Biomedical subjects
Publications and source records attributed to N C Price.
Corneal endothelial cell density was measured in normal subjects and in a group of monozygotic and dizygotic twins. Cell density was found to decline with age and to be similar in right and left eyes in normal subjects. Both monozygotic and dizygotic twins were found to have very similar mean endothelial cells densities and particularly so in like-sided eyes.
A contact specular microscope was compared with a noncontact endothelial camera on 20 eyes of 10 consecutive patients presenting for cataract surgery. No difference was found in the percentage of photographs acceptable for cell counting or the time for photography by one or other camera. A close correlation was found in cell density estimations by the 2 methods. Most patients prefer the noncontact mode of examination.
Lens implant surgery has a significant complication rate, although most complications are treatable and occur within the first 2 months after operation. The short-term visual results are comparable with the published results of intracapsular extraction alone. Six cases of bullous keratopathy developed in 138 cases in 7 1/2 years. When endothelial cell density fell below 500/mm2, corneal oedema developed within the year. However, a more recent study showed that endothelial cell loss was much reduced by the closed chamber technique of insertion and by serum coating of the implant.
1. The refolding of rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) which had been denatured in 3 M guanidine hydrochloride was monitored by studying the regain of enzyme activity. Full activity could be regained provided that the residual denaturant concentration was less than or equal to 0.1 M. 2. The refolded product was shown by a number of criteria (CD, kinetic parameters and polyacrylamide gel electrophoresis) to be identical with the native enzyme. 3. The rate of regain of enzyme activity was studied as a function of protein concentration. It was found that 70% of the activity was regained in a rapid, first-order process. The remaining activity was regained more slowly. In the rapid phase the number of reactive thiol groups per subunit declined from four to two; the further decline to one per subunit occurred more slowly. 4. It was found that the presence of the reducing agent dithiothreitol was not necessary for the regain of full activity, provided that the chelating agent EDTA was present. 5. The subunit structure of the enzyme during refolding was studied using dimethylsuberimidate as a cross-linking agent. From these experiments, a pathway for the refolding process could be proposed.
The reaction of rabbit muscle creatine kinase with diethyl pyrocarbonate was studied. It was found that up to five of the sixteen histidine groups per enzyme subunit could be modified, and under the conditions employed, there was no evidence for formation of the disubstituted derivative of histidine. Evidence was obtained for small but significant amounts of modification of lysine and cysteine groups; tyrosine groups were not modified. Modification of the enzyme led to inactivation; this could be protected against by inclusion of substrates or, more effectively, by inclusion of the combination MgADP plus creatine plus nitrate, which is thought to produce a 'transition-stage-analogue' complex. Analysis of data on the rates of inactivation and the stoicheiometry of modification suggested that there was one essential histidine group per enzyme subunit, modification of which led to inactivation.
The dimeric enzyme creatine kinase from rabbit muscle was treated with three derivatives of iodoacetamide that are capable of introducing fluorescent groups into the enzyme. All the three reagents (4-iodoacetamidosalicylate (IAS), 5-[N-(iodoacetamidoethyl)amino]-naphthalene-1-sulphonate (IAEDANS) and 6-(4-iodoacetamidophenyl)aminonaphthalene-2-sulphonate (IAANS)) were shown to react at the same single thiol group on each enzyme subunit, leading to complete inactivation of the enzyme. The reaction with IAS was extremely rapid by comparison with the reaction with iodoacetamide or iodoacetate, but various lines of evidence suggest that IAS is not a true affinity label. However, kinetic and binding studies indicate that salicylate itself probably binds at the nucleotide-binding site on the enzyme. As the size of the modifying reagent increased, the first thiol group reacted more rapidly than the second; this trend was more pronounced at 0 degree C than at 25 degree C. With the largest modifying reagent used (IAANS), the pronounced biphasic nature of the modification reaction permitted the preparation of a hybrid enzyme in which only one subunit was modified, but a study of the thiol-group reactivity showed that this hybrid enzyme preparation underwent subunit rearrangement.
Dimeric creatine kinase (EC 2.7.3.2) from rabbit skeletal muscle can be immobilized via a single subunit to CNBr-activated Sepharose 4B and subsequently treated with guanidine hydrochloride followed by renaturation to yield a catalytically active matrix-bound subunit derivative. The importance of the intact dimeric structure in the activation of the enzyme by acetate was demonstrated. Immobilization did not appear to alter the pH optimum of the enzyme, and the kinetic parameters fot the matrix-bound derivatives were generally similar to those for the soluble enzyme, but the matrix-bound derivatives showed higher thermal stability and greater resistance to denaturation than did the soluble enzyme. The rates of reaction of thiol groups of the matrix-bound derivatives with iodoacetamide in the absence and in the presence of combinations of substrates were similar to those of the soluble enzyme. Studies with 5,5'-dithiobis-(2-nitrobenzoic acid) and with iodoacetamide revealed the presence of an additional reactive thiol group in the matrix-bound subunit derivative, which is presumably masked in the dimeric derivatives.
Substrate- and ligand-induced conformational changes were studied in a series of thiol-modified derivatives of rabbit muscle creatine kinase that retained different amounts of enzymic activity. The results indicate that the 'reactive' thiol group of the enzyme is required for the conformational changes associated with formation of a 'transition-state analogue' complex.
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2-Nitro-5-thiocyanatobenzoic acid has been proposed as a reagent for converting thiol groups in proteins into their S-cyano derivatives. Evidence was obtained for formation of both the S-cyano derivative and the mixed disulphide derivative. Formation of the S-cyano derivative can be promoted by addition of excess of CN-to the reaction mixture.
1. The dimeric enzyme creatine kinase (ATP: creatine N-phospotransferase, EC 2.7.3.2) from rabbit muscle was reacted with three separate reagents, each of which specifically modifies one thiol group per subunit. 2. The reactions of the enzyme with these reagents (4-chloro-7-nitrobenzofurazan, 5,5'-dithiobis-(2-nitrobenzoic acid) and iodoacetate) all behave as normal second-order processes. This indicates that the thiol groups on the two subunits of the enzyme react at the same rate as each other in all three cases. 3. The effects of various ligands (Mg2+, ADP and creatine, and combinations of these) on the kinetics of the reactions were studied. In all cases the reactions behave as normal second-order processes. 4. In the presence of the ligand combination Mg2+ plus ADP plus creatine plus nitrate, which has been postulated to form a "transition state analogue" complex with the enzyme, the reactions of the thiol group show considerable deviation from second-order kinetics. This indicates that the thiol groups on the two subunits react at different rates from each other. A similar effect is also noted in the presence of the combination ADP plus creatine plus nitrate. 5. The binding of ADP to the enzyme (studied by equilibrium dialysis) is hyperbolic in the absence of other ligands or in the presence of Mg2+ or Mg2+ plus creatine. The dissociation constant is similar in all three cases. 6. In the presence of creatine plus nitrate (with or without Mg2+) the bindings of ADP to the enzyme is tightened considerably and the binding plots indicate the presence of either negative interactions between the subunits or two distinct types of binding sites. 7. Possible causes for the observed non-identical behaviour of the two subunits of the enzyme are discussed.
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