The relations between the kinetic parameters for both sorbitol oxidation and fructose reduction by sheep liver sorbitol dehydrogenase show that a Theorell-Chance compulsory order mechanism operates from pH 7.4 to 9.9. This is supported by many parallels with the kinetics of horse liver alcohol dehydrogenase, which operates by this classical mechanism.An isotope-exchange study using U-('H8)sorbitol confirmed the existence of ternary complexes and that, under maximum velocity conditions, their interconversion is not rate-determining. Substrate inhibition at high concentrations of o-sorbitol or D-fructose confirmed rate-determining enzymecoenzyme product dissociation, slowed by the existence of more stable abortive ternary enzymccoenzyme product complexes with substrate. The effect of the inhibitor/activator 2,2,2-tribromoethanol showed the existence of enzyme-NAD-CBr3CH20H complexes inhibiting the first phase of reaction and enzyme-NADH-CBr3CH20H complexes dissociating more rapidly than the usual ratedetermining enzyme-NADH coenzyme product dissociation in the final phase. Inhibition studies with dithiothreitol also confirmed an ordered binding of coenzymes and second substrates to sorbitol dehydrogenase. Neither o-sorbitol nor D-fructose had any effect on enzyme inactivation by the affinity labelling reagent n~-2-bromo-3-(5-imidazolyl)propionic acid, thus giving no evidence for their existence as binary enzyme-substrate complexes.Several alternative polyol substrates for sorbitol dehydrogenase gave the same maximum velocity as sorbitol. This indicated a common rate-limiting binary enzyme-NADH product dissociation and a similarity of mechanism.An enzyme assay for pH 7.0 and 9.9 is given which enables the concentration of sorbitol dehydrogenase to be determined from initial rate measurements of enzyme activity.Sorbitol dehydrogenase (SDH) and aldose reductase constitute the sorbitol or polyol pathway, which functions as an important bypass to glycolysis and the pentose phosphate pathway in the metabolism of glucose, via u-sorbitol to Dfructose [l, 21. Operation of the sorbitol pathway has been implicated in the accumulation of sorbitol in the lens, leading to diabetic cataractogenesis [3], as well as other diabetic complications [4]. Because of the sorbitol pathway, the elucidation of the kinetic mechanism of action of sorbitol dehydrogenase is of particular interest.Sorbitol dehydrogenase catalyzes the reversible reaction

Analysis of amyloid fibril material associated with familial amyloidotic cardiomyopathy revealed that it contains a mixture of transthyretin-related polypeptides. The major protein band in SDS/polyacrylamide gel corresponding to a molecular mass of 14.5 kDa, consists of transthyretin fragments starting at positions 46, 49 and 59, the latter not previously identified, and one blocked fragment derived from the N-terminal part of transthyretin. In reverse-phase HPLC, the major fragment recovered was that starting at Thr49, indicating a trypsin-like cleavage (Lys at position 48). Two minor bands, corresponding to 17 kDa and 35 kDa, contained proteins with blocked N-termini, and migrated as monomeric and dimeric transthyretin, respectively. A 13-kDa protein band was found to contain transthyretin with a ragged N-terminus, mainly starting at positions 2 and 5. Three more bands, corresponding to 10, 25 and 29 kDa, consist of transthyretin molecules with blocked N-termini and most likely of aggregates of truncated molecules. A point mutation of amyloid transthyretin was identified at position 111 (Met instead of Leu in normal serum transthyretin) which confirms the mutation found for Danish siblings with familial amyloidotic cardiomyopathy. However, the presence of a non-variant amyloid transthyretin was also observed, indicating that the Danish kindred is heterozygous with respect to this point mutation. Isoelectric focusing of the amyloid fibril material resolved multiple protein bands ranging over pH 4.5-6.5, confirming heterogeneities. Methanol extraction of the cardiac amyloid fibril material prior to the purification steps reveals a methanol-soluble substance amounting to about 10% (by mass dry material) of the amyloid fibril material. A yellow substance in this fraction shows absorbance maxima (270, 280 and 430 nm) similar to those observed for transthyretin in normal serum. Gas chromatography/mass spectrometry of the methanol extract revealed the presence of saturated fatty acids (C14:0, C16:0 and C18:0 in the corresponding ratio 2:8:5) and polyunsaturated fatty acids (C16:1, C18:1, C18:2 and C20:4 in the corresponding ratio of 1:2:1:1) as further constituents of the amyloid fibril material.

Reversible inhibition and activation, as well as protection against affinity labelling with DL-2-bromo-3-(5-imidazolyl)propionic acid, of sheep liver sorbitol dehydrogenase have been studied. The results presented are discussed in terms of enzyme active-site properties and may have potential applications for drug design.Kinetics with mainly sorbitol competitive inhibitors reveals that aliphatic thiols are generally the most potent inhibitors of enzyme activity. Inhibition and inactivation by heterocyclics parallel that seen previously with sorbitol dehydrogenase from other sources as well as with alcohol dehydrogenase from yeast. However, there are significant differences in relation to the structurally similar horse liver alcohol dehydrogenase, as the catalytic zinc of sorbitol dehydrogenase is more easily removed by chelating molecules. Several aldose reductase inhibitors are shown to also inhibit sorbito1 dehydrogenase, but at concentrations unlikely to be reached clinically. Enzyme activation has been observed with various compounds, in particular halo-alcohols and detergents.Several inhibitors provide competitive protection against enzyme inactivation by ~~-2 -b r o m o -3-(5-imidazolyl)propionic acid. This enables the dissociation constants for binary enzyme-inhibitor complexes to be determined. NADH protects noncompetitively against inactivation.The presence of some binary and ternary enzyme-NADH complexes is indicated from fluorescence emission spectra, as a shift in the fluorescence maximum and intensity is observed due to their formation.Sorbitol dehydrogenase (SDH) is present in various tissues [ l ] and is an important diagnostic marker for pathogenesis in the liver and testis [2-41. SDH catalyzes the reversible oxidation of several polyols to their corresponding ketoses with the concomitant reduction of NAD [l, 51: Polyol + NAD' + Ketose + NADH + H' Aldose reductase and sorbitol dehydrogenase constitute the sorbitol or polyol pathway, which catalyzes the conversion of glucose to fructose via sorbitol in vivo [l, 6, 71. In diabetes and galactosemia, high intracellular concentrations of aldoses causes the accumulation of the polyols sorbitol and galactitol. Since these molecules penetrate cell membranes poorly [8, 91, this results in hypertonicity of the tissue and osmotic complications. Polyol accumulation has been implicated in pathological changes in various tissues, includCorrespondence to J. S.

There is a recognised need for methods that permit rapid estimation of the sanitary quality of water e.g. during raw water monitoring and emergencies involving water treatment failure or main breaks in a distribution network. In this study, two models for predicting the level of faecal contamination of water were studied. The first format, based on measurement of beta-galactosidase activity by the automated Colifast analyser, detected faecal contamination of high levels, corresponding to > 15 thermotolerant coliforms (FC)/5 mL, in 1-3 h, in a format that allowed for semi-quantification of results. By setting up a cut-off level, the system could be used as an operational tool identifying random increases in faecal contamination during routine raw water monitoring. A second Presence-Absence format was dependent upon the growth of low levels of FC with subsequent detection in the Colifast analyser. 95% of water samples containing 1-15 FC/sample volume showed positive detection after 11 h.

A protein transfer method which allows elution and immobilization of polypeptides onto a polyvinylidene difluoride (PVDF) membrane has been developed. The protein band in a gel is eluted by centrifugation. The centrifuge-blotting procedure involves the following steps: (i) visualization of the protein in a sodium dodecyl sulfate (SDS)-polyacrylamide gel with 1 M KCl, (ii) excision of the protein band and equilibration for 15 min in a solution of 0.05% SDS/5% methanol/0.02% dithiothreitol in distilled water, (iii) placing the gel piece in direct contact with the PVDF membrane in the receptacle, (iv) centrifugation at 3000 g for 1 h. A 10 kDa cut-off dialysis membrane is placed beneath the PVDF membrane to retain nonimmobilized protein. The N-terminal sequence of the immobilized protein on the PVDF membrane was determined. For proteins with a molecular mass less than 30 kDa, an overall yield between 10%-30% has been obtained.