Patricia Mulcahy

FURTHER STUDIES ON THE BIOAFFINITY CHROMATOGRAPHY OF NAD+ -DEPENDENT DEHYDROGENASES USING THE LOCKING-ON EFFECT

Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic.

Lactate dehydrogenase in plants: Distribution and function

Abstract Contrary to previous reports, lactate dehydrogenase (LDH; EC 1.1.1.27) occurs in all green land plants ranging from flowering plants to mosses. Conditions must be carefully designed and monitored, however, to achieve detection and extraction, and even then the specific activity is always orders of magnitude lower than that typically encountered in animal tissues. Green algae contain even lower levels of an NAD-dependent LDH activity not clearly identified as EC 1.1.1.27. Red and brown algae seem to lack LDH activity entirely. A striking feature of all the flowering plants investigated was a prominent peak of expression of LDH in stem tissue, at, or immediately above, soil level which was paralleled by a similar peak of alcohol dehydrogenase (ADH; EC 1.1.1.1) activity. The possible functional significance of this, and the function(s) of plant LDH generally, are discussed.

Purification and substrate kinetics of plant lactate dehydrogenase

Abstract Lactate dehydrogenase (LDH) from turnip ( Brassica rapa ; Cruciferae), purified to electrophoretic homogeneity using affinity chromatography, has a native M r , of 157 × 1O 3 and a subunit M r , of 38 × 10 3 . The LDH from turnip shows the same relative effectiveness (relative V max and K m values) as the mammalian H 4 and M 4 isoenzymes with pyruvate, lactate and glyoxylate (oxoacetate and dihydroxyacetate) as substrates. All three LDH types show no activity with glycolate (hydroxyacetate). The affinities for these and a range of competitive inhibitory analogues shows a consistent pattern of highest affinity for the H 4 mammalian isoenzyme, medium affinity for the M 4 form and lowest affinity for the plant enzyme, in a ratio of about 10:3:1, respectively. The catalytic mechanism of the plant enzyme is very similar to that of the mammalian forms. The major physiological activity of the plant LDH is considered to be pyruvate reduction, rather than the disproportionation of glyoxylate that has been proposed as a plant cell pH-stat.

Plant lactate dehydrogenase: NADH kinetics and inhibition by ATP

Abstract Lactate dehydrogenase (LDH), isolated from either turnip ( Brassica rapa , Cruciferae) or from leek ( Allium porrum , Liliaceae) shows normal Michaelis-Menten kinetics with NAD + in the lactate dehydrogenase direction, but non-hyperbolic kinetics with NADH in the pyruvate reductase direction. These kinetics are not ‘sigmoidal’ and appear consistently as two intersecting straight lines in reciprocal plots, representing a sharp decline in the effectiveness of NADH as a substrate in its lower concentration range. The overall apparent affinity for NADH decreases with decreasing pH. Competitive inhibition by ATP, which is much stronger than that characteristic of mammalian LDH, also displays kinetics that seem to be biphasic, but the K i value does not vary significantly with pH in the physiological range. The counter-inhibitory effect of Mg 2+ ions is shown to be due to the formation of non-inhibitory Mgue5f8ATP complexes. The dissociation of this is strongly pH dependent and this results in most of the ATP being complexed, and therefore non-inhibitory at pH 7.4 (the normal pH in plant cytosol) but largely uncomplexed and inhibitory at pH 6.4 (probably a lower cytosolic pH limit). These factors, and particularly the last, combine to inhibit the enzyme more strongly as the pH drops (as a result of lactate-terminating anaerobic glycolysis) and may act as an effective negative feed-back mechanism in plant cells, allowing a pH-activated switchover to ethanol-terminating glycolysis while preventing ‘overshoot’ into harmful acidosis.

BOTH D- AND L-SPECIFIC LACTATE DEHYDROGENASES CO-EXIST IN INDIVIDUAL CEPHALOPODS

Abstract Both d -lactate-specific and l -lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l -specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.

Application of kinetic-based biospecific affinity chromatographic systems to ATP-dependent enzymes: studies with yeast hexokinase

This study is concerned with the development of kinetic-based bioaffinity chromatographic systems for purification of ATP-dependent kinases, with a particular focus on the allosteric yeast hexokinase enzyme (EC 2.7.1.1). Synthesis and characterization of highly substituted N(6)-linked and S(6)-linked immobilized ATP derivatives are described using a rapid solid-phase modular approach. Evaluation of the new immobilized ATP derivatives has been carried out using model chromatographic studies with yeast hexokinase, employing specific substrate analogues (N-acetyl-D-glucosamine and suramin) to promote biospecific adsorption, in the presence and absence of citrate (a so-called allosteric activator of hexokinase activity). In this paper, successful bioaffinity chromatography systems were developed for yeast hexokinase and, as a result, interesting binding and catalytic properties of the enzyme were highlighted and explored. The overall results confirm the potential for extrapolation of the kinetic locking-on tactic, a general kinetic-based bioaffinity approach already developed for the NAD(P)(+)-dependent dehydrogenases, to ATP/ADP-dependent enzymes. However, in view of the enhancement of the intrinsic ATPase activity of hexokinase with glucosamine derivatives, and the coincidental hydrolysis of immobilized ATP to immobilized ADP, future developments necessary to support adaptation of the approach to ATP-dependent enzymes are discussed.