Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Ruptured fission yeast walls

Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis. As shown previously (Johnson et al.,Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length. The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986). The median length of cells was found to decline through the log phase in an unbalanced manner.     

Amino acid accumulation in frog muscle. II. Are cycloleucine fluxes consistent with an adsorption model for concentrative uptake of amino acid?

Cycloleucine accumulation by frog muscle was studied at o °C and 25 °C. At external concentrations less than 5 mM the distribution ratio of cycloleucine is higher at 0 °C than at 25 °C. At concentrations greater than 5 mM the converse is true due to apparent exclusion of cycloleucine from a larger portion of the cell water at 0 °C.The steady state data are consistent with an absortion model for amino acid accumulation. Flux studies provide a means to rule out this model if all the possible rate-limiting steps in the movement of amino acid into and out of the cell are considered. These steps include intra-cytoplasmic diffusion, desorption from cytoplasmic or membrane sites and passage through the cell membrane. The assumption is made that the rate-limiting step for influx and efflux is the same, allowing the use of either influx or efflux data to examine the model.Diffusion-limited flux is ruled out on the basis of“influx profile analysis” of the time course of cycloleucine entry at both 0 °C and 25 °C.At least 95% of all intracellular cycloleucine leaves frog muscle cells with a single exponential time course at both 0 °C. The rate constant of efflux does not vary with cellular concentration.These findings are shown to be incompatible with desorption-limited efflux. They are compatible with membrane-limited efflex only if (i) adsorption sites are located on membranes with direct access to the extracellular space and (ii) the rate constant for desorption is equal to the rate constant of membrane-limited efflux of free amino acid. It is considered unlikely that such a coincidence would occur at both 0 °C and 25 °C. Therefore, an absorption model for cycloleucine accumulation in frog muscle appears to be untenable.     

Acid-catalyzed hydration of reduced nicotinamide adenine dinucleotide and its analogues.

The rate of the primary acid modification reaction of 1,4-dihydronicotinamide adenine dinucleotide (NADH) and 1,4-dihydro-3-acetylpyridine adenine dinucleotide (APADH) and their analogues has been studied over a wide pH range (pH 1-7) with a variety of general acid catalysts. The rate depends on [H+] at moderate pH and becomes independent of [H+] at low pH. This behavior is attributed to substrate protonation at the carbonyl group (pK of NADH = 0.6). The reaction is general acid catalyzed; large solvent deuterium isotope effects are observed for the general acid and lyonium ion terms. Most buffers cause a linear rate increase with increasing buffer concentration, but certain buffers cause a hyperbolic rate increase. The nonlinear buffer effects are due to complexation of the buffer with the substrate, rather than to a change in rate-limiting step. The rate-limiting step is a proton transfer from the general acid species to the C5 position of the substrate. Anomerization is not a necessary first step in the case of the primary acid modification reaction of beta-NADH, in which beta to alpha anomerization takes place.     

Wiring diagrams of MAPK regulation by MEKK1, 2, and 3.

Mitogen-activated protein kinase (MAPK) pathways are activated by a plethora of stimuli. The literature is filled with papers describing the activation of different MAPKs by almost any stimulus or insult imaginable to cells. In this review, we use signal transduction wiring diagrams to illustrate putative upstream regulators for the MAPK kinase kinases, MEKK1, 2, and 3. Targeted gene disruption of MEKK1, 2, or 3 defined phenotypes for each MEKK associated with loss of specific MAPK regulation. Genetic analysis of MEKK function clearly defines specific components of the wiring diagram that require MEKK1, 2, or 3 for physiological responses. We propose that signal transduction network wiring diagrams are valuable tools for hypothesis building and filtering physiologically relevant phenotypic responses from less connected protein relations in the regulation of MAPK pathways.     

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.     

Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.