PubMed Health⌕ Search

Biomedical subjects

M F Roberts

Publications and source records attributed to M F Roberts.

At least 55 records · Page 3Linked to original sources
PubMed1996

Phospholipases: structural and functional motifs for working at an interface.

Phospholipases form a ubiquitous class of enzymes optimized to catalyze the hydrolysis of phospholipids. Because their products are often second messengers, they are highly regulated by the cell. For a given ester bond, there are separate secreted as well as cytoplasmic phospholipases with different substrate specificities and modes of regulation. As it becomes available, structural information provides a view of interfacial catalysis for several of these phospholipases on a molecular level. Recent structural advances include solution structures of a pancreatic phospholipase A2 in the absence and presence of a micellar interface, crystal structures of a bacterial phosphatidylinositol-phospholipase C whose active site is reminiscent of ribonuclease, and a Ca2+ lipid binding domain with high homology to regions in several cytoplasmic phospholipases that can model the way those proteins interact with the membrane surface. Phospholipases also have a wide and complex array of regulatory mechanisms involving cytoplasmic proteins, notably G-proteins, as well as different effector lipids (e.g., phosphatidylinositol-4,5-biphosphate, or PIP2) or Ca2+. Deconvolution of these interactions is necessary to understand their roles in different signal transduction pathways.-Roberts, M. F. Phospholipases: structural and functional motifs for working at an interface.

Animals↗
PubMed1995

Phospholipase A2 domain formation in hydrolyzed asymmetric phospholipid monolayers at the air/water interface.

Phospholipase A2 (PLA2) catalyzed hydrolysis of asymmetric 1-caproyl-2-palmitoyl-phosphatidylcholine (6,16-PC) and 1-palmitoyl-2-caproyl-phosphatidylcholine (16,6-PC) lipid monolayers at the air/water interface was investigated. Surface pressure isotherms, surface potential and fluorescence microscopy at the air/water interface were used to characterize the asymmetric monolayer systems. Cobra (N. naja naja) and bee venom PLA2 exhibit hydrolytic activity towards 16,6-PC monolayers at all surface pressures up to monolayer collapse (37 mN m-1). Pancreatic PLA2 hydrolytic activity, however, was observed to be blocked at a lateral surface pressure of approx. 18 mN m-1 for both 6,16-PC and 16,6-PC monolayers. For 6,16-PC monolayers, fluorescence microscopy revealed that monolayer hydrolysis by PLA2 from cobra, bee, and bovine pancreatic sources all produced monolayer microstructuring. Fluorescence microscopy also showed that PLA2 is bound to these monolayer microstructures. Very little PLA2-induced microstructuring was observed to occur in 16,6-PC monolayer systems where caproic acid (C6) hydrolysis products were readily solubilized in the aqueous monolayer subphase. Surface potential measurements for 16,6-PC monolayer hydrolysis indicate dissolution of caproic acid reaction products into the monolayer subphase. Monolayer molecular area as a function of 6,16-PC monolayer hydrolysis time indicates the presence of monolayer-resident palmitic acid reaction products. With bovine serum albumin present in the monolayer subphase, PLA2 domain formation was observed only in hydrolyzed 6,16-PC monolayers. These results are consistent with laterally phase separated monolayer regions containing phospholipid and insoluble fatty acid reaction products from PLA2 monolayer hydrolysis electrostatically driving PLA2 adsorption to and enzyme domain formation at the heterogeneous, hydrolyzed lipid monolayer interface.

Animals↗
PubMed1995

Anthranilate synthase in microorganisms and plants.

The enzymology of anthranilate synthase (EC 5.4.99.6) in microorganisms and plants is reviewed. Aminoacid sequences of the enzyme subunits in different species are compared, and the mechanism of reaction is discussed.

Amino Acid Sequence↗
PubMed1995

Internalization of Sucrose by Methanococcus thermolithotrophicus.

When sucrose is present in the external medium, it is internalized by Methanococcus thermolithotrophicus. Sucrose internalization, as determined by both natural abundance (sup13)C nuclear magnetic resonance spectroscopy and [(sup14)C]sucrose uptake, is directly proportional to external sucrose levels. The uptake is energy independent and exhibits kinetic behavior consistent with a simple passive diffusion process. In the presence of 0.2 M sucrose, methanogenesis is inhibited as the NaCl concentration in the external medium is increased. Growth, as determined by protein content, is inhibited by 0.2 M sucrose when the external NaCl concentration is 1.4 M. These results are important because they show that (i) sucrose cannot be used as a noncharged solute to replace NaCl in experiments to evaluate how external osmotic strength affects the internal solute composition of M. thermolithotrophicus, and (ii) sucrose cannot be used as an impermeable marker for the extracellular volume in experiments to measure the intracellular volume of M. thermolithotrophicus.

Journal Article↗
PubMed1994

Fluorescence studies of phosphatidylcholine micelle mixing: relevance to phospholipase kinetics.

Two fluorescent micellar phospholipid probes (1-hexanoyl-2-(1-pyrenebutyroyl)phosphatidylcholine and 1-octanoyl-2-(1-pyrenebutyroyl)phosphatidylcholine) have been synthesized, characterized, and used to monitor the dynamics of lipid/amphiphile exchange in a variety of detergents and phospholipid micelles using both steady-state and stopped-flow fluorescence techniques. The ratio of the pyrene monomer to excimer band is a good indicator of the extent of lipid mixing at equilibrium. Following the time dependence of increase in the monomer band with stopped-flow methodology provides a rate constant for this exchange process (most systems were well fit with a single exponential). Short-chain pyrene-labeled phosphatidylcholine mixing with Triton X-100 micelles is extremely fast and follows a concentration dependence indicative of the importance of micelle collisions for the exchange process. Submicellar amounts of Triton have no effect on the fluorescent dynamics of the probe molecule. Other detergents such as beta-octyl glucoside and deoxycholate are also effective at higher concentrations, although significant differences exist in the extent of probe mixing. Short-chain diacylphosphatidylcholine and lysophosphatidylcholine mixing rates are moderately fast with mixing times that decrease as the hydrophobicity/chain length of the diluent matrix increases. The rate constants for lipid exchange can be compared to turnover rates of several phospholipases in these assay systems. Anomalous mixing behavior of unusual micelle forming lipids [bolaforms and omega-carboxylate phosphatidylcholines [Lewis, K. A., Bian, J., Sweeny, A., & Roberts, M. F. (1994) Biochemistry 29, 9962-9970] and polymerizable phosphatidylcholines [Soltys, C. E., Bian, J., & Roberts, M. F. (1993) Biochemistry 32, 9545-9551] is particularly helpful in understanding kinetics of water-soluble phospholipases on these systems.

Colloids↗
PubMed1994

Annexin V binding to the outer leaflet of small unilamellar vesicles leads to altered inner-leaflet properties: 31P- and 1H-NMR studies.

Calcium-dependent binding to phospholipid membranes is closely associated with annexin functional properties. In these studies, 31P- and 1H-nuclear magnetic resonance (NMR) experiments have been performed to study the effects of binding of recombinant rat annexin V to sonicated small unilamellar vesicles (SUVs). High-resolution 31P-NMR spectra of SUVs containing mixtures of synthetic phosphatidic acid (PA) and phosphatidylcholine (PC) show resolvable resonances corresponding to the inner-leaflet PA, outer-leaflet PA, and PC phosphoryl groups. When annexin binding occurs, the outer-leaflet PA 31P resonance shifts while that of PC is unaffected, consistent with selective binding of the protein to the phosphoryl moiety of the PA component. Further, annexin V binding to membrane outer-leaflet phospholipids has a measurable effect on inner-leaflet phospholipids of intact vesicles. 1H-NMR T1 relaxation measurements of SUVs containing acyl-chain-perdeuterated PC show no effects on the PA hydrocarbon-chain segmental motions upon annexin binding. Circular dichroism measurements indicate that the protein does not undergo a significant conformational change upon binding to the vesicles. The observed NMR changes do not correspond to proton or calcium gradients, nor to lateral segregation of extended patches of homogeneous phospholipids. The combined evidence suggests that selective, peripheral annexin-membrane interactions influence the environment of the inner vesicular surface. The mechanism proposed is a protein-induced change in vesicle morphology that corresponds to reduced curvature.

Animals↗
PubMed1994

Effect of vesicle composition and curvature on the dissociation of phosphatidic acid in small unilamellar vesicles--a 31P-NMR study.

Sonicated small unilamellar vesicles (SUVs) containing phosphatidic acid (PA) give two PA 31P-NMR resonances corresponding to PA molecules in the inner and outer leaflets of the bilayer. This NMR differentiation between the two monolayers is not due to a pH gradient across the membrane but instead reflects differential packing in the inner and outer leaflets imposed by the highly curved SUV surface. The apparent pKa of the outer-leaflet PA increases with decreasing surface curvature and with increasing PA content. The estimated relationship between the apparent pKa of the outer-leaflet PA headgroup and vesicle curvature may provide a qualitative probe for effects related to surface curvature in these model-membrane systems.

Hydrogen-Ion Concentration↗
PubMed1994

Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases.

A series of mixed-chain diacyl-PCs which contain an omega-COOH on the sn-2 chain [1-Cx-2-Cy-(COOH)-PC] and bolaform (1-Cx-2,2'-Cy-1'-Cx-PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A2 (Naja naja naja) and C (Bacillus cereus). There is very little detectable phospholipase A2 activity toward pure micellar 1-acyl-2-acyl-(omega-COOH) species. In addition, when these same omega-COOH species are present at concentrations above their CMCs, they are potent inhibitors of phospholipase A2 hydrolysis of other micellar lipids. In contrast, phospholipase C hydrolysis of the same 1-acyl-2-acyl-omega-COOH)-PC species proceeds with rates comparable to that of diheptanoyl-PC. The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C8-2,2'-C12-1'-C8-PC) display quite different kinetic results. Under limiting Ca2+ conditions (100 microM) all the available sn-2 acyl bonds of the dimer are hydrolyzed. However, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followed by much slower rate. This is consistent with only the micellar 1-acyl-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A2 hydrolysis of the dimer acting as an inhibitor of subsequent phospholipase A2 activity. Phospholipase C hydrolysis of the PC dimer and the sn-2 omega-COOH PC is rapid, with both available glycerophosphate groups cleaved at presumably the same rate. These results are discussed in terms of the unique physical properties (as measured by NMR and fluorescence experiments) of these phospholipids.

Carbon Isotopes↗
PubMed1994

Occurrence and Role of Di-myo-Inositol-1,1'-Phosphate in Methanococcus igneus.

Methanococcus igneus, a hyperthermophilic marine methanogen (optimum growth temperature of 88 degrees C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with l-alpha-glutamate and beta-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-myo-inositol-1,1'-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (l-alpha-glutamate, beta-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by CO(2)-pulse-CO(2)-chase experiments. The results indicated that both the beta-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was l-alpha-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-myo-inositol-1,1'-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when M. igneus was grown at temperatures of >/=80 degrees C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.

Journal Article↗
PubMed1994

Identification, Biosynthesis, and Function of 1,3,4,6-Hexanetetracarboxylic Acid in Methanobacterium thermoautotrophicum DeltaH.

An unusual compound, 1,3,4,6-hexanetetracarboxylic acid, was identified by H and C two-dimensional nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as one of the major components of the small-molecule pool in Methanobacterium thermoautotrophicum DeltaH under optimal conditions of cell growth. Incorporation of C- and H-labeled acetates was consistent with the biosynthesis of this tetracarboxylic acid from alpha-ketoglutarate, two molecules of acetyl-coenzyme A, and one molecule of CO(2), as established for the tetracarboxylic acid moiety of methanofuran. CO(2) pulse- CO(2) chase methodology was used to establish the turnover rate for this compound. In contrast to the two other major solutes in this bacterium, cyclic 2,3-diphosphoglycerate and glutamate, which are key metabolic intermediates, this free tetracarboxylic acid was metabolically inactive, with a half-life that exceeded the cell doubling time. Hence, this molecular pool cannot serve as a metabolic intermediate in cell biosynthesis. The functional role of free tetracarboxylate as a conservative part of a system that maintains high positive internal osmotic pressure in this bacterium is proposed.

Journal Article↗
PubMed1994

Halotolerance of Methanobacterium thermoautotrophicum delta H and Marburg.

Methanobacterium thermoautotrophicum delta H and Marburg were adapted to grow in medium containing up to 0.65 M NaCl. From 0.01 to 0.5 M NaCl, there was a lag before cell growth which increased with increasing external NaCl. The effect of NaCl on methane production was not significant once the cells began to grow. Intracellular solutes were monitored by nuclear magnetic resonance (NMR) spectroscopy as a function of osmotic stress. In the delta H strain, the major intracellular small organic solutes, cyclic-2,3-diphosphoglycerate and glutamate, increased at most twofold between 0.01 and 0.4 M NaCl and decreased when the external NaCl was 0.5 M. M. thermoautotrophicum Marburg similarly showed a decrease in solute (cyclic-2,3-diphosphoglycerate, 1,3,4,6-tetracarboxyhexane, and L-alpha-glutamate) concentrations for cells grown in medium containing > 0.5 M NaCl. At 0.65 M NaCl, a new organic solute, which was visible in only trace amounts at the lower NaCl concentrations, became the dominant solute. Intracellular potassium in the delta H strain, detected by atomic absorption and 39K NMR, was roughly constant between 0.01 and 0.4 M and then decreased as the external NaCl increased further. The high intracellular K+ was balanced by the negative charges of the organic osmolytes. At the higher external salt concentrations, it is suggested that Na+ and possibly Cl- ions are internalized to provide osmotic balance. A striking difference of strain Marburg from strain delta H was that yeast extract facilitated growth in high-NaCl-containing medium. The yeast extract supplied only trace NMR-detectable solutes (e.g., betaine) but had a large effect on endogenous glutamate levels, which were significantly decreased. Exogenous choline and glycine, instead of yeast extract, also aided growth in NaCl-containing media. Both solutes were internalized with the choline converted to betaine; the contribution to osmotic balance of these species was 20 to 25% of the total small-molecule pool. These results indicate that M. thermoautotrophicum shows little changes in its internal solutes over a wide range of external NaCl. Furthermore, they illustrate the considerable differences in physiology in the delta H and Marburg strains of this organism.

2,3-Diphosphoglycerate↗
PubMed1993

Polymerizable phosphatidylcholines: importance of phospholipid motions for optimum phospholipase A2 and C activity.

Cross-linkable short-chain phosphatidylcholines with thiols at the chain terminus have been synthesized and characterized. These micelle-forming species were used to investigate two water-soluble phospholipases. When reduced, the thiol lipids were excellent substrates for phospholipase A2. Once cross-linked, they became extremely poor substrates. This is consistent with a mechanism in which a key step is the partial extraction of the substrate phosphatidylcholine from an aggregate. In contrast, phospholipase C activity was slightly enhanced if the product diglyceride was tethered to the aggregate through disulfide formation. For this enzyme such a kinetic effect is consistent with the hydrophobic diglyceride biasing the enzyme to the interface.

Elapid Venoms↗
PubMed1993

Substrate requirements of bacterial phosphatidylinositol-specific phospholipase C.

A series of symmetric short-chain phosphatidylinositols (PI), including dihexanoyl-PI, diheptanoyl-PI (racemic as well as D and L forms), and 2-methoxy inositol-substituted diheptanoyl-PI, have been synthesized, characterized, and used to investigate key mechanistic questions about phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis. Key results include the following: (i) bacterial PI-PLC exhibits a 5-6-fold "interfacial activation" when its substrate is present in an interface as opposed to existing as a monomer in solution (in fact, the similarity to the activation observed with nonspecific PLC enzymes suggests a similarity in activation mechanisms); (ii) the 2-OH must be free since the enzyme cannot hydrolyze diheptanoyl-2-O-methyl-PI (this is most consistent with the formation of inositol cyclic 1,2-phosphate as a necessary step in catalysis); (iii) the inositol ring must have the D stereochemistry (the L-inositol attached to the lipid moiety is neither a substrate nor an inhibitor); and (iv) the presence of noninhibitory L-PI with the D-PI substrate relieves the diacylglycerol product inhibition detected at approximately 30% hydrolysis.

Bacillus thuringiensis↗
PubMed1993

Effect of glpT and glpD mutations on expression of the phoA gene in Escherichia coli.

In vivo 31P nuclear magnetic resonance analysis of Escherichia coli cells showed that the intracellular concentration of P(i) remained constant in wild-type and in a glpT mutant strain whether the cells were grown on excess (2 mM) P(i) or sn-glycerol-3-phosphate as a phosphate source. The function of the phoA promoter (measured by beta-galactosidase activity in a phoA-lacZ fusion strain) was repressed when glpT+ cells were utilizing sn-glycerol-3-phosphate as the sole source of phosphate. These cells were devoid of alkaline phosphatase activity. However, the phoA promoter was fully active in a glpT mutant. These results indicated that the repression of the enzyme synthesis was not due to a variation in the level of cytoplasmic P(i) but was due to the P(i) excreted into the periplasm and/or to the medium.

Alkaline Phosphatase↗
PubMed1993

Cyclic 2,3-diphosphoglycerate as a component of a new branch in gluconeogenesis in Methanobacterium thermoautotrophicum delta H.

A unique compound, cyclic 2,3-diphosphoglycerate (cDPG), is the major soluble carbon and phosphorus solute in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth. It is a component of an unusual branch in gluconeogenesis in these bacteria. [U-13C]acetate pulse-[12C]acetate chase methodology was used to observe the relationship between cDPG and other metabolites (2-phosphoglycerate and 2,3-diphosphoglycerate [2-PG and 2,3-DPG, respectively]) of this branch. It was demonstrated that cells could grow exponentially under conditions in which 2-PG and 2,3-DPG, rather than cDPG, were the major solutes. While the total concentration of these three phosphorylated molecules was maintained, rapid interconversion of 13C label among them was observed. Label flow from 2-PG to 2,3-DPG to cDPG to polymer is the usual direction in this pathway in exponentially growing cells, while the reverse reactions sometimes predominate in the stationary phase. Evidence of the presence of a polymeric compound in this pathway was provided by 13C nuclear magnetic resonance (one-dimensional and two-dimensional INADEQUATE) studies of solubilized cell debris.

2,3-Diphosphoglycerate↗
PubMed1992

Multinuclear NMR studies of the trp-repressor.

The binding of the corepressor, L-tryptophan, to the Escherichia coli trp-aporepressor in solution has been examined by 13C- and 19F-NMR spectroscopy. The binding of a number of tryptophan analogues have been studied by equilibrium dialysis. Evidence is presented that support the crystallographic studies (Schevitz, R. W., Otwinowski, Z., Joachimiak, A., Lawson, C. L. and Sigler, P. B. (1985) Nature 317, 782-786) that Val-58 is within the ring currents of the bound tryptophan and also close in space to the indole 5'-position, on the basis of heteronuclear 19F(1H)-NOE experiments. The tryptophan carboxylate is in hydrogen-bonding distance to a highly positively charged residue, probably Arg-54 and this bond strengthens on formation of the trp-repressor-DNA complex.

Cell Nucleus↗