Interaction of purple membrane with solvents. II. Mode of interaction.

Using the solubility parameter mapping technique (Eisenbach, M., Caplan, S.R. and Tanny, G (1979) Biochim. Biophys. Acta 554, 269-280) we studied spectroscopically the mode of interaction between the purple membrane of Halobacterium halobium and pure organic solvents or solvent mixtures. Although the interacting solvents formed a well-defined closed region in the interaction maps, mapping the modes of interaction did not reveal a closed region for each spectrally classifiable type. A suggested interpretation for this is that interaction with the purple membrane chromophore requires that a solvent (or solvent mixture) possess apolar groups in order to obtain access to the chromophore, together with a polar character and hydrogen-bonding capacity. The mode of interaction, however, is dependent on the specificity of the reactive group of the solvent for retinal, and this has nothing to do with membrane properties. We also examined the influence of the duration of the interaction and of illumination. Some solvents appeared to react more sluggishly than others, but no generalization in terms of the solubility parameter mapping was found, probably because the map describes thermodynamic rather than kinetic phenomena. The only effect of illumination was to enhance the reaction of some of these solvents. It did not change the solubility parameters of purple membrane.     

Paramagnetic ion effects on the nuclear magnetic resonance spectrum of transfer ribonucleic acid: assignment of the 15--48 tertiary resonance.

We have studied the effects of Co2+ and Mn2+ ions on the low-field nuclear magnetic resonance (NMR) spectra of pure class 1 transfer ribonucleic acid (tRNA) species. With 1.2 mM tRNA in the presence of 15 mM MgCl2 discrete paramagnetic effects were observed for Co2+ at concentrations in the range 0.02--0.1 mM and for Mn2+ in the range 0.002--0.01 mM, indicating fast exchange of these cations with tRNA. Both of these cations paramagnetically relax the s4U8--A14 resonance as well as other resonances from proximal base pairs. The Co2+ site appears to be the same site on G15 which was observed crystallographically [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315-328]; the initially occupied tight Mn2+ site is the cation site involving the phosphate of U8. There are three base pairs within 10 A of both sites, namely, G15--C48, A14--s4U8, and C13--G22; this has led to the assignment of the G15--C48 and C13--G22 resonances in the NMR spectrum [Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315--328; Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, Sung-Hou (1977) Nucleic Acids Res. 4, 2811--2820; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64--68].     

Modeling the cellular basis of altered excitation-contraction coupling in heart failure

Ca transients measured in failing human ventricular myocytes exhibit reduced amplitude and slowed relaxation [Beuckelmann, D.J., Nabauer, M., Erdmann, E., 1992. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85, 1046-1055; Gwathmey, J.K., Copelas, L., MacKinnon, R., Schoen, F.J., Feldman, M.D., Grossman, W., Morgan, J.P., 1987. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ. Res. 61, 70-76; Kaab, S., Nuss, H. B., Chiamvimonvat, N., O'Rourke, B., Pak, P.H., Kass, D.A., Marban, E., Tomaselli, G.F., 1996. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. 78(2); Li, H.G., Jones, D.L., Yee, R., Klein, G.J., 1992. Electrophysiologic substrate associated with pacing-induced hert failure in dogs: potential value of programmed stimulation in predicting sudden death. J. Am. Coll. Cardiol. 19(2), 444-449; Vermeulen, J.T., McGuire, M.A., Opthof, T., Colonel, R., Bakker, J.M.T.d., Klopping, C., Janse, M.J., 1994. Triggered activity and automaticity in ventricular trabeculae of failing human and rabbit hearts. Cardiovasc. Res. 28, 1547-1554.] and blunted frequency dependence [Davies, C.H., Davia, K., Bennett, J.G., Pepper, J.R., Poole-Wilson, P.A., Harding, S.E., 1995. Reduced contraction and altered frequency response of isolated ventricular myocytes from patients with heart failure. Circulation, 92, 2540-2549; Hasenfuss, G., Reinecke, H., Studer, R., Meyer, M., Pieske, B., Holtz, J., Holubarsch, C., Posival, H., Just, H., Drexler, H., 1994. Relation between myocardial function and expression of sarcoplasmic reticulum Ca-ATPase in failing and nonfailing human myocardium. Circ. Res. 75, 434-442; Hasenfuss, G., Reinecke, H., Studer, R., Pieske, B., Meyer, M., Drexler, H., Just, H., 1996. Calcium cycling proteins and force-frequency relationships in heart failure. Basic Res. Cardiol. 91, 17-22; Monte, F.D., O'Gara, P., Poole-Wilson, P.A., Yacoub, M., Harding, S.E., 1995. Cell geometry and contractile abnormalities of myocytes from failing human left ventricle. Cardiovasc. Res. 30, 281-290; Philips, P.J., Gwathmey, J.K., Feldman, M.D., Schoen, F.J., Grossman, W., Morgan, J.P., 1990. Post-extrasystolic potentiation and the force-frequency relationships: differential augmentation of myocardial contractility in working myocardium from patients with end-stage heart failure. J. Mol. Cell. Cardiol. 22, 99-110; Pieske, B., Hasenfuss, G., Holubarsch, C., Schwinger, R., Bohm, M., Just, H., 1992. Alterations of the force-frequency relationship in the failing human heart depend on the underlying cardiac disease. Basic Res. Cardiol. 87, 213-221.]. Analyses of protein levels in these failing hearts reveal that the SR Ca-ATPase is down-regulated on average by 50% and that the Na/Ca exchanger is up-regulated on average by a factor of two. In this paper, we test the hypothesis that this altered pattern of expression of Ca handling proteins is sufficient to account for changes in excitation-contraction coupling properties measured experimentally at the cellular level. To do this, we present an integrated model of excitation-contraction coupling in the guinea pig ventricular cell. The model is used to determine the effects of SR Ca-ATPase down-regulation and Na/Ca exchanger up-regulation on action potential duration, Ca transient shape and amplitude, and isometric force. Model analyses demonstrate that changes in Ca handling proteins play a direct and critical role in prolongation of action potential duration, and in reduction of contractile force in heart failure.     

Quantitative physiological study of the fast dynamics in the intracellular pH of Saccharomyces cerevisiae in response to glucose and ethanol pulses

Considering the effects of pH on many aspects of cell metabolism, such as its role in signaling processes and enzyme kinetics, it is indispensable to include the measurement of the dynamics of the intracellular pH, when studying the fast dynamic response of cells to perturbations. It has been shown previously that the intracellular pH rapidly drops following an increase in external glucose concentration [Kresnowati, M.T.A.P., Suarez-Mendez, C., Groothuizen, M.K., Van Winden, W.A., Heijnen, J.J., 2007. Measurement of fast dynamic intracellular pH in Saccharomyces cerevisiae using benzoic acid pulse. Biotechnol. Bioeng. 97, 86-98; Ramos, S., Balbin, M., Raposo, M., Valle, E., Pardo, L.A., 1989. The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae. J. Gen. Microbiol. 135, 2413-2422; Van Urk, H., Schipper, D., Breedveld, G.J., Mak, P.R., Scheffers, W.A., Van Dijken, J.P., 1989. Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621. Biochim. Biophys. Acta 992(1), 78-86]. The mechanism for this fast intracellular acidification, however, has not been elucidated yet. This paper presents a metabolome-based analysis to reveal the physiological phenomena that cause the fast intracellular acidification following either a glucose pulse or an ethanol pulse to carbon-limited chemostat cultures of Saccharomyces cerevisiae. This quantitative study, which includes the determination of intracellular buffering capacity, the calculation of electric charge balance and the quantification of weak organic acid transport shows that none of the previously suggested mechanisms, i.e. increase in glucose phosphorylation and accumulation of CO(2), is sufficient to explain the measured decrease in intracellular pH following a glucose pulse.     

Integrins regulate the intracellular distribution of eukaryotic initiation factor 4E in platelets. A checkpoint for translational control.

Recent evidence from our laboratory demonstrates that platelets synthesize numerous proteins in a signal-dependent fashion (Pabla, R., Weyrich, A. S., Dixon, D. A., Bray, P. F., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1999) J. Cell Biol. 144, 175-184; Weyrich, A. S., Dixon, D. A., Pabla, R., Elstad, M. R., McIntyre, T. M., Prescott, S. M., and Zimmerman, G. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5556-5561). Protein synthesis in platelets is controlled at the translational level; however, the mechanisms of regulation are not known. Here we demonstrate that translation initiation factors are redistributed to mRNA-rich areas in aggregated platelets, an event that induces protein synthesis. Interrogation of cDNA arrays revealed that platelet-derived mRNAs are primarily associated with the cytoskeletal core. In contrast, eukaryotic initiation factor 4E (eIF4E), the essential mRNA cap-binding protein that controls global translation rates, is localized in the membrane skeleton and soluble fraction of platelets, physically separated from most mRNAs. Platelet activation redistributes eIF4E to the cytoskeleton and increases interactions of eIF4E with mRNA cap structures. Redistribution of eIF4E to the mRNA-rich cytoskeleton coincides with a marked increase in protein synthesis, a process that is blocked when intracellular actin is disrupted. Additional studies demonstrated that beta(3) integrins are the primary membrane receptor that distributes eIF4E within the cell. These results imply that integrins link receptor-mediated pathways with mRNA-rich cytoskeletal domains and thereby modulate the organization of intracellular translational complexes. They also indicate that the functional status of eIF4E is regulated by its intracellular distribution.     

Control of cytochrome oxidase activity. A transient spectroscopy study.

The kinetics of cytochrome oxidase reconstituted into small phospholipid vesicles (COV) has been followed by transient optical spectroscopy under steady-state and pre-steady-state conditions, in the presence and absence of ionophores. The effect of valinomycin on the activity of reconstituted cytochrome oxidase is shown to depend on the absolute concentration of the ionophore and on the number of turnovers elapsed by the enzyme; this novel observation, which escaped previous investigations, may account for important differences in results and therefore in interpretation of the mechanism of control of the enzyme activity as between Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), Gregory and Ferguson-Miller (Gregory, L., and Ferguson-Miller, S. (1989) Biochemistry 28, 2655-2662) and Capitanio et al. (Capitanio, N., De Nitto, E., Villani, G., Capitanio, G., and Papa, S. (1990) Biochemistry 29, 2939-2944). Quantitative analysis of the optical spectra acquired within 10 ms over a large wavelength and time range (500-650 nm and 5 ms to 60 s) under different experimental conditions, indicates that the electrical component of the transmembrane electrochemical gradient controls the rate of the internal electron transfer from cytochrome a-CuA to cytochrome a3-CuB as well as the cytochrome c to cytochrome a electron transfer. The slow down of cytochrome oxidase activity observed in the presence of valinomycin after several (greater than 10) turnovers is attributed to alkalinization of the vesicle interior, which affects the internal electron transfer rate. These two mechanisms of control act most likely independently. A "cubic scheme," which illustrates the effect of the electrochemical gradient on two states of cytochrome oxidase characterized by different redox and proton pumping activities is presented and discussed.     

Books

Books reviewed in this article:
B aker . K. 1993. Identification Guide to European Non-passerines.
B arnard . C., G ilbert , F. & M c G regor
B askett , T.S., S ayre . M.W., T omlinson , R.E. & M irarchi .
B ezzel . E. 1993. Kompendium der Vögel Mitteleuropas.
B right , M. 1993. The Private Life of Birds.
C ook , M. 1992. The Birds of Moray and Nairn.
D avison . G.W.H. 1992. Birds of Mount Kinabalu. Borneo.
E rritzoe . J. 1993. The Buds of CITES and How to Identify Them.
F arner , D.S., K ing , J.R. & P arkes , K.C.
G ibbons , D.W., R eid , J.B. & C hapman . R.A. (eds). 1993. The New Atlas of Breeding Birds in Britain and Ireland.
H illman , J.C.
H uxley . E.
J ackson . C.E. 1993. Great Bird Paintings of the World.
J ohnsgard . P.A. 1993. Cormorants, Darters and Pelicans of the World.
M adge . S. & B urn , H. 1994. Crows and Jays. A Guide to the Crows, Jays and Magpies of the World.
N icolai . B. (ed.).
P ower , D.M. (ed.).
P riklonskiy . S.G. (ed.).
R alph . R. 1993. William MacGillivray.
R obinson , D. & C hapman , A.
S harp . P.J. 1993. Avian Endocrinology.
S mith , K.W., D fe , C.W., F earnside . J.D., F letcher , E.W. & S mith , R.N.
S olomon . D. & W illiams , J.
S ørensen , S., B loch . D. & L angvad . S.
Z immerman , J.L.     

NMR studies of ion binding to Escherichia coli tRNAPhe

The effects of magnesium, spermine, and temperature on the conformation of Escherichia coli tRNAPhe have been examined by proton and phosphorus nuclear magnetic resonance spectroscopy. In the low-field proton NMR spectra we have characterized two slowly interconverting conformations of this tRNA at low magnesium ion concentrations. The relative proportion of the conformers is ion dependent but not ion specific. Magnesium affects protons in all the stems of tRNA while spermine effects are localized near the s4U-8-A-14 and G-15-C-48 tertiary bonds. The effects seen in the proton NMR spectra are compared and correlated with those observed in the phosphorus spectra to give assignments of some of the resolved signals from the phosphate groups. The phosphorus spectra are compared with those of yeast tRNAPhe [Gorenstein, D. G., Goldfield, E. M., Chen, R., Kovar, K., & Luxon, B. A. (1981) Biochemistry 20, 2141; Salemink, P. J. M., Reijerse, E. J., Mollevanger, L., & Hilbers, C. W. (1981) Eur. J. Biochem. 115, 635], and the ion effects are discussed with reference to the magnesium and spermine sites found in the crystal structures of yeast tRNAPhe [Holbrook, S. R., Sussman, J. L., Warrant, R. W., Church, G. M., & Kim, S.-H. (1977) Nucleic Acids Res. 4, 2811; Quigley, G. J., Teeter, M. M., & Rich, A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 64; Jack, A., Ladner, J. E., Rhodes, D., Brown, R. S., & Klug, A. (1977) J. Mol. Biol. 111, 315].     

Interleukin-1 receptor antagonist competitively inhibits the binding of interleukin-1 to the type II interleukin-1 receptor.

The interleukin-1 receptor antagonist (IL-1ra) inhibits the binding of interleukin-1 (IL-1) to T-cell lines possessing the type I IL-1 receptor; evidence has been published (Carter, D. B., Deibel, M. R. J., Dunn, C. J., Tomich, C. S., Laborde, A. L., Slightom, J. L., Berger, A. E., Bienkowski, M. J., Sun, F. F., McEwan, R. N., Harris, P. K. W., Yem, A. W., Waszak, G. A., Chosay, J. G., Sieu, L. C., Hardee, M. M., Zurcher-Neely, H. A., Reardon, I. M., Heinrickson, R. L., Truesdell, S. E., Shelly, J. A., Eessalu, T. E., Taylor, B. M., and Tracey, D. E. (1990) Nature 344, 633-638; Hannum, C. H., Wilcox, C. J., Arend, W. P., Joslin, F. G., Dripps, D. J., Heimdal, P. L., Armes, L. G., Sommer, A., Eisenberg, S. P., and Thompson, R. C. (1990) Nature 343, 336-340) that IL-Ira does not bind to the type II IL-1 receptor (IL-1RtII). In this study we examined the ability of human recombinant IL-1ra to block the binding of IL-1 to the IL-1RtII on human polymorphonuclear leukocytes (PMN) and Raji human B-lymphoma cells. The binding of 125I-IL-1 beta to PMN was competively inhibited by IL-1ra. IL-1 beta was more potent in inhibiting the binding of 125I-IL-1 beta than IL-1ra. Incubating PMN with 125I-IL-1ra in the presence of increasing concentrations of IL-1 beta or IL-1ra showed that IL-1 beta was an approximately 40-fold more potent inhibitor of binding of 125I-IL-1ra than unlabeled IL-1ra. The IL-1ra was approximately 500-fold less potent in inhibiting the binding of 125I-IL-1 alpha than IL-1 alpha. IL-1ra was also able to competitively inhibit binding of 125I-IL-1 beta to Raji cells. PMN or Raji cells were also incubated with 125I-IL-1 in the absence or presence of IL-1 or IL-1ra. After cross-linking of IL-1 to cells followed by specific immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a band at 85 kDa corresponding to the 68-kDa IL-1RtII. However, in the presence of an excess of either unlabeled IL-1 or IL-1ra, the 85-kDa IL-1.IL-1RtII complex was not present. These findings demonstrate that the IL-1ra recognizes and blocks IL-1 binding to the IL-1RtII.     

Differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In two recent papers, we reported the effects of several point mutations on the thermodynamics of the thermal unfolding of the lysozyme of phage T4 as determined by differential scanning calorimetry. The mutants studied were R96H [Kitamura, S., & Sturtevant, J.M. (1989) Biochemistry 28, 3788-3792] and T157 replaced by A, E, I, L, N, R, and V [Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J.M. (1991) Biochemistry 30, 1887-1891]. Here we report the results of a similar study of the single mutations A82P, A93P, and G113A and the double mutation C54T:C97A. The three single mutants all show small apparent stabilization at pH 2.5 and 46.2 degrees C (the denaturational temperature of the wild-type protein), amounting to -0.5 +/- 0.4 kcal mol-1 in free energy, whereas the double mutant shows a weak apparent destabilization, +0.8 +/- 0.4 kcal mol-1. As in all our previous studies of mutant proteins, the enthalpy changes produced by these mutations are in general of much larger magnitude than the corresponding free energy changes and frequently of opposite sign.     

Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.

NIH 3T3 cells were transfected with plasmids containing Moloney murine leukemia virus long terminal repeats and either chicken c-src or v-src genes. In contrast with the effects observed after transfection with plasmids containing c-src and avian retrovirus or simian virus 40 promoter-enhancers (H. Hanafusa, H. Iba, T. Takeya, and F. R. Cross, p. 1-8, in G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, ed., Cancer Cells, vol. 2, 1984; H. Iba, T. Takeya, F. R. Cross, T. Hanafusa, and H. Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 81:4424-4428, 1984; R. C. Parker, R. Swanstrom, H. E. Varmus, and J. M. Bishop, p. 19-26, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; R. C. Parker, H. E. Varmus, and J. M. Bishop, Cell 37:131-139, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, p. 9-17, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, Proc. Natl. Acad. Sci. U.S.A. 81:7071-7075; and K. C. Wilhelmsen, W. G. Tarpley, and H. M. Temin, p. 303-308, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984), we found that both types of Moloney murine leukemia virus long terminal repeat-src expression plasmids induced focus formation, although c-src induced only 1% as many foci as v-src. The focus-selected c-src overexpressed cells had altered morphology and limited growth in soft agarose but were not tumorigenic in vivo. Cleveland digests, comparative in vitro kinase assays, secondary transfections, and immunoprecipitations indicated that focus formation was caused by rare transfection events that resulted in very high-level pp60c-src expression rather than by mutations of the transfected c-src genes. These results suggest that pp60v-src induced transformation is not a completely spurious activity which is unrelated to the function of pp60c-src but that it represents a perturbation of already existent molecular control processes involving pp60c-src.     

The Stanford Microarray Database

The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.     

Analysis of DsRed Mutants. Space around the fluorophore accelerates fluorescence development.

Earlier mutagenesis of the red fluorescent protein drFP583, also called DsRed, resulted in a mutant named Fluorescent Timer (Terskikh, A., Fradkov, A., Ermakova, G., Zaraisky, A., Tan, P., Kajava, A. V., Zhao, X., Lukyanov, S., Matz, M., Kim, S., Weissman, I., and Siebert, P. (2000) Science 290, 1585--1588). Further mutagenesis generated variants with novel and improved fluorescent properties. The mutant called AG4 exhibits only green fluorescence. The mutant, called E5up (V105A), shows complete fluorophore maturation, eventually eliminating residual green fluorescence present in DsRed. Finally, the mutant, called E57 (V105A, I161T, S197A), matures faster than DsRed as demonstrated in vitro with purified protein and in vivo with recombinant protein expressed in Escherichia coli and Xenopus leavis. Comparative analysis of the mutants in the context of the crystal structure of DsRed suggests that mutants with free space around the fluorophore mature faster and more completely.     

Books

Book reviews in this article:
A ntarcitc E cology . R. M. Laws (ed.).
S eals O f T he W orld . J. E. King.
R eproduction I n W hales , D olphins and P orpoises . W. F. Perrin, R. L. Brownell, Jr., and D. P. DeMaster, eds.
E volution I n T he G alapagos I slands . R. J. Berry (ed.).
H istorical W haling R ecords . M. F. Tillman and G. P. Donovan (eds.).
D iving and A sphyxia , A C omparative S tudy O f A nimals and M an . R. Elsner and B. Gooden.     

Evolutionary potential of (beta/alpha)8-barrels: stepwise evolution of a "new" reaction in the enolase superfamily

The molecular details of the processes involved in divergent evolution of "new" enzymatic functions are ill-defined. Likely starting points are either a progenitor promiscuous for the new reaction or a progenitor capable of catalyzing the new reaction following a single substitution that results from a single base change. However, the molecular (sequence) pathway by which the selective advantage provided by this protein can be improved and ultimately optimized is unclear. In the mechanistically diverse enolase superfamily, we discovered that a monofunctional progenitor could acquire the ability to catalyze a "new" reaction by a single base change: the D297G mutant of the monofunctional l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli catalyzed a low level of the o-succinylbenzoate synthase (OSBS) reaction as well as a reduced level of the AEE reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387-8393]. We then discovered that the selective advantage and OSBS activity of the D297G mutant are both enhanced by the I19F substitution [Vick, J. E., Schmidt, D. M. Z., and Gerlt, J. A. (2005) Biochemistry 44, 11722-11729]. Both the D297G and I19F substitutions are positioned to alter the substrate specificity so that the substrate for the OSBS reaction is more productively positioned vis a vis the active site catalytic groups. We now report that both the selective advantage and OSBS activity of the D297G/I19F double mutant are enhanced by the R24C (one base change from the wild type Arg codon), R24W (two base changes from the wild type Arg codon and one base change from the R24C codon), and L277W (one base change from the wild type Leu codon) substitutions. The effects of the R24C and L277W mutants are "additive" in the D297G/I19F/R24C/L277W mutant. The greatest selective advantage and OSBS activity are associated with the D297G/I19F/R24W mutant. These "new" substitutions that enhance both the selective advantage and kinetic constants are positioned in the active site where they can alter the specificity, highlighting that the evolution of the "new" OSBS function can be accomplished by changes in substrate specificity.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).     

SOLVING TAXONOMIC AND NOMENCLATURAL PROBLEMS IN PACIFIC GIGARTINACEAE (RHODOPHYTA) USING DNA FROM TYPE MATERIAL

Molecular data obtained by a procedure for extracting PCR-amplifiable nuclear and chloroplast DNA from old and formalin-fixed red algal herbarium specimens were used to elucidate problems in the systematics of Pacific Gigartinaceae. Correspondence between nucleotide sequences of the internal transcribed spacer 1 region or the RUBISCO spacer from type specimens and modern collections supports the following conclusions. (1) The type of Fucus cordatus Turner, now Iridaea cordata (Turner) Bory, came from the southern hemisphere (probably from Isla de los Estados, Argentina) rather than from Banks Island, B.C., Canada. (2) The type of Iridaea heterocarpa P. et R. [Mazzaella heterocarpa (P. et R.) Fred.] represents the tetrasporangial phase of a species of Chondrus, possibly C. crispus Stackh. (3) The types of Iridaea lilacina P. et R., I. phyllocarpa P. et R., and Iridophycus furcatum S. et G. represent a single species from Alaska, Mazzaella phyllocarpa (P. et R.) Perest., currently but incorrectly called M. heterocarpa. (4) The type of Iridophycus oregonum Doty represents the tetrasporangial phase of the species from southern Alaska to southern California known incorrectly as M. heterocarpa. (5) Mazzaella splendens (S. et G.) Fred. is more closely related to M. linearis (S. et G.) Fred. than it is to M. flaccida (S. et G.) Fred. (6) Iridophycus coriaceum S. et G. is conspecific with M. splendens, whereas Rhodoglossum coriaceum E.Y. Dawson is an independent species: Mazzaella coriacea (E.Y. Dawson) Hughey. (7) Iridaea cornucopiae P. et R. is conspecific with Mazzaella laminarioides (Bory) Fred., and the type probably came from Chile rather than from the North Pacific. (8) Plants attributed to Iridaea cornucopiae in Pacific North America are referable to Mazzaella parksii (S. et G.) comb. nov. (9) Rhodoglossum parvum G. M. Smith et Hollenb. is an independent species: Mazzaella parva (G. M. Smith et Hollenb.) comb. nov. (10) Grateloupia squarrulosa S. et G., Grateloupia johnstonii S. et G., and Gigartina pectinata E.Y. Dawson represent a single species: Chondracanthus squarrulosus (S. et G.) comb. nov.     

Probing of the coupling site of the beta-adrenergic receptor. Competition between different forms of the guanyl nucleotide binding protein for interaction with the receptor

The interaction of the beta-adrenergic receptor (R) with different forms of the regulatory protein (G) was studied. For this purpose, the reconstituted system formed from separate soluble preparations of R and G was employed (Citri, Y., and Schramm, M. (1980) Nature (Lond.) 287, 297-300). Soluble preparations of the native nonactive G, to which GDP is bound (GGDP), and the persistently active G, to which guanyl-5'-yl-imidophosphate (Gpp(NH)p) is bound GGPP(MH)P), were prepared. Reconstituted systems made of GGDP, GGPP(NH)P, and R were used to test whether each of the two forms of G can affect the other's interaction with R. GGPP(NH)P was found to compete with GGDP for the interaction with R. However, GGDP was unable to interfere with the interaction of R with GGPP(NH)P. Similar experiments were carried out with another persistently active G (GGTP gamma S). In sharp contrast to GGPP(NH)P, GGTP gamma S was found incapable of competing with GGDP for R. The above experiments suggest that GGDP and GGPP(NH)P interact with the same coupling site on R. However, the degree of recognition of G by R depends strongly on the guanyl nucleotide which is bound to G. The relative order of recognition being GGPP(NH)P greater than GGDP greater than GGTP gamma S approximately 0. These findings may also explain the known irreversible effects of GTP gamma S in the adenylate cyclase system.