Biomedical subjects

S R Martin

Publications and source records attributed to S R Martin.

At least 91 records · Page 5Linked to original sources

PubMed1988

Alpha-helix in the carboxy-terminal domains of histones H1 and H5.

Although the carboxy-terminal domains of histones H1 and H5 exist as random-coil in aqueous solution, secondary structure prediction suggests that this region has a high potential for alpha-helix formation. We have measured CD spectra in various conditions known to stabilize alpha-helices, to determine whether this potential can be realized in an appropriate environment. Trifluoroethanol increases the helix contents of H1, H5 and their carboxy-terminal fragments, presumably through promotion of axial hydrogen bonding. Sodium perchlorate is also effective and better than sodium chloride, suggesting stabilization by binding of bulky perchlorate ions rather than simple charge screening. Extrapolating from these measurements in solution, and taking into account the occurrence of proline residues throughout the carboxy-terminal domain, we propose that binding to DNA stabilizes helical segments in the carboxy-terminal domains of histones H1 and H5, and that it is this structured form of the domain that is functionally important in chromatin.

Animals↗

PubMed1988

Studies on the structure of the influenza virus haemagglutinin at the pH of membrane fusion.

At the pH required to trigger the membrane fusion activity of the influenza virus haemagglutinin (HA) the soluble ectodomain of the molecule, BHA, which is released from virus by bromelain digestion, aggregates into rosettes. Analyses of soluble proteolytic fragments derived from the rosettes indicated that aggregation is mediated by association of the conserved hydrophobic amino-terminal region of BHA2, the smaller glycopolypeptide component of each BHA subunit. Further analyses of the structure of the soluble fragments and of HA in its low pH conformation by electron microscopy, spectroscopy and in crosslinking experiments showed that, although the membrane distal globular domains lose their trimer structure at the pH of fusion, the central fibrous stem of the molecule remains trimeric and assumes a more stable conformation. The increase in length of BHA2 at low pH observed microscopically appears to result from movement of the amino-terminal region to the membrane proximal end of the molecule and in virus incubated at low pH the amino terminus may insert into the virus membrane. The consequences of these possibilities for the mechanism of membrane fusion are discussed.

Bromelains↗

PubMed1988

Membrane fusion by peptide analogues of influenza virus haemagglutinin.

We have studied the interactions of synthetic peptides corresponding to the sequence of the amino terminus of the HA2 subunit of influenza virus haemagglutinin with artificial lipid membranes. The peptides could fuse cholesterol-free liposomes at neutral as well as acid pH; however, liposomes containing cholesterol could only be fused below pH 6. The fusion process caused leakage of aqueous liposomal contents. Peptides with amino acid substitutions had fusion properties similar to whole haemagglutinin molecules with the corresponding sequence changes. Non-fusogenic peptides still interacted with the membrane but did not cause leakage of liposomal contents. A correlation between the alpha-helical content of peptide and its fusogenicity was noted, but this was not absolute. The results reported here support suggestions for a role of the amino terminus of HA2 in virus-endosome fusion.

Hemagglutinins, Viral↗

PubMed1987

Dynamic properties of microtubules at steady state of polymerisation.

Microtubules of tubulin dimer, polymerised in vitro to steady-state are shown to incorporate tubulin rapidly and extensively. The method involves adding [3H]-GTP to microtubules at steady state, and analysing for non-exchangeable [3H]-GDP in the presence of a GTP regenerating system. The rate and extent of this exchange process is dependent on the length distribution of the microtubules, and is notably faster with sheared microtubules. We simulate all these features of the exchange kinetics, together with the length redistributions occurring at steady state of polymerisation, using a simple model based on a limited number of kinetic parameters deriving from the measurements of microtubule dynamics by Horio and Hotani. The observed exchange kinetics thus provide a direct experimental criterion of 'dynamic instability' of microtubules at steady state of polymerisation.

Guanosine Diphosphate↗

PubMed1987

On the relationship between nucleotide hydrolysis and microtubule assembly: studies with a GTP-regenerating system.

The assembly of pure tubulin dimer has been studied in two buffer systems (containing low and high glycerol/Mg), using a regeneration system protocol to assess the amount of GDP-tubulin in the assembling polymer. For both assembly systems studied, the GDP content is effectively stoichiometric with tubulin throughout assembly. This indicates a high degree of coupling between assembly and GTP-hydrolysis, giving a hydrolysis rate at least 10-fold faster than previously deduced. The steady state GTP hydrolysis rate is quantitatively consistent with this finding. We conclude that the extent of any GTP-tubulin cap is below the detectable limit, both during elongation and at steady state.

Animals↗

PubMed1987

Magnesium ion effects on microtubule nucleation in vitro.

Much interest has currently been attached to the length distribution of microtubules polymerized in vitro and the related question of their possible 'dynamic instability'. Fundamental to this question is the mechanism of microtubule nucleation, which controls the rates of assembly and disassembly of microtubule protein in vitro. These kinetics are affected by a number of factors, including both the guanine nucleotides, GTP and GDP, and magnesium ion. Mg2+ exerts complex effects, as indicated by the existence of an optimal Mg2+ concentration for the maximum assembly rate of microtubule protein, and we investigate these effects in this report. At [Mg2+] greater than 0.5 mM, the characteristic lag-phase is substantially increased and the rate of assembly is greatly reduced without affecting the critical concentration significantly. We show that increasing [Mg2+] has two effects on the assembly process: nucleation is less efficient and the intrinsic rate constant for the elongation reaction is reduced. Lowering [Mg2+] (less than 0.5 mM) also inhibits nucleation. These effects of varying [Mg2+] can be explained predominantly in terms of enhanced stability of the microtubule-associated protein-containing oligomeric species present in the microtubule protein preparation. [Mg2+] is thus found to be a further important factor in microtubule nucleation, and hence, in determining length distributions in assembling microtubules.

Animals↗

PubMed1987

Effects of GDP on microtubules at steady state.

We have re-examined the effect of varying GDP concentrations on the kinetics of GTP-induced assembly of microtubules from microtubule protein, and on the elongation of pre-existing microtubules subjected to a temperature jump relaxation from 21.5 to 37 degrees C. The assembly kinetics follow a simple model for assembly which involves a fast equilibrium of tubulin-GTP and tubulin-GDP coupled to the elongation process due to tubulin-GTP. The initial rate of the relaxation process is found to be dependent upon the GTP/GDP ratio, in confirmation of the results of Engelborghs and Van Houtte (Biophys. Chem. 14 (1981) 195). As an alternative to the interpretation previously advanced by them, involving modification of the reactivity of microtubule ENDs by GDP, we show that this result is consistent with the above model with one reasonable modification, namely, that the ratio of the affinities of tubulin for GTP and GDP should vary with temperature. The analysis shows that a decrease in this ratio of approx. 2-fold at 37 degrees C accounts for the observed effects. We conclude that more complex mechanisms involving consideration of modification of the reactivity of microtubule ENDs by GDP are not required to explain these results. This finding has important implications for current models of GDP-induced microtubule disassembly.

Animals↗

PubMed1986

Kinetics of cadmium and terbium dissociation from calmodulin and its tryptic fragments.

The kinetics of cadmium and terbium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by stopped-flow fluorescence methods, using the calcium indicator Quin 2. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, have clearly identified cadmium binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. Thus the site preference of cadmium is the same as that of calcium. For terbium, however, sites I and II are the high-affinity sites and sites III and IV are the low-affinity sites. Thus, the site preference or terbium is not the same as that of calcium and cadmium. In contrast to previous studies with calcium, we observe two kinetic processes for dissociation from sites III and IV for experiments with both cadmium and terbium. Possible models for the binding of metal ions are discussed.

Binding Sites↗

PubMed1986

Solution structure of human growth hormone releasing factor. Combined use of circular dichroism and nuclear magnetic resonance spectroscopy.

The solution structures of two human growth hormone releasing factor analogues, 27Leu45Gly-hGHRF(1-45)OH and 27Nle-hGHRF(1-29)NH2, are investigated by means of circular dichroism and nuclear magnetic resonance spectroscopy. Using circular dichroism spectroscopy, it is shown that both peptides adopt ordered structures at low concentrations of trifluoroethanol (approximately 30%). Quantitative analysis of the circular dichroism spectra indicates that the same number of residues, approximately 23 to 25, are in a helical state in both peptides. Using two-dimensional nuclear magnetic resonance methods all proton resonances of the 27Nle-hGHRF(1-29)NH2 fragment are assigned and its secondary structure is determined from a qualitative interpretation of the nuclear Overhauser enhancement data. Two distinctive regions of alpha-helix are present extending from residues 6 to 13 and 16 to 29.

Amino Acid Sequence↗

PubMed1986

The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study.

Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.

Animals↗

PubMed1986

N-alkylation of exogenous haem analogues caused by drugs in isolated hepatocytes. Structural isomerism and chirality of the resulting porphyrins.

Isolated rat hepatocytes incubated with two suicide substrates of cytochrome P-450, 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine(4-ethyl-DD C), convert exogenous mesohaem and deuterohaem into N-alkylated mesoporphyrins and deuteroporphyrins respectively. The N-alkylated mesoporphyrins can be separated by h.p.l.c. from the corresponding N-alkylated protoporphyrins originating from endogenous haem; in this way the contribution of both endogenous and exogenous pools of haem can be studied in the same experiment. N-Alkylated mesoporphyrin exhibits chiral properties, and its isomeric composition and/or amount are dependent on the particular cytochrome P-450 enzyme predominating in the cell. These findings provide additional and more direct evidence that exchangeable haem is taken up by cytochrome P-450 before being N-alkylated.

Acetamides↗

PubMed1986

Inhibition of microtubule elongation by GDP.

GDP reduces both the rate and amplitude of GTP-induced assembly of microtubules from tubulin dimer or from microtubule protein, and promotes disassembly from microtubules at the steady state. One interpretation postulates that added GDP modifies microtubule ENDS so that tubulin-GTP, the species involved in steady state elongation of microtubules, cannot bind to a microtubule END containing tubulin-GDP. This concept has been used in subsequent models of assembly which treat the 'dynamic instability' of microtubules. We question this interpretation on the basis of the published experimental data and the results reported here. Using a relatively simple model for microtubule assembly, we show by numerical simulation that the quantitative effects of GDP on the rate and amplitude of microtubule assembly and inhibition of steady state GTPase activity are well accounted for by the nucleotide exchange equilibrium of tubulin-GDP and tubulin-GTP. We therefore conclude that the effect of added GDP on elongation of MAP-containing microtubules and on steady state GTPase activity does not indicate modification of the activity of microtubule ENDs but depends on the tubulin-GTP/tubulin-GDP equilibrium. Additional evidence argues that microtubule ENDS containing GDP can indeed accept elongation by tubulin-GTP.

Animals↗

PubMed1986

Protein conformation of potato (Solanum tuberosum) lectin determined by circular dichroism.

The structure of potato (Solanum tuberosum) lectin, which is a hydroxyproline-rich glycoprotein, has been investigated by circular dichroism. The spectra of the native lectin, and of the oxidized, reduced and carboxymethylated and deglycosylated derivatives were examined, as was a hydroxyproline-rich glycopeptide and its deglycosylated derivative. It is concluded that the lectin contains about 35% polyproline II conformation, 34% type II beta-turn and 31% irregular conformation. No indications were found for the presence of alpha-helix or beta-sheet conformations. The polyproline II conformation is heat-stable, but is markedly destabilized by deglycosylation. The type II beta-turn is destabilized by cleavage of disulphide bonds.

Amino Acids↗

PubMed1986

Conformational changes in the hemagglutinin of influenza virus which accompany heat-induced fusion of virus with liposomes.

Incubation of X-31 influenza virus at 62 degrees results in changes in hemagglutinin structure which cause the virus to fuse with liposomes at neutral pH. Mutants of X-31 which contain different hemagglutinins and as a consequence fuse with liposomes at higher pH than wild-type virus also fuse at a lower temperature. The heat-induced changes in hemagglutinin structure were monitored by CD, fluorescence spectroscopy, electron microscopy, and proteolytic digestion and compared with the characteristic changes which occur at low pH required for hemagglutinin-mediated membrane fusion at physiological temperature. The results indicate that the heat-induced changes in hemagglutinin which allow fusion activity result in partial denaturation of the molecule. By comparison the changes in structure required for fusion at low pH and normal temperature are specifically restricted.

Hemagglutinins, Viral↗

PubMed1986

Interaction between chloramphenicol and acetaminophen.

Acetaminophen has been reported either to prolong or not to affect the clearance of chloramphenicol. To confirm one of these findings we studied the clearance of chloramphenicol and its metabolites using high pressure liquid chromatography in five patients (ages 2.5 to 5 years) before and during oral treatment with acetaminophen (50 mg/kg/day). Significant differences were observed in mean (SD) peak serum chloramphenicol concentration (-9.7 (3.2) mg/l), mean (SD) apparent volume of distribution (+225 (162) ml/kg), mean (SD) chloramphenicol half life (-1.9 (1.1) hours), mean (SD) chloramphenicol clearance (+236 (94) ml/kg/h), mean (SD) area under the curve (-83.5 (33.0) mg/l/h), and mean (SD) elimination constant (+0.34 (0.13) h-1) between samples obtained before and during treatment with acetaminophen. Acetaminophen, when given orally for several days, increased the clearance of chloramphenicol, perhaps by increased glucuronidation. This report re-emphasises the need for therapeutic drug monitoring whenever these two drugs are used together.

Acetaminophen↗

PubMed1986

Retrobulbar anesthesia and orbicularis akinesia.

After noting that simple retrobulbar injection often caused orbicularis akinesia, we evaluated this phenomenon prospectively. A total of 50 patients were given a standard injection using 3 cc of a 50-50 mixture of 0.75% bupivacaine and 2% lidocaine with epinephrine and hyaluronidase. The entire 3 cc was injected into the muscle cone through an inferolateral lid entry using a 35-mm retrobulbar needle of either 23- or 27-gauge. Injection was considered successful if orbicularis function was sufficiently reduced to proceed with an intraocular case after a single retrobulbar injection. If facial nerve block was necessary or if the patient's lid moved during surgery, then the procedure was considered a failure. Of the 50 patients, 44 achieved adequate akinesia of the orbicularis after retrobulbar injection alone, giving a success rate of 88%. Benefits of this method include lower drug volume. decreased chance for direct nerve damage during a facial nerve block, less pain, less ecchymosis, and less edema following surgery.

Anesthesia, Local↗

PubMed1985

Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.

The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.

Aminoquinolines↗

PubMed1985

The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins.

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.

Animals↗