Purification, Properties and Immunological Relationship of l(+)‐Lactate Dehydrogenase from Lactobacillus casei

Abstract

The fructose-1,6-bisphosphate-activated L-lactate dehydrogenase (EC 1.1 .I .27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue -Sephadex-G-200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an M , of 132000-135000 with a subunit M , of 34000. The pH optimum was found to be 5.4 in sodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 "C to 70 "C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6-bisphosphate. In the presence of 20 pM fructose 1,6-bisphosphate a K, of 1 .OmM for pyruvate was obtained, whereas fructose 1,6-bisphosphate had no effect on the K,,, of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6-phosphogluconate are strong inhibitors.Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. casei ATCC 393 L( +)-lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross-react :Fructose 1,6-bisphosphate activation of lactate dehydrogenase is found widely in several streptococcal groups [I -61. This phenomenon also exists among bifidobacteria [7], fermentative mycoplasmas [S, 91, certain coagulase-negative and mannitol-negative staphylococci [lo], and an actinomycete [l 11. However, Lactobacillus casei is the only member of its genus which possesses a fructose-l,6-bisphosphate-activated lactate dehydrogenase [12,32], with the enzyme being specific for L(+)-lactate [12]. This lactate isomer is also the major end-product of glucose fermentation [13]. Immunological and hence evolutionary links have been established between the isofunctional malic enzyme from Streptococcus fuecalis and L. cusei [14, 151. Further evidence of a close relationship between these two genera was obtained [16] using an antiserum prepared from purified S. faecalis fructose-l,6-bisphosphate aldolase. Difficulties in assaying and purifying L. casei lactate dehydrogenase [17] precluded this lactic acid bacterium from an immunological study of the relationships among lactate dehydrogenase from lactobacilli and leuconostocs [18].The presented paper reports the purification of L( +)-lactate dehydrogenase from L. casei to a homogenous preparation and establishes the kinetic characteristics of this enzyme. It also investigates the effects of various divalent metal ions, reaction products, metabolic intermediates and adenylates in an effort to elucidate the metabolic control of glucose utilization as well as the immunological relationship to the lactate dehydrogenases of other lactic acid bacteria.

Distinct Forms of Lactate Dehydrogenase Purified from Ethanol- and Lactate-producing Cells of Clostridum thermohydrosulfuricum

“…The strong activation of LDHE by phosphate is unusual (see Garvie, 1980). More commonly the FBP-dependent enzymes such as that from Lactobacillus cusei (Holland & Pritchard, 1975;Gordon & Doelle, 1976) are inhibited.…”

l‐Lactate dehydrogenase from Thermus caldophilus GK24, an extremely thermophilic bacterium

“…Recently, several studies on such subjects have been reported [lo-181. On Lactobacillus casei L-lactate dehydrogenase, which is activated by fructose 1,6-bisphosphate and Mn2 + [6,7], some important investigations including primary structure [ 171, lower-resolution crystallography [16] and affinity labelling analysis [18] have been achieved [I0 -181. It was reported that the structure of L. casei enzyme is very similar to that of the non-allosteric vertebrate enzymes [I61 and the allosteric site of L. casei enzyme corresponds to the anion-binding site of the vertebrate enzymes [ …”

Some Lactobacillus l -Lactate Dehydrogenases Exhibit Comparable Catalytic Activities for Pyruvate and Oxaloacetate

“…While the enzyme was relatively active toward 2-ketobutylate and phenylpyruvate, it was less active toward 2-ketoglutarate and 2-ketoacid substrates with long aliphatic chains, such as 2-ketovalerate, 2-ketocaproate, and 2-ketoisocaproate (Table 1), which are favorable substrates for the L-2-hydroxyisocaproate dehydrogenase from L. confusus (27). Unlike the L. pentosus enzyme, the L. casei enzyme is a representative allosteric L-LDH that requires Fru(1,6)P 2 to exhibit its activity (14,18,20). Under acidic conditions such as pH 5.0, the L. casei enzyme shows marked catalytic activity even in the absence of Fru(1,6)P 2 , giving a sigmoidal saturation curve for substrate pyruvate but a hyperbolic saturation curve in the presence of Fru(1,6)P 2 .…”

“…Under neutral conditions such as pH 7.0, on the other hand, it is known that the L. casei enzyme absolutely requires Fru (1,6)P 2 for activity, and the activation function of Fru(1,6)P 2 is markedly improved in the presence of certain divalent cations such as Mn 2ϩ (14,18,20 a Kinetic parameters were calculated using the data for Ͻ10 mM oxaloacetate, since oxaloacetate was insoluble above 20 mM under these conditions. exhibited no apparent catalytic activity toward oxaloacetate in the absence or in the presence of a nonsaturating concentration (5 mM) of Fru(1,6)P 2 , where pyruvate gives a sigmoidal saturation curve for the enzyme reaction (14,18).…”

“…exhibited no apparent catalytic activity toward oxaloacetate in the absence or in the presence of a nonsaturating concentration (5 mM) of Fru(1,6)P 2 , where pyruvate gives a sigmoidal saturation curve for the enzyme reaction (14,18). Like the activity toward pyruvate, however, the activity toward oxaloacetate was also greatly enhanced by addition of 10 mM MnSO 4 in the presence of 5 mM Fru(1,6)P 2 , giving a hyperbolic saturation curve with about 1.4-and 1.2-fold reduced K m and V max values, respectively, compared with those for pyruvate (Table 2).…”