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Biomedical subjects

S R Martin

Publications and source records attributed to S R Martin.

At least 109 records · Page 6Linked to original sources
PubMed1984

The kinetics of calcium binding to calmodulin: Quin 2 and ANS stopped-flow fluorescence studies.

The rate of calcium dissociation from bovine testis calmodulin was measured by fluorescence stopped-flow using the calcium indicator Quin 2 or the fluorescence probe 8-anilinonaphthalene sulphonate. Two processes are resolved with Quin 2 corresponding to dissociation from the high affinity sites (kdiss 2 to 9 s-1 for T = 11 to 28 degrees C) and from the low affinity sites (kdiss 293 to 550 s-1 for T = 11 to 19 degrees C). These rates and the activation parameters as determined for the slow process delta H not equal to = 59 +/- 10 kJ X mol-1 and delta S not equal to = 30 +/- 30 JK-1 X mol-1 are in good agreement with values determined from the 43Ca NMR exchange rates. These experiments provide confirmation that the calcium induced conformational change cannot be resolved kinetically from the calcium binding or dissociation, and by inference this conformational change is not a rate-limiting process in the function of calmodulin.

Aminoquinolines↗
PubMed1983

Specific DNA binding of the cyclic AMP receptor protein to a synthetic oligodeoxyribonucleotide. A circular dichroism study.

The interaction of the cAMP receptor protein (CRP) of Escherichia coli with a synthetic DNA undecamer (11 mer) comprising a portion of the specific target site in the gal operon and containing 8 basepairs out of the 10 basepair concensus making up specific CRP sites, has been studied by circular dichroism spectroscopy. The binding constants for the interaction of CRP with the 11 mer in the presence and absence of cAMP have been determined, and it is shown that CRP, both in the presence and absence of cAMP, induces a B-C transition in the conformation of the 11 mer.

Circular Dichroism↗
PubMed1983

Molecular flexibility in microtubule proteins: proton nuclear magnetic resonance characterization.

Bovine microtubule protein preparations have been examined by proton nuclear magnetic resonance (1H NMR) spectroscopy at 270 MHz. Sharp resonances have been identified as deriving from microtubule-associated proteins. These resonances persist after self-assembly of microtubule protein. Brief tryptic treatment of assembled microtubules, specifically cleaving the microtubule-associated protein HMW2 (Mr = 270 000), releases the pendant portion of HMW2 (Mr = 240 000), three-quarters of which is in a flexible conformation. Isolated tau protein and HMW2 protein both show substantial flexibility; on recombination with tubulin dimer, tau shows considerable decrease in flexibility whereas HMW2 is unaffected. The observations may have important implications for the interactions between microtubules and other cytoskeletal structures.

Amino Acids↗
PubMed1983

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin.

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.

Adenosine Triphosphatases↗
PubMed1983

Evidence for a repeating cross-beta sheet structure in the adenovirus fibre.

The amino acid sequence of the adenovirus fibre protein reveals an approximately repeating motif of 15 residues. A diagonal comparison matrix established that these repeats extended from residue 43 to residue 400 of the 581 residue sequence. Assignment of secondary structure combined with model building showed that each 15-residue segment contained two short beta-strands and two beta-bends, one of which incorporated an extra residue in a beta-bulge of the Gx type. The 44 strands together gave a long (210 A) narrow, amphipathic beta-sheet, which could be stabilised by dimer formation to give the shaft of the fibre. The knob could arise from a dimer of the C-terminal 180 residue segment, predicted to be an 8-10 stranded beta-sandwich. This model is consistent with the electron micrographs of the fibre and it was supported by measurements of c.d. and of electron diffraction from microcrystals. The latter gave a pair of wide angle arcs, corresponding to a repeat of 4.7 A, oriented appropriately for a cross-beta structure. The relation of this structure to globular structures is discussed and a folding pathway is proposed. In its general features the structure resembles that proposed for the tail fibre of bacteriophage T4.

Adenoviruses, Human↗
PubMed1983

Differential promotion and suppression of Z leads to B transitions in poly[d(G-C)] by histone subclasses, polyamino acids and polyamines.

The right-handed (B) conformation of poly[d(G-C)] in 7.5 mM sodium cacodylate and 25% ethylene glycol can be readily converted to the left-handed (Z) conformation by the addition of 250 microM MnCl2 and this transition can be reversed by chelation of the Mn ions with EDTA or by addition of NaCl. This ability to obtain such reversible transitions in solvent and solute conditions which allow DNA-protein interactions and their assessment by c.d. permitted an analysis of the effect of purified histones, polyamino acids, protamine and polyamines on these transitions. Individual core histones H3, H4, H2a and H2b or protamine stabilised the Mn-induced Z form and prevented the transition to B DNA normally observed after chelation with EDTA or on dialysis to physiological salt concentrations. A similar suppression of Z leads to B transition was also achieved with poly-L-arginine (but not with poly-L-lysine). In contrast, histones H1 and H5 promoted the Z leads to B transition. Polyamines (spermine and spermidine) converted the B form to another right-handed (A) form which transformed to the Z form after the addition of EDTA and this Z form was restored to the B conformation on the addition of NaCl. These results suggest that sequence-dependent variations in the conformation of natural DNA may be modulated by interaction with histones and other basic cellular components and may provide a conformational basis for nucleosome formation and possibly for the control of gene expression.

Animals↗
PubMed1982

Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain.

1. The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol has been studied by near-u.v. circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 have been correlated with the known oligomeric composition of microtubule protein preparations, as determined by the sedimentation behaviour of this preparation [Bayley, Charlwood, Clark & Martin (1982) Eur. J. Biochem.121, 579-585]. 2. The formation of 30S oligomeric ring species, either by decreasing ionic strength at pH6.5 or by changing pH in the presence of 0.1m-NaCl, correlates with a significant change in tubulin c.d. Formation of 18S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. 3. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. 4. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH6.4 than at pH6.9. 5. Oligomeric complexes of tubulin with microtubule-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.

Animals↗
PubMed1982

A circular-dichroism study of epidermolytic toxins A and B from Staphylococcus aureus.

The far-u.v. circular-dichroism spectra of the two epidermolytic toxins was analysed into fractional contributions of 0.09 helix and 0.46 beta-sheet to each toxin structure. Trifluoroethanol perturbation caused an initial increase in dichroic absorption at 205 nm and then a change characterized as a beta-sheet-to-alpha-helix transition. The intense near-u.v. spectra suggested that the toxins have unusually rigid, though different, aromatic-side-chain arrangements.

Bacterial Toxins↗
PubMed1982

Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion.

A conformational change in the hemagglutinin glycoprotein of influenza virus has been observed to occur to pH values corresponding to those optimal for the membrane fusion activity of the virus. CD, electron microscopic, and sedimentation analyses show that, in the pH range 5.2-4.9, bromelain-solubilized hemagglutinin (BHA) aggregates as protein-protein rosettes and acquires the ability to bind both lipid vesicles and nonionic detergent. Trypsin treatment of BHA in the pH 5.0-induced conformation indicates that aggregation is a property of the BHA2 component and that the conformation change also involves BHA1. The implications of these observations for the role of the glycoprotein in membrane fusion are discussed.

Bromelains↗
PubMed1982

The binding of NADH and NADPH to bovine-liver glutamate dehydrogenase. Spectroscopic characterisation.

1. The binding of NADH and NADPH to bovine liver glutamate dehydrogenase has been studied using circular dichroism (CD) and fluorescence spectroscopy, under comparable conditions of enzyme concentration. Spectroscopic titrations have been analysed using non-linear least-squares techniques to determine dissociation constants and parameters for the different chromophoric portions of the bound coenzymes, according to different models for the binding equilibria. 2. For NADPH, enhancement of dihydronicotinamide fluorescence and positive CD at 340 nm reflect binding of the coenzyme at the active site (site I) via the dihydronicotinamide moiety with a dissociation constant K1 of 50 microM; negative CD at 260 nm reflects the binding of a second molecule of coenzyme at the non-active site (site II) via the adenine moiety with a KII of 650 microM. 3. For NADH, the same spectroscopic assignment is made; however, site II affinity is much greater (KII 20 microM) and exceeds the affinity at the active site (KI 50 microM). 4. In no case was spectroscopic evidence obtained with either coenzyme to support the concept of non-identical active site binding as three sites IA and three sites IB per hexamer. 5. Coenzyme affinities are approximately twofold higher in triethanolamine buffer (0.05 + 0.1 M KCl, pH 7.6) compared with phosphate buffer (0.1 M at pH 7.0). 6. ADP enhances the CD of bound NADPH at 340 nm by up to twofold; phosphate ion also enhances the CD and binds with a Kd of 60-80 mM. GTP causes strong negative CD at 340 nm but only for the NADPH bound at site II. These results are consistent with independent binding sites for ADP (site II) and GTP (site III); phosphate ion is apparently able to affect certain properties of all three sites.

Animals↗
PubMed1981

Conformation and assembly characteristics of tubulin and microtubule protein from bovine brain.

The conformational requirements for the efficient assembly of bovine brain tubulin into microtubules have been investigated by using near-UV circular dichroism. Microtubule protein was prepared by the assembly-disassembly method of Shelanski et al. [Shelanski, M. L., Gaskin, F., & Cantor, C. R. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 765-768]. Tubulin dimer, isolated from this multiprotein complex by phosphocellulose ion-exchange chromatography in the presence and absence of Mg2+, was compared with tubulin dimer (WT dimer) prepared by the method of Weisenberg & Timasheff [Weisenberg, R. C., & Timasheff, S. N. (1970) Biochemistry 9, 4110-4116]. The tubulin from both dimer preparations showed identical electrophoretic patterns in which high molecular weight protein was undetectable. However, reproducible and significant differences were found in the near-UV CD spectra. Phosphocellulose-treated tubulin resembles the original microtubule protein more closely than does WT dimer, although this latter material has been widely accepted as being representative of the native protein. The phosphocellulose-treated tubulin and WT dimer are not readily interconvertible by simple physical or chemical treatments. The assembly capability of the various tubulin dimer preparations was compared by measuring the enhancement by tubulin dimer of assembly of ring fraction (isolated from microtubule protein by gel filtration of Sepharose 6B). Again phosphocellulose-treated tubulin is found to have more like native microtubule protein than does WT dimer.

Animals↗