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R H Pain

Publications and source records attributed to R H Pain.

At least 73 records · Page 4Linked to original sources
PubMed1981

The existence of a precursor of cathepsin D: evidence from autolysis, denaturation and activation studies.

This report evidences that the single polypeptide chain of cathepsin D undergoes in vitro autolysis resulting in heavy (Mr about 30000) and light (Mr about 15000) polypeptide chains. These two chains are held together through non-covalent interaction, thus constituting a stable active conformation. Fluorescence and circular dichroism measurements demonstrate the irreversible denaturation of cathepsin D. The existence of cathepsin D precursor, cathepsinogen D, of about 50000 molecular weight was proved. Cathepsinogen D is converted to the active enzyme by intramolecular activation, releasing activation-inhibitory peptide(s).

Animals↗
PubMed1980

Time resolved spectroscopy of tryptophyl fluorescence of yeast 3-phosphoglycerate kinase.

The tryptophyl fluorescence emission of yeast 3-phosphoglycerate kinase decreases from pH 3.9 to pH 7.2 following a normal titration curve with an apparent pK of 4.7. The fluorescence decays have been determined at both extreme pH by photocounting pulse fluorimetry and have been found to vary with the emission wavelength. A quantitative analysis of these results according to a previously described method allows to determine the emission characteristics of the two tryptophan residues present in the protein molecule. At pH 3.9, one of the tryptophan residues is responsible for only 13% of the total fluorescence emission. This first residue has a lifetime tau 1 = 0.6 ns and a maximum fluorescence wavelength lambda 1max = 332 nm. The second tryptophan residue exhibits two lifetimes tau 21 = 3.1 ns and tau 22 = 7.0 ns (lambda 2max = 338 nm). In agreement with the attribution of tau 21 and tau 22 to the same tryptophan residue, the ratio beta = C21/C22 of the normalized amplitudes is constant along the fluorescence emission spectrum. At pH 7.2, the two tryptophan residues contribute almost equally to the protein fluorescence. The decay time of tryptophan 1 is 0.4 ns. The other emission parameters are the same as those determined at pH 3.9. We conclude that the fluorescence quenching in the range pH 3.9 to pH 8.0 comes essentially from the formation of a non emitting internal ground state complex between the tryptophan having the longest decay tie equilibrium constant of the internal complex can be estimated. The quenching group is thought to be a carboxylate anion. Excitation transfers between the two tryptophyl residues of the protein molecule appear to have a small efficiency.

Hydrogen-Ion Concentration↗
PubMed1978

Conformation of a stable intermediate on the folding pathway of Staphylococcus aureus penicillinase.

The partly unfolded intermediate (state H) of penicillinase from Staphylococcus aureus PC1 is stable in 0.8 M guanidinium chloride at pH 7.0. It has been characterized by measurements of intrinsic viscosity, sedimentation and diffusion coefficients, leading to an equivalent hydrodynamic volume of five times that of the native penicillinase molecule. Values of alpha-helix content calculated from circular dichroism were 27% for penicillinase and 16% for state H. A multi-domain model is proposed for penicillinase in which the domains can separate without appreciable change in secondary structure. This model is important in understanding the means by which the enzyme activity can be controlled, and corresponds to a kinetic pathway of folding.

Circular Dichroism↗
PubMed1976

The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies.

1. A homogeneous preparation of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

Guanidines↗
PubMed1976

The mechanism of folding of globular proteins. Equilibria and kinetics of conformational transitions of penicillinase from Staphylococcus aureus involving a state of intermediate conformation.

1. The thermodynamically reversible unfolding and refolding of penicillinase between the native and fully unfolded states were followed by using guanidinium chloride as denaturant. 2. The equilibria, studied by optical rotation, u.v. absorption, viscosity and enzyme activity, show the presence of a state of intermediate conformation, termed state H, which is stable at 20 degrees C in 0.8 M-guanidinium chloride. 3. The physical properties of this state show that it is slightly expanded with an intrinsic viscosity of 8 ml-g-1, that the 13 tyrosine residues, which are distributed through the primary sequence, are maximally exposed to the solvent and that the helix content is the same as that of the native state. 4. The kinetics of the transition between the native state, state H and the fully unfolded state were followed by u.v. absorption and by optical rotation. They are interpreted as showing that state H lies on the folding pathway between the native and fully unfolded states. 5. The transition between the native state and state H exhibits monophasic unfolding kinetics and biphasic refolding kinetics. This indicates that there must be at least two intermediate states in this process, at least one of which lies on the folding pathway which may also involve cul-de-sac paths. 6. The results are discussed in terms of a mechanism involving rapid stabilization of nucleation regions in a moderately compact but internally solvated structure, with 'native format' [Anfinsen (1973) Science 181, 233-230] secondary structure stabilized by tertiary interaction. The final and rate-limiting step in refolding involves shuffling of these structural elements into the native state. 7. This model is discussed in relation to folding in vivo.

Guanidines↗
PubMed1975

The active site of penicillinase from Staphylococcus aureus PC1. Isolation of a specific covalent complex with the substrate quinacillin.

1. The penicillinase-catalysed hydrolysis of quinacillin was quenched by addition of 5 m-guanidinium chloride or 1% (w/v) sodium dodecyl sulphate, and the quenched reaction mixture was dialysed exhaustively against solutions of the denaturant. 2. Irreversibly bound quinacillin was shown by titration with HgCl2 to be covalently attached to the protein by the beta-lactam carboxyl group. 3. The derivative was found to be stable over the pH range 3.5-8.5. 4. Chymotryptic hydrolysis of the product and subsequent fractionation showed that quinacillin was bound to one or possibly two peptides.

Binding Sites↗
PubMed1974

Conformational changes in gastric mucoproteins induced by caesium chloride and guanidinium chloride.

1. Caesium chloride and guanidinium chloride were shown to cause conformational changes in the high-molecular-weight mucoprotein A of water-soluble gastric mucus with no change in molecular weight. 2. Increasing concentrations of CsCl decrease the viscosity of the mucoprotein bringing about a transition which is essentially complete in 0.1m-CsCl. The shear-dependence of viscosity of the mucoprotein is abolished by low concentrations of CsCl. The normally highly expanded molecule becomes contracted in CsCl to a molecule having the same symmetry but a smaller volume and decreased solvation, in keeping with an increased sedimentation coefficient (18.7S-->33S). 3. This contracted form does not revert to the native conformation on removal of the CsCl. 4. A mechanism is discussed in terms of the effect of the Cs(+) and Cl(-)ions on water structure and the water-mucoprotein interaction. 5. Guanidinium chloride causes the CsCl-treated material to expand, in keeping with a decrease in s(0) (25,w) (33S-->26S). This is analogous to the known unfolding effect of guanidinium chloride on proteins and suggests that guanidinium chloride solubilizes groups involved in stabilizing the contracted structure. Removal of the guanidinium chloride results in a limited aggregation of four mucoprotein molecules. 6. These results show that caution must be exercised before interpreting the physical properties of mucoproteins which have been treated with CsCl and/or guanidinium chloride.

Animals↗
PubMed1974

Analysis of the code relating sequence to conformation in globular proteins. Development of a stereochemical alphabet on the basis of intra-residue information.

1. The relation of primary sequence to all residue backbone conformations was explored to test out starting conformations for protein folding. 2. Information theory was used to obtain measures of information which quantitate the role of each residue in determining its own conformation; i.e. intra-residue information. 3. The information measures are plotted as a function of varphi, psi peptide-backbone angles and varphi, psi contour maps obtained for each of the 20 amino acids. These show characteristic differences between residues. 4. To find practical ways of relating sequence to varphi, psi angles, several types of stereochemical alphabet were investigated. The value of these was tested by using them to predict the varphi, psi angles of nine different proteins. 5. A difference plot was constructed to show regions of the sequence that require little or no information extra to the intra-residue information in order to predict a correct conformation. These regions are suggested to be candidates for nucleating sites in the protein.

Amino Acid Sequence↗