controlled by the cis----trans isomerization of Pro-117. The above mutant shows only one native conformation in NMR experiments. To test the hypothesis that the biexponential fluorescence decay of Trp-140 of nuclease can also be related to the existence of these conformational states of the protein, we have compared the decay patterns of the wild type and mutant. Essentially no difference was observed, which indicates that there is some other basis for the nonexponential decay of Trp-140. We have used global nonlinear least squares analysis to link the fit of data at several temperatures.     

Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata to identified volatile constituents from calling males

Fifty-six compounds from the odor of calling, sexually mature, laboratory reared males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) were isolated by headspace trapping on Tenax columns and identified using GC/MS techniques (69 total compounds were detected). Electroantennogram responses (EAGs) to 54 of the 56 identified compounds as well as 5 analogs were tested on both sexes. Significant differences between the sexes in their responsiveness were found in 9 of the 54 identified compounds tested. There was no correlation between the amplitude of the EAG response and the relative abundance of compound identified from headspace analysis. Of the five major identified components, three elicited relatively small EAG responses, while two elicited large EAGs compared to the hexan-1-ol standard. The relative ranking of EAG responses were: methyl and ethyl hexenoates and hexanoates > C₄–C₆ esters and/or acetates > ethyl and methyl octenoates > monoterpenes > sesquiterpenes > C₂–C₅ acetates, alcohols and ketones. Behavioral bioassays on each of the five major identified components as well as a blend of six of the compounds showed some degree of attractancy to virgin females which in some cases approached the response to a pheromonal standard (male odors absorbed onto filter paper). These results are discussed in relationship to the insect's antennal sensitivity to putative pheromone components and/or allomonal components and to other reported C. capitata pheromone studies.

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Résumé Cinquante-six composés de l'odeur de mâles de C. capitata Weidemann, élevés en laboratoire, sexuellement mûrs et en appel, ont été isolés par piégeage sur colonnes tenax et identifiés par la technique GC/MS (69 composés avaient été détectés en tout). Les électroantennogrammes (EAGs) ont été examinés chez les deux sexes pour 54 des 56 composés identifiés et 5 de leurs analogues. Des différences significatives entre les sexes ont été observées pour 9 des 54 composés identifiés. Il n'y avait pas de corrélation entre l'ampleur de l'EAG et l'abondance relative du composé lors de son isolement. Pour les 5 principaux composés identifiés, 3 ont induit des EAGs relativement faibles, tandis que 2 étaient importants, par comparaison avec l'Hexane-1-ol utilisé comme témoin. Le classement relatif des EAG a été: hexénoates et hexanoates d'éthyl et de méthyl C₄–C₆ esters et/ou acétates octénoates d'éthyl ou de méthyl monoterpènes sesquiterpènes C₂–C₅ acétates, alcools et kétones. Les expériences de comportement avec chacun des 5 composés principaux identifiés, comme avec des mélanges de 6 composés ont mis en évidence une attraction des femelles vierges qui dans quelques cas avoisine la réponse à la phéromone témoin (odeur du mâle absorbée sur papier filtre). Ces résultats sont discutés en fonction de la sensibilité de l'antenne d'insexte aux composés supposés de la phéromone et aux composés allomonaux, et en fonction des autres études connues sur les phéromones de C. capitata.

     

Stereoselective, strong inhibition of ribonucleotide reductase from E. coli by cisplatin

Using ribonucleotide reductase (EC 1.17.4.1) purified from E. coli clones with overproducing plasmids for the B1 and B2 subunits, respectively, studies have been carried out of the inhibition of this enzyme by cisplatin. Under anaerobic conditions, using the dithiol, reduced form of the enzyme, it was found that ribonucleotide reductase is extremely sensitive to cisplatin: greater than 90% inhibition was achieved with 2-fold molar excess of platinum reagent even at 10(-8)M enzyme. Inhibition was essentially instantaneous and irreversible to G-25 gel filtration. The site of inhibition was found to be the B1 subunit. Transplatin was much less effective. Inhibition of the enzyme by cisplatin (molar ratio cisplatin:B1 = 4.3) led to a decrease in thiol titre corresponding to approximately 1 thiol group per dimer of B1 subunits under conditions leading to 94% inactivation of the ribonucleotide reductase activity.