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Biomedical subjects

R H Pain

Publications and source records attributed to R H Pain.

At least 55 records · Page 3Linked to original sources
PubMed1987

Temperature and macromolecular structure and function.

Stability is frequently a knife-edge phenomenon and it is this aspect which is both essential for the effective involvement of macromolecules in the living cell and also provides the basis for the sensitivity of some macromolecular systems to temperature. The response of proteins and nucleic acids is relatively simple at the phenomenological level, with rather sharp 'melting' transitions occurring. Since however the thermodynamic stability of both depends on the cooperation of a variety of non-covalent interactions which are qualitatively well understood but quantitatively difficult to assess, the full understanding and prediction of structural stability still evades us. We shall consider the effects of temperature on the different non-covalent interactions and how far these can account for protein and nucleic acid denaturation at elevated temperatures and also the cold inactivation of proteins. The latter has recently been shown to involve unsuspected complications in terms of protein conformational change. The increased stability of proteins and nucleic acids from thermophiles will be discussed. Stability is important not only in native folded proteins but also with respect to intermediate structures which occur during the folding of the newly synthesized polypeptide chain into the native, active protein. Through studies of protein folding the molecular basis of the phenomenon of temperature sensitive synthesis has been revealed. Given a stable molecular structure, its function will frequently be subject to variation with temperature. The deceptively simple temperature dependence of enzyme activity will involve the non-covalent interactions considered above for interaction between enzyme and substrate and for stability of the transition state complex. This complexity again makes the temperature dependence difficult to interpret. Further, the fact that proteins are dynamic structures is becoming recognized as an important feature factor in determining function. A balance has to be struck between on the one hand dynamic mobility which is essential for catalytic activity and on the other thermodynamic stability which holds the molecule in a potentially functional conformation under the given conditions of temperature and pressure. Readjustment of thermostability stability, as in thermophiles (or vice versa?), must also involve readjustment of dynamic mobility.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Reversible deactivation of beta-lactamase by quinacillin. Extent of the conformational change in the isolated transitory complex.

Conditions have been established where the deactivation of the beta-lactamase from Staphylococcus aureus PC1 by the penicillin substrate, quinacillin, is close to complete but fully reversible. The temperature-dependence of the rate of re-activation indicated a half-life of about 170 min for the deactivated state at 0 degrees C. Measurement of the relative viscosity of mixtures of enzyme and quinacillin at 8.4 degrees C ruled out any significant difference in shape or solvation between the deactivated and the normal enzyme. C.d. measurements of the deactivated protein, separated from excess quinacillin, showed that the quinacillin side-chain chromophore was bound in an asymmetric environment. The ellipticity associated with the bound quinacillin chromophore decreased with the same first-order rate constant as that for reappearance of enzyme activity. These findings support the accumulation of a deactivated state that contains bound quinacillin or a derivative. Quinacillin caused a 3-fold increase in the rate of 3H exchange-out (at a rate that was low compared with that for the substantially unfolded or expanded protein). However, there was rapid exchange-out of about 50 3H atoms on addition of 1 M-urea to the deactivated enzyme, whereas the same concentration had no effect on the exchange-out of 3H from native enzyme. The interpretation that quinacillin increases the susceptibility of the native state to unfolding in the presence of urea is supported by the demonstration that SO4(2)- ions decreased the rate and extent of deactivation but had no effect on the rate of re-activation, as predicted from the observation that SO4(2)- ions, in competition with urea, stabilize the native state relative to the partially unfolded state H [Mitchinson & Pain (1985) J. Mol. Biol. 184, 331-342].

Circular Dichroism↗
PubMed1986

The effect of substrates on the inter-domain interactions of the hinge-bending enzyme 3-phosphoglycerate kinase.

A hinge-bending mechanism has been proposed for phosphoglycerate kinase, in which the two domains bend about the connecting 'waist' region. In partially denaturing concentrations of guanidinium chloride the substrate 3-phosphoglycerate stabilises one domain against denaturation and destabilises the other. The reduction of mutual stabilisation of the two domains on binding substrate indicates a freeing of the hinge to allow the protein to take up other states rather than a directive mechanism. The stabilisation of both domains at higher concentrations of ATP at which the enzyme is inhibited supports this mechanism.

Adenosine Triphosphate↗
PubMed1985

The folding and mutual interaction of the domains of yeast 3-phosphoglycerate kinase.

Analysis of the reversible unfolding of yeast phosphoglycerate kinase leads to the conclusion that the two lobes are capable of folding independently, consistent with the presence of intermediates on the folding pathway with a single domain folded. The domains have different free energies of stabilisation. Immunological cross-reactivity, circular dichroism and thiol reactivity provide evidence for cyanogen bromide peptide 1-173, which comprises five-sixths of the N-terminal domain, containing sufficient information to refold into a native-like structure which dimerises.

Circular Dichroism↗
PubMed1985

Single amino acid mutations block a late step in the folding of beta-lactamase from Staphylococcus aureus.

Two single amino acid mutant proteins of beta-lactamase PC1 from Staphylococcus aureus, P2 Thr40----Ile and P54 Asp146----Asn, have been investigated using urea-gradient polyacrylamide gel electrophoresis, circular dichroism and sedimentation velocity. Investigation of the folded states of the mutants has shown that compared to wild-type PC1 they are slightly more expanded, and have reduced aromatic circular dichroism, but the same content of secondary structure as PC1. The mutants exhibit fast refolding kinetics to the folded state, in contrast to PC1, which refolds only slowly. We conclude from these results that the folded mutants are in a state close to but distinct from the native state of PC1 and have certain properties in common with the compact intermediate in the folding of beta-lactamase. Therefore, these single amino acid substitutions result in a folding pathway blocked at a point located after collapse of the already folded structural units into a globular shape, and close to the final reshuffling step that leads to the native state of the wild-type enzyme.

Amino Acid Sequence↗
PubMed1985

Effects of sulphate and urea on the stability and reversible unfolding of beta-lactamase from Staphylococcus aureus. Implications for the folding pathway of beta-lactamase.

The reversible denaturation by urea of beta-lactamase from Staphylococcus aureus was followed in the presence and absence of ammonium sulphate by circular dichroism studies, difference absorption spectroscopy and measurement of enzyme activity. The multiple unfolding and refolding transitions demonstrate the existence of a thermodynamically stable state of intermediate conformation in equilibrium with the native (N) and fully unfolded (U) states. Its physical properties show that it is identical to the state H found on denaturation by guanidinium chloride. State H is 10.1 (+/-1.5) kJ mol-1 less stable than the native state and 10.1 (+/-1.6) kJ mol-1 more stable than the unfolded state. Ammonium sulphate shifts both the N in equilibrium H and H in equilibrium U transitions to concentrations of urea higher by 5.3 M per mole of sulphate. It has markedly different effects on the thermodynamic stabilities of states N and H, making delta G'N-H, O and delta G'H-U, O more negative by 41 kJ mol and 20 kJ mole, respectively, per mole of ammonium sulphate. The change in equilibrium constant for the N-H transition is reflected almost exclusively in a dramatic change of the unfolding rate constant, which is decreased by a factor of 10(11) on addition of 1.4 M-sulphate. The presence of the substrate benzyl penicillin has little effect on the equilibria or kinetics of the N-H transition. The results are discussed in terms of the nature of the N-H transition and of the ordering of intermediate states on the folding pathway.

Ammonium Sulfate↗
PubMed1985

A reinvestigation of the cross-reactivity between Klebsiella and HLA-B27 in the aetiology of ankylosing spondylitis.

The existence of cross-reactivity between Klebsiella antigens and cells from donors who are HLA-B27 positive and exhibit ankylosing spondylitis (AS) has been reinvestigated. Cells and antisera from different laboratories have been tested together using simultaneously microcytoxicity, chromium release and enzyme linked immunosorbent assays (ELISA). No reproducible interaction has been found. Mitogenic stimulation did not induce cross-reactivity and 'transformation' of B27+AS- cells by Klebsiella culture supernatants failed. Two transformed cell lines from B27+ AS+ donors exhibited specific cross reaction with two anti-Klebsiella antisera but only by chromium release. Immunoprecipitation with these cells and antisera showed the absence of any AS+ -specific antigen. It is concluded that the involvement of Klebsiella in ankylosing spondylitis through simple immunological cross-reactivity or through interaction with HLA-B27 is unlikely.

Antigens, Bacterial↗
PubMed1984

The reversible deactivation of beta-lactamase from Staphylococcus aureus by quinacillin and cephaloridine and its modification by antibodies.

The effect of antibody on the reversible deactivation of the beta-lactamase (penicillin amino-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus has been studied using quinacillin and cephaloridine as substrates. The latter has been shown to exhibit the characteristics of an A-type substrate Citri, N., Samuni, A. and Zyk, N. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052) and reversibly to lower the activity of the enzyme towards benzylpenicillin in a manner analogous to quinacillin. Both divalent and monovalent antibodies reduce the activity of the lactamase to 60% of the native value in the absence of substrate. The reduction by monovalent antibody is slow (t1/2 approximately equal to 25 min). Both divalent and monovalent antibodies modify the time-course of reversible deactivation independently of being added before or subsequent to deactivation by substrate. The full recovery of activity is delayed in the case of quinacillin and accelerated for cephaloridine. The activity against benzylpenicillin in the deactivated states is unaffected. These effects are shown to reflect the changed rates of hydrolysis of the two substrates in the presence of antibody. The effect of antibody is mediated by minor conformational change. Continuous assays for following the hydrolysis of quinacillin and cephaloridine by optical rotation are reported.

Cephaloridine↗
PubMed1984

Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies.

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

Animals↗
PubMed1983

Identification by n.m.r. spectroscopy of a stable intermediate structure in the unfolding of staphylococcal beta-lactamase.

The unfolding of beta-lactamase (penicillinase) from Staphylococcus aureus by guanidinium chloride was followed by using n.m.r. spectroscopy. On the basis of the observation of resonances corresponding to histidine, tyrosine and other amino acid side chains, the existence of a stable partially folded species was demonstrated. These experiments provide detailed characterization of the intermediate that confirms and extends previous characterization by absorption and c.d. spectroscopy and by flow properties. In addition, they show that residues in the N-terminal third of the molecule are affected by the native-to-intermediate transition. Persistent non-equivalence of the two imidazole C2 proton resonances at high guanidinium chloride concentrations is discussed in terms of local sequence effects on the chemical shift.

Guanidine↗
PubMed1983

The relative effectiveness of guanidinium and some biguanide salts as denaturants. Assessment against penicillinase.

Penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) has been used as a model for quantitating the effectiveness of several guanidine-derived denaturants. It was chosen because the mechanism of its denaturation by urea and guanidinium chloride has been worked out in detail, because it has a low thermodynamic stability bringing it in the range of weak denaturants, and because its denaturation can readily be followed by enzyme activity as well as by spectroscopic probes. Contrary to previous reports, biguanide HCl is found to be no more effective than guanidinium chloride. The denaturant effectiveness of the various compounds studied is found to increase in the order: guanidinium chloride identical to biguanide HCl less than propylbiguanide HCl less than hexylbiguanide HCl much less than n-decylbiguanide HCl. n-Decylbiguanide HCl is a particularly powerful denaturant, unfolding penicillinase at a concentration of less than 0.015 M.

Biguanides↗
PubMed1982

Partial purification of a low molecular weight inhibitor of epidermal DNA synthesis.

An attempt has been made to purify factors present in aqueous extracts of pig epidermis which inhibit epidermal cell proliferation. A lipophilic factor of low molecular weight (less than 10,000), has been shown to inhibit DNA synthesis as measured by the uptake of tritiated thymidine in mouse ear epidermis. Purification by alcohol precipitation, ethyl acetate extraction and silicic acid column chromatography produced a fifteen-fold increase in the specific activity of the inhibitory action. It seems likely that aggregation or absorption of this low molecular weight factor may explain the high molecular weight of epidermal cell proliferation inhibitors previously studied, as well as the difficulty in their characterization.

Animals↗