Genetic studies among Kanet and Koli of Kinnar district in Himachal Pradesh,India

Data are presented on serological and electrophoretic variants of 18 systems of red cells in 228 individuals belonging to a scheduled tribe (Kanet) and a scheduled caste (Koli) of Kinnar district in Himachal Pradesh, India. Differences in gene frequencies clearly indicate biological distinction in the local population. The possible cause of this genetic heterogeneity is discussed.     

L-lactate dehydrogenase from leaves of Capsella bursa-pastoris (L.) Med.

By ammonium sulfate fractionation and gel filtration an enzyme preparation which catalyzed NAD⁺-dependent L-lactate oxidation (10^-4 kat kg^-1 protein), as well as NADH-dependent pyruvate reduction (10^-3 kat kg^-1 protein), was obtained from leaves of Capsella bursa-pastoris. This lactate dehydrogenase activity was not due to an unspecific activity of either glycolate oxidase, glycolate dehydrogenase, hydroxypyruvate reductase, alcohol dehydrogenase, or a malate oxidizing enzyme. These enzymes could be separated from the protein displaying lactate dehydrogenase activity by gel filtration and electrophoresis and distinguished from it by their known properties. The enzyme under consideration does not oxidize D-lactate, and reduces pyruvate to L-lactate (the configuration of which was determined using highly specific animal L-lactate dehydrogenase). Based on these results the studied Capsella leaf enzyme is classified as L-lactate dehydrogenase (EC 1.1.1.27). It has a K_m value of 0.25 mmol l^-1 (pH 7.0, 0.3 mmol l^-1 NADH) for pyruvate and of 13 mmol l^-1 (pH 7.8, 3 mmol l^-1 NAD⁺) for L-lactate. Lactate dehydrogenase activity was also detected in the leaves of several other plants.Abbreviation FMN flavin adenine mononucleotide     

Photosynthetic induction in wheat protoplasts and chloroplasts. Autocatalysis and light activation of enzymes

Abstract Unlike wheat chloroplasts, wheat protoplasts showed a pronounced restoration of the induction phase after a short period of darkness. This difference was used to investigate the relative roles of light-induced reductive activation of enzymes and the auto-catalytic increase in the level of substrates in the control of the rate of photosynthesis during induction. Light activation and dark inactivation of ribulose 5-phosphate kinase, fructose 1,6-biphosphatase and NADP⁺-specific glyceraldehydephosphate dehydrogenase were measured. In this respect there was no appreciable difference between protoplasts and chloroplasts. In contrast, the level of photosynthetic intermediates remained constant in darkened isolated chloroplasts, but declined rapidly in chloroplasts isolated from darkened protoplasts. When fructose 1,6-bisphosphatase was pre-activated by treating protoplasts with dithiothreitol the lag was only slightly shortened. These results are discussed in terms of control of the rate of the photosynthesis during the lag by substrates rather than limitation imposed by activity of any of the enzymes measured.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Properties of enzymes from bacteria grown in the 70–100°C range

In chemostat cultures of Bacillus caldolyticus, adaptation in a single step from 70–100°C was followed under aerobic and oxygen-limited conditions and was found to proceed more smoothly under the latter circumstances. Variations of the medium (e.g. yeast extract or silicate concentrations) showed that growth at 100°C is in all respects similar to that of cultures at moderate temperatures.Enzyme preparations derived from cultures at 5°C intervals between 70 and 100°C were used to determine the temperature range. For all nine enzymes tested, the optimum temperature was found to be 67°C; the latter was independent of the growth temperature. Differences were found, however, with respect to the maximum temperature of individual enzymes, and three groups, with maxima between 70 and 80°C, 80 and 90°C and 90 and 100°C can be distinguished. Again, there was no correlation with the growth temperature.Stability experiments also revealed that enzymes from the same organism can have different thermal properties: Some were found to be quite thermolabile (e.g. the pyruvate kinase), while others (e.g. hexokinase and glutamate-pyruvate transaminase) exhibited a high thermostability. These properties were not related to the growth temperature within the 70–100°C range, too.Six of the enzymes tested could be stabilized by their respective substrates, but the degree of protection varied for individual enzymes. Three enzymes (acetate kinase, glutamate dehydrogenase and myokinase) could not be stabilized by their substrates.Comparative experiments with the hexokinase suggested, that the thermal integrity of the enzymes is better protected within the cell as compared to the stability of the enzyme preparations.Abbreviations used AK acetate kinase - Ala-DH alanine dehydrogenase - Ald aldolase - GIDH glutamate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GTP glutamate-pyruvate transaminase - HK hexokinase - ICDH isocitrate dehydrogenase - MK myokinase - PK pyruvate kinase     

Specificity of two isolated wheat carboxypeptidases

The substrate specificity of two purified carboxypeptidases from germinated wheat has been examined. Both enzymes were active on a wide variety of carbobenzoxy substituted peptides but inactive with unsubstituted dipeptides. Neither enzyme was active upon endoprotease or amidase substrates and only low levels of esterase activity were evident. In time course studies, both enzymes gave rapid non-specific sequential release of amino acids, including proline, from the carboxyterminal of proteins and polypeptides of known amino acid sequence.     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Starch breakdown in the spadix of Arum maculatum

The aim of this work was to discover the pathway of starch breakdown during thermogenesis in the club of the spadix of Arum maculatum. The conventional α-amylase of higher plants could not be demonstrated in extracts of clubs although such extracts did exhibit considerable hydrolytic activity towards starch. This activity had an action pattern characteristic of an endo-amylase, was destroyed by heating to 70°, and was not inhibited by either 7 mM ethylenediaminetetra-acetic acid or 100 mM N-ethyl maleimide. Measurements of this hydrolytic activity, and of the maximum catalytic activities of starch phosphorylase, phosphoglucomutase and hexokinase, were made at different stages of club development. These measurements were compared with estimates of the rate of starch breakdown at thermogenesis. This comparison indicates that phosphorolytic cleavage does not play a large role in such starch breakdown, and that this process is mediated, mainly, by the hydrolytic activity, described above, and by hexokinase.