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The emulsifying properties of three genetic variants of β -lactoglobulin ( β -lac) (the A, B and C variants) are investigated as a function of protein concentration. Differences in the emulsifying properties and emulsion stability are explained in view of the known differences in physico-chemical and structural/conformational properties of the β -lac variants. β -lac A forms the finest emulsion droplets, and β -lac C the largest droplets. The order of decreasing emulsifying ability (A>B>C) can be explained in terms of differences in the molecular structure, and conformational stability of the variant proteins. The creaming stability, when compared at the same particle size, is greatest for β -lac C, with β -lac A and B having a similar and lower stability. The differences in creaming stability may arise from a higher surface coverage for the β -lac C droplets at an equivalent particle size. The storage stability is lower for β -lac A than for β -lac B and C, which both show a similar behaviour. Storage stability differences are discussed in terms of differences in molecular structure, conformational stability, interfacial viscosity and surface coverage for the three variants.

beta-Lactoglobulin is a whey protein that affects milk composition and product functionality and which can be present in up to eight genetic variant forms. A free zone capillary electrophoresis method has been developed to separate and identify the beta-lactoglobulin A, B and C variants. Three buffer systems [borate, 2-(N-morpholino)-ethanesulphonic acid (MES) and bis(2-hydroxyethyl)imino-tris(hydroxymethyl)methane (Bistris)] were examined over a range of pH values and with the addition of the separation buffer modifiers Tween 20 and/or ethanolamine. The most successful combination of these was 50 mM MES at pH 8.0 with the addition of 0.1% Tween 20 which clearly resolved the three variants from both each other and from the other whey proteins even though the MES buffer was acting well outside its pKa range (pH 5.3-7.3). The retention times and identification of the individual variants were verified by spiking with commercially purified beta-lactoglobulin A and B proteins and a beta-lactoglobulin AC whey. The method was then used to phenotype beta-lactoglobulin in a sample population of New Zealand Jersey cows.

Sheep liver cytoplasmic aldehyde dehydrogenase was labelled by reaction with the substrate p-nitrophenyl di[14C]methylcarbamate. After tryptic digestion and peptide fractionation the labelled residue was identified as Cys-302. This is the first unequivocal identification of the essential enzymic nucleophile in the esterase activity of aldehyde dehydrogenase. By implication, Cys-302 is probably also the residue that is acylated by aldehyde substrates and the first residue that is modified by disulfiram.

The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.

Compliance with prescribed treatment remains a major problem in the control of tuberculosis worldwide. We describe a simple method of improving patient compliance with hospital-based treatment. Eighty-two patients paid a deposit at the start of their treatment which entitled them to cheaper drugs and was refundable on completion of the prescribed course. Sixty-two per cent of patients completed the course compared with 23% of retrospective controls. A direct relationship was found between the amount of deposit paid and the rate of completion. Reasons why poor patients (who paid a lower deposit) may default are discussed, as are the merits of short-course drug regimens. It is recommended that similar schemes be assessed elsewhere.

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

Although the oxidation of aldehydes by aldehyde dehydrogenase is usually considered to be irreversible, Hart & Dickinson (1978) have shown that anhydrides of carboxylic acids could be reduced to aldehydes by enzyme.NADH mixtures. In a later study Motion et al (1988) demonstrated that acylation of aldehyde dehydrogenase.NADH complexes by acetic anhydride led to the production of acetaldehyde and NAD+ in stoichiometric amounts. It is clear from these results, and the fact that 80% of the initial NADH concentration can be oxidised by anhydrides at an almost constant rate, that there is no intrinsic thermodynamic barrier to the reverse reaction once the appropriate acyl enzyme species is formed. The mechanism for reduction of aldehydes is presumably the reverse of that for aldehyde oxidation (Scheme I).

p-Chloromercuribenzoate (PCMB) at stoichiometric levels reacts with a thiol group of the binary NAD+ complex of sheep liver cytoplasmic aldehyde dehydrogenase (E.NAD+) faster than with the corresponding thiol group of either the free enzyme or the binary enzyme. NADH complexes. High concentrations of propionaldehyde have a protective effect against modification of the enzyme with PCMB in steady-state assays. This protection arises from a reduction in the concentration of the E.NAD+ binary complex rather than competition for a common binding site. PCMB has three major effects on aldehyde dehydrogenase. First, rapid reaction with a high-affinity thiol group in the E.NAD+ binary complex causes activation of the steady-state rate. The activation results from an increase in the rate of NADH release from the enzyme. This modification simultaneously protects against dilution-induced dissociation of enzyme tetramers. Second, premodification of the high-affinity thiol group leads to inhibition of the steady-state rate at high propionaldehyde concentrations, because of the increased affinity of the free enzyme for propionaldehyde with the resultant formation of an enzyme-aldehyde dead-end complex. Third, when higher ratios of PCMB to enzyme (> 3:1) are used, one or more other thiol groups are also modified, causing enzyme dissociation and subsequent inactivation. Since modification of the high-affinity thiol by PCMB causes activation, clearly it cannot be the active site acylation center involved in propionaldehyde oxidation. The different amplitudes of the proton burst at high and low propionaldehyde concentrations for the PCMB modified enzyme provide support for a second binding site for propionaldehyde on the enzyme.

Although studies on the effect of pH on aldehyde dehydrogenase have been carried out in a number of laboratories, the results of these studies do not give a complete picture of the mechanism of the aldehyde dehydrogenase catalyzed oxidation of propionaldehyde over an extended pH range. The enzyme catalyzed oxidation reaction at pH 7.0 and 7.6 is generally agreed to occur by the following ordered mechanism: $$E \rightleftharpoons E.NA{D^ + } \rightleftharpoons E.NA{D^ + }.ALD \rightleftharpoons E.NADH.acyl \to *E.NADH \rightleftharpoons E.NADH \rightleftharpoons E$$ Scheme I At least at low propionaldehyde concentrations, the rate of the reaction is controlled by the slow conformational change which occurs during the release of NADH from the enzyme (Blackwell et al., 1987; Dickinson, 1985).

The cytosolic (Class 1) aldehyde dehydrogenase (AIDH) from sheep liver has been crystallized in a form suitable for X-ray diffraction studies. The crystals, grown by vapour diffusion using 6·5 to 7·5% methoxypolyethylene glycol 5000 as precipitant, at pH 6·5, are orthorhombic with cell dimensions a = 80·7, b = 92·5, c = 151·6 A, space-group P 2 1 2 1 2 1 , and one dimer in the asymmetric unit. The crystals diffract to at least 2·8 A resolution. Although unmodified AIDH crystallized readily, a key factor in obtaining diffraction-quality crystals was the covalent attachment of an active site reporter group, provided by 3,4-dihydro-3-methyl-6-nitro-2 H -1,3-benzoxazin-2-one.

The release of NADH from the enzyme·NADH complexes was rate limiting at 37°, for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase. Marked substrate activation was observed at this temperature as was activation by p -(chloromercuri)benzoate. Activation of enzymic activity may be of importance in vivo .

EDITOR,—The consensus statement for good practice and audit measures in the management of hypothyroidism and hyperthyroidism recommends surveillance of patients at high risk of iatrogenic hypothyroidism and suggests this as a point for future audit.1 We have recently audited the follow up of thyrotoxicosis and thyroidectomy in a general practice population of 10 761 patients. Previously we did not have a systematic method of surveillance for such patients but checked their thyroid function opportunistically. Our audit …

1. The pig enzyme was purified to homogeneity and was found to be a tetramer of apparently identical subunits. 2. The pig enzyme was found to contain 1 mol NADH/mol enzyme which is tightly bound, which is not directly involved in catalysis and which so far has not been removed from the enzyme so as to produce an active apoenzyme. 3. The pig enzyme seems to contain only one functioning active site/tetramer. 4. The pig and sheep enzymes are compared in respect of NADH binding, substrate specificity, immunological response and surface charge.