Tissue distribution of mammalian lactate dehydrogenase isoenzymes.

system of Jervis [7] is used here.) Previous electrophoretic examination of the LDH of most other plants was carried out with extracts from leaves [1,2j. Such a study of potato leaves would not have revealed the existence of isoenzymic forms in this species. We have, therefore, examined extracts from various regions of a number of plants: roots, stem-base, upper stems, leaves and any special tissue, such as the tubers of the potato. The plants used were grown under normal horticultural conditions, harvested before flowering, and immediately extracted. They were first washed thoroughly, but quickly, with ice-cold water. and representative portions were finely chopped and ground in a pestle and mortar with acid-washed sand and 50 mwpotassium phosphate buffer, pH 7.4, containing an 8% (w/v) suspension of polyvinylpyrollidone ( 1-4 volumes of this buffer were used, depending on the consistency of the homogenate). The homogenates were then centrifuged at 8000 g for 30 min and the supernatants were subjected to ammonium sulphate fractionation (largely to concentrate the LDH activity, which is orders of magnitude lower in plants than in animals and bacteria). The fraction precipitating between 30 and 70% saturation of ammonium sulphate was redissolved in a minimal volume of 50 mMpotassium phosphate buffer, pH 7.4, and subjected to electrophoresis in Tris/glycine buffer, pH 8.6, in 7.5% (w/v) polyacrylamide gels either by the ‘disc-gel’ method of Davis [9] or using the ‘Mighty Small’ I1 SE 250 slab gel apparatus (Hoefer Scientific Instruments, San Francisco, CA, U.S.A.). The LDH activity was visualized using the zymogram-staining ‘cocktail’ of Epstein et ai. [lo]. We have experienced problems with many plants due, apparently, to interfering substances that may adsorb the LDH or otherwise interfere with its extraction or electrophoresis. With special extraction procedures we have been able to demonstrate LDH activity in extracts from all higher plants [ll], even those previously thought to lack this enzyme (see, e.g. [ 11). However, we have been less successful in overcoming interference with electrophoresis. The LDH activity in extracts from many plants did not penetrate properly into the polyacrylamide gels, remaining as a diffuse streak near the point of application and this limited the number of species we could usefully include in this study. Of these, the majority showed only a single electrophoretically identical form in all tissues. Such species include onion (Al l ium cepa), leek (Allium porrum) and turnip (Brassica rapa). But multiple forms were found in potato (Solanum tuberosum) and in the related tomato (Lycopersicum esculentum). Other members of the Solanaceae, Capsicum annuum and Solanum dulcamara, were also examined, but although extracts from the tissues of these species have LDH activity, we were unable to ‘find’ it on electropherograms, only diffuse activity near the origin being detectable, as described above. The same problem, probably attributable to interfering factor(s) in the extracts, was encountered with extracts from leaves of the two tomato cultivars studied, ‘Moneymaker’ and ‘Hildares’, but the other tissues gave clear electrophoretic patterns with up to four LDH bands, the number and intensity of the bands varying with tissue and with cultivar. In addition to the leaves and tubers mentioned above, other regions of potato plants showed variations of isoenzyme pattern. With the cultivar ‘Kerr’s Pink‘, the tubers showed all five isoenzymes, with isoenzymes 2 and 3 greatly predominating. In the stems and roots, there was a predominance of isoenzymes 1 and 2 , but the others were also detectable, while only isoenzyme-1 could be detected in extracts from the leaves. In another potato cultivar, ‘British Queen’, the leaf extracts again had predominantly isoenzyme1, but also a trace of isoenzyme-2, while isoenzyme-5 appeared missing from the tubers and other tissues whose LDH patterns were otherwise similar to those found in the ‘Kerr’s Pink’ cultivar. Only three isoenzymes could be detected in tubers of the cultivar ‘Homeguard’. Such variation among cultivars seems odd, if the isoenzymic forms fulfil any important differential function. A recent survey by Hoffman et ui. [ 121 of LDH isoenzymes in barley seedlings shows somewhat similar variation among cultivars. More significantly, these authors describe one strain that seemed to have only a single isoenzymic form of LDH without suffering any apparent disadvantage. This seems to cast doubt on any important physiological role for the isoenzymic multiplicity in barley. We have considered the possibility that the variation of isoenzyme pattern among potato tissues and cultivars might be an artifact produced by the interfering substances so evident in our experiments with other plants. However, we have been unable to find any evidence for this. Co-extraction of tissues having different isoenzyme patterns (e.g. a half-and-half mixture of leaf and tuber tissue) gave seemingly additive isoenzyme patterns.