Secretion of Hexosaminidase Isozymes by Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A₁ and B₁, and 2 minor forms, A₂ and B₂, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B₁ has a molecular weight of ?93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A₁ has a molecular weight of ?170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-β-D-galactosaminide than the B forms. Neither hexosaminidases A₁ or B₁ has any endo-β-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete ?20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxy-benzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

Retina-specific LDH isozyme in blueback herring, Alosa aestivalis (Mitchell), and alewife, Aiosa pseudoharengus (Wilson)

The retina of 390 Alosa aestivalis and 410 Alosa pseudoharengus have been examined by means of starch-gel electrophoresis. The retina-specific E₄ isozyme has been found to occur in all the fish examined. This study demonstrates for the first time that the E₄ isozyme occurs in A. aestivalis. Because the E₄ isozyme is not polymorphic and has an identical mobility in A. pseudoharengus and in A. aestivalis it is neither suitable for use as a species identification characteristic nor a population marker. Alosa aestivalis and Alosa pseudoharengus are two commercially important ana-dromous species of fish in New Brunswick, Canada. These two species may occur together in the same spawning runs but w^rhile A. pseudoharengus has a wide distribution along the East coast of North America (Leim & Scott, 1966) A. aestivalis occurs in a very limited area in Canada where it is at the northern limit of its range and because of increasing threat of pollution has been listed by McAllister (1970) as one of 17 endangered species. These two species of fish are morphologically very similar and can only be separated by the colour of the abdominal peritoneum (Leim & Scott, 1966). McKenzie (1973) has compared these two species by means of protein electrophoresis and found that while the muscle myogen patterns were species specific, the LDH patterns were the same in both species. He described these two species as five isozyme fish showing the LDH isozymes A₄, A₃B, A₂B₂, AB₃ and B₄. Since Horowitz & Whitt (1972) have reported the presence of the E₄ isozyme in the retina of some teleosts including. A. pseudoharengus but not including A. aestivalis, I considered it worth-while to re-examine A. aestivalis and A. pseudoharengus to find out whether A. aestivalis possessed this isozyme and, if so, whether the mobility of the isozyme could be used as a species identification characteristic and as a population marker. The fish used in this study were collected from five different locations during the 1971 spring migration period and held deep frozen for eight months before they were examined. They were identical to the specimens used for the study reported by McKenzie (1973) where the collection dates, numbers of fish and geographic locations are given. One eyeball from each of 390 A. aestivalis and 410 A. pseudoharengus was removed. Each eyeball was homogenized in 10 drops of distilled water and allowed to stand for one hour at 4^oC. The samples were then centrifuged for 10 min at 12 000 g. The supernatants were used immediately for vertical starch-gel electrophoresis. The apparatus used was that described by Boyer & Hiner (1963). The conditions of electrophoresis were the same as used by Saunders & McKenzie (1971). The LDH bands were stained by the deposition of blue formazan dyes in the regions of LDH activity. The stain formula and details of methods are given in Whitt (1970). The LDH isozyme patterns of all the fish examined were identical. The location of the isozymes A₄, A₃B, A₂B₂, AB₃ and B₄ are indicated in Fig. 1. A₄ is abundant in muscle while B₄ is abundant in heart. Because the LDH subunits assemble preferentially into homodimeric pairs before forming tetramers (Markert & Ursprung, 1971). A₄. A₂B₂ and B₄ show up in electropherograms as strong bands while A₃B and AB₃ show up as weak bands. The retina-specific isozyme E₄ is shown between B₄ and AB₃. Whitt (1970) has already demonstrated that A. pseudoharengus is a five isozyme fish. This has been confirmed by McKenzie (1973) who also compared both A. pseudoharengus and A. aestivalis and found these five isozymes had identical mobilities in both species of fish. The present study demonstrates for the first time that the retina-specific E₄ occurs in A. aestivalis where it has the same electro-phoretic mobility as that of A. pseudoharengus. The patterns are the same and do not appear to vary with geographic origin of the fish. The reason why the presence of the E₄ isozyme was demonstrated in the present study and not in the previous one (McKenzie, 1973) is because of the method used. In that study the migration length was not long enough to sufficiently separate the enzymes. In the present study vertical starch-gel electrophoresis which allows for long migration distances was used. As has already been shown for the A-B isozymes (McKenzie, 1973), the E₄ isozyme is not polymorphic in these two species of fish. It therefore has no use as apopulation marker. Because the E₄ isozyme has an identical mobility in both species of fish, it cannot be used as a species identification characteristic.     

Studies on Vitamin B6 Metabolism in Microorganisms

Escherichia freundii alkaline phosphatase was found in a membrane fraction and was purified by procedures involving spheroplast formation with lysozyme and EDTA, and DEAE-cellulose and Sephadex G-150 column chromatographies. Then this enzyme along with other phosphatases was investigated on the ability to transfer the phosphoryl group from p-nitrophenyl phosphate to pyridoxine. It was found that the ability of the transphosphorylation varied with these phosphatases. The transphosphorylation to hydroxy compounds such as alcohols, sugars and nucleosides was also compared. Escherichia freundii acid phosphatase showed the highest activity of transphosphorylation among phosphatases tested. The mechanism of transphosphorylation was discussed.

An enzyme, pyridoxamine 5′-phosphate transaminase, was purified from the cell-free extract of Clostridium kainantoi. The purification procedures involved ammonium sulfate fractionation, protamine sulfate treatment and, DEAE-cellulose, hydroxylapatite, DEAE-Sephadex and Sephadex G-200 column chromatographies. The purified enzyme, which had approximately 2700-fold higher specific activity over the original extract, showed a single schlieren pattern in the ultracentrifuge. From the spectral analysis, it seemed that pyridoxamine 5′-phosphate transaminase did not contain pyridoxal 5′-phosphate as a prosthetic group. It was recognized that the transamination was accelerated by the addition of amino acid and was inhibited by diisopropyl phosphofluoride. Glutamic acid formed in the reaction was identified to be a D-isomer. A study on the substrate specificity showed that the enzyme might be possible to be specific for pyridoxamine 5′-phosphate.

The extracellular formation of vitamin B₆ was searched in marine and terrestrial microorganisms. Two bacterial strains were selected and were identified as Vibrio and Flavobacterium, respectively. Marine microorganisms showed the considerable formation of vitamin B₆ and the presence of vitamin B₆ in sea water was also recognized. The cultural and reaction conditions for vitamin B₆ formation by these strains were investigated. Glycerol was commonly the most effective compound on vitamin B₆ formation among the compounds tested. It was suggested that both bacteria did not have the control system on vitamin B₆ biosynthesis by the amount of possible end products.     

Life's utilization of B vitamins on early Earth

Coenzymes are essential across all domains of life. B vitamins (B₁‐thiamin, B₂‐riboflavin, B₃‐niacin, B₅‐pantothenate, B₆‐pyridoxine, B₇‐biotin, and B₁₂‐cobalamin) represent the largest class of coenzymes, which participate in a diverse set of reactions including C₁‐rearrangements, DNA repair, electron transfer, and fatty acid synthesis. B vitamin structures range from simple to complex heterocycles, yet, despite this complexity, multiple lines of evidence exist for their ancient origins including abiotic synthesis under putative early Earth conditions and/or meteorite transport. Thus, some of these critical coenzymes likely preceded life on Earth. Some modern organisms can synthesize their own B vitamins de novo while others must either scavenge them from the environment or establish a symbiotic relationship with a B vitamin producer. B vitamin requirements are widespread in some of the most ancient metabolisms including all six carbon fixation pathways, sulfate reduction, sulfur disproportionation, methanogenesis, acetogenesis, and photosynthesis. Understanding modern metabolic B vitamin requirements is critical for understanding the evolutionary conditions of ancient metabolisms as well as the biogeochemical cycling of critical elements such as S, C, and O.     

Distribution Patterns of 14C in the Labelled Vitamin B6 Prepared with the Cell-suspension of Achromobacter cycloclastes

The biosynthetic pathway of vitamin B₆ (abbreviated as Be) has been studied with the cell-suspension of B₆-producing bacteria, Achromobacter cycloclastes A.M.S. 6201. The distribution of ¹⁴C in the Be molecule prepared with the cell-suspensions containing glycerol-1,3-¹⁴C, glycerol-2-¹⁴C or γ-aminobutyric acid-U-¹⁴C was investigated by using three novel degradation methods. The results showed that carbon skeletons of glycerol and γ-aminobutyric acid were used for the formation of the major part of B₆ carbon skeleton respectively. The implication of these compounds as precursors of B₆ was discussed.     

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: Comparison of kinetic properties among the three isozymes

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25^oC, the major and minor components of the E3 isozyme catalyzed the reaction with V_max of 1.1 and 0.8 μmol NADH min⁻¹ mg⁻¹ protein, respectively, compared to 0.067 and 0.060 μmol NADH min⁻¹ mg⁻¹ protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K_m for methylglyoxal of 8.6 μM, the lowest compared to 46 μM for E1 and 586 and 552 μM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with K_i values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 μM for E1 and E2 isozymes, respectively.     

Enhancement of vitamin B6 levels in rice expressing Arabidopsis vitamin B6 biosynthesis de novo genes

Vitamin B₆ (pyridoxine) is vital for key metabolic reactions and reported to have antioxidant properties in planta. Therefore, enhancement of vitamin B₆ content has been hypothesized to be a route to improve resistance to biotic and abiotic stresses. Most of the current studies on vitamin B₆ in plants are on eudicot species, with monocots remaining largely unexplored. In this study, we investigated vitamin B₆ biosynthesis in rice, with a view to examining the feasibility and impact of enhancing vitamin B₆ levels. Constitutive expression in rice of two Arabidopsis thaliana genes from the vitamin B₆ biosynthesis de novo pathway, AtPDX1.1 and AtPDX2, resulted in a considerable increase in vitamin B₆ in leaves (up to 28.3‐fold) and roots (up to 12‐fold), with minimal impact on general growth. Rice lines accumulating high levels of vitamin B₆ did not display enhanced tolerance to abiotic stress (salt) or biotic stress (resistance to Xanthomonas oryzae infection). While a significant increase in vitamin B₆ content could also be achieved in rice seeds (up to 3.1‐fold), the increase was largely due to its accumulation in seed coat and embryo tissues, with little enhancement observed in the endosperm. However, seed yield was affected in some vitamin B₆‐enhanced lines. Notably, expression of the transgenes did not affect the expression of the endogenous rice PDX genes. Intriguingly, despite transgene expression in leaves and seeds, the corresponding proteins were only detectable in leaves and could not be observed in seeds, possibly pointing to a mode of regulation in this organ.     

Studies on the Biosynthesis of Vitamin B6

The production of vitamin B₆ (B₆) compounds with a cell-suspension of Achromobacter cycloclastes A.M.S. 6201 under various conditions were examined. An obvious accumulation of B₆ compounds in the incubation medium with a cell-suspension of the organism harvested at the middle to later part of exponential phase of growth was observed. γ-Aminobutyric acid or β-alanine was found to stimulate the B₆ production markedly, when they were added to the incubation mixture together with glycerol. Some discussion on the implication of these compounds as precursors of B₆ was presented.     

Effects of vitamin B6 supplementation in rats during lactation on vitamin B6 concentration and transaminase activities in the offspring

The aim of the present investigation was to study the effect of a varying maternal vitamin B₆ supplementation during lactation period on vitamin B₆ levels in blood, liver and total body, and on the activity of two transaminase enzymes in the offspring. Therefore, eighty female Sprague‐Dawley rats were fed a semi‐synthetic diet (0.2 mg vitamin B₆ per kg) which was supplemented during gravidity with 5 mg vitamin B₆ per kg diet. During the following lactation period the rats were assigned to one of 10 vitamin B₆ treatment groups (supplementation of 0, 3, 6, 9, 12, 15, 18, 36, 360, 3600 mg vitamin B₆ per kg diet). At day 14 of lactation the pubs of all dams were decapitated and blood, liver, and carcass were used for analysis of vitamin B₆ concentration, activities of two transaminases, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in plasma, erythrocytes, and liver, and of haematological parameters.

While the liver and total body wet weights as well as the haematological parameters (red blood cells, haemoglobin concentration, hematocrit, middle corpuscular cell volume, middle corpuscular haemoglobin, middle corpuscular haemoglobin concentration) did not differ within the experimental groups, the present data clearly show that in blood, liver and total body of the offspring exists a slight dose‐response relationship between the maternal dietary vitamin B₆ supplementation and the vitamin B₆ concentration. Concerning the activities of the transaminases a dietary supplementation above 3mg vitamin B₆ per kg diet had no influence on the AST and ALT activities in offspring plasma. In the erythrocytes no statistical significant influence of the vitamin B₆ supplementation during lactation on the activities of AST and ALT was found. The activities of ALT and AST in liver were not consistently altered by the vitamin B₆ supplementation of the dams during lactation. In conclusion these results indicate that a minimal maternal dietary vitamin B₆ supply of 3.1 mg per kg diet is necessary with regard to health and development of their offspring. But not all of the analysed parameters as the liver and total body weights, the activities of AST and ALT in the erythrocytes, and the haematological parameters were influenced by a deficient maternal dietary vitamin B₆ supply.     

Murine liver arylsulfatase B processing influenced by region on chromosome 17

SM/J liver arylsulfatase B has a more rapid electrophoretic mobility and occurs as a series of more acidic isozymes following electrofocusing in narrow pH gradients than the liver enzyme from C57BL/6J mice. The SM/J and C57BL/6J electrofocusing patterns were both converted to a single isozyme with similar isoelectric points by pretreatment with neuraminidase, suggesting that the SM/J and C57BL/6J isozymes differed with respect to their sialic acid content. Arylsulfatase B electrofocusing and thermostability phenotypes segregated independently among progeny of SM/J×C57BL/6J crosses, suggesting that the electrofocusing phenotypes were not determined by different alleles at As-1, the putative structural locus for arylsulfatase B. Comparison of the joint segregation of hepatic acid phosphatase electrophoretic patterns and liver arylsulfatase B electrofocusing profiles revealed that the electrofocusing profiles may be determined by a region on chromosome 17 near or identical to Apl. Kidney, brain, and spleen arylsulfatase B electrofocusing patterns did not appear to differ between SM/J and C57BL/6J mice.This research was supported in part by Biomedical Sciences Research Support Grant RR-07030, by NIGMS Grant 1-RO1GM27707-01, and by Grant 1–570 from the National Foundation/March of Dimes.     

Incorporation of 14C-Labelled Glycerol and γ-Aminobutyric Acid into Vitamin B6 in a Cell-suspension of Achromobacter cycloclastes

Incorporation of ¹⁴C from various ¹⁴C-labelled compounds into vitamin B₆ (abbreviate as B₆) in a cell-suspension of B₆-producing bacteria, Achromobacter cycloclastes A.M.S. 6021, was studied by using a newly designed purification procedure of the radioactive B₆. The procedure consisted of Sephadex G–25 gel filtration, Dowex 50W–X8 column chromatography, Amberlite CG–50 column adsorption, powdered cellulose partition column chromatography, crystallization and sublimatography. It was observed that the labels both from 1,3- and from 2-labelled glycerol were clearly incorporated into B₆ and the label of ¹⁴C-labelled γ-aminobutyric acid was also incorporated. The incorporation of ¹⁴C from ¹⁴C-labelled glycerol or γ-aminobutyric acid was affected by the addition of cold γ-aminobutyric acid or glycerol. The implication of these compounds as precursors of B₆ was discussed.     

Concanavalin A-Sepharose Chromatography of Hexosaminidase Isozymes Secreted By Tetrahymena*

Tetrahymena pyriformis strain HSM secretes 4 isozymes of hexosaminidase. Purified isozymes B₁ and B₂ are eluted from the void volume of a concanavalin A-Sepharose column, suggesting that they are not glycosylated. Purified isozymes A₁ and A₂ bind to the column and are eluted at ～0.1 M α-methylmannoside, suggesting that these isozymes are glycoproteins. In agreement with earlier deductions based on a differential kinetic assay for the A and B isozymes, the elution pattern of hexosaminidase activity from material secreted by cells grown to early and late stationary phase was consistent with these secretions containing primarily the B and the A isozymes, respectively.     

Studies on the vitamin B12 auxotrophy of Rhodocyclus purpureus and two other vitamin B12-requiring purple nonsulfur bacteria

The vitamin B₁₂ requirement of Rhodocyclus purpureus 6770, Rhodospirillum tenue 1/67, and Rhodopseudomonas palustris G 53/2 was determined. A wide variety of biogenetic precursors of the vitamin including cobinamide, cobyric acid, cobinic acid and several partially amidated cobyrinic acids showed growth-promoting activity in all three strains. In R. purpureus vitamin B₁₂ could even be substituted by cobyrinic acid which is the first cobalt-containing precursor of vitamin B₁₂ so far established. Neither methionine, deoxynucleosides, dimethylbenzimidazole nor increased amounts of cobalt could replace vitamin B₁₂ as growth factor.Cupribalamin, which is a strong antimetabolite of vitamin B₁₂ in Escherichia coli 113-3 and Lactobacillus leichmannii ATCC 7830, exhibited only a weak antagonistic effect on growth of R. purpureus and R. tenue. Growth of R. palustris was not inhibited by cupribalamin. The cells of all three strains were shown to contain metal-free corrinoids in addition to cobalt-containing corrinoids. The principal products were identified as 5-deoxyadenosylcobalamin and hydrogenobalamin, the metal free analogue of vitamin B₁₂. The latter does not originate from the vitamin by removal of cobalt but is de novo biosynthesized as could be demonstrated in the case of R. purpureus by a labelling experiment with [¹³C] methyl-l-methionine.     

Effect of vitamin B6 availability on serine hydroxymethyltransferase in MCF-7 cells

Folate-activated one-carbon units are derived from serine through the activity of the pyridoxal-phosphate (PLP)-dependent isozymes of serine hydroxymethyltransferase (SHMT). The effect of vitamin B(6) availability on the activity and expression of the human mitochondrial and cytoplasmic SHMT isozymes was investigated in human MCF-7 cells. Cells were cultured for 6 months in vitamin B(6) replete (4.9 microM pyridoxine) minimal essential medium (alphaMEM) or vitamin B(6)-deficient medium containing 49, 4.9 or 0.49 nM pyridoxine. Total cellular PLP levels and SHMT activity were reduced 72% and 7%, respectively, when medium pyridoxine was decreased from 4.9 microM to 49 nM. Cells cultured in medium containing 4.9 nM pyridoxine exhibited 75%, 27% and 60% reduced levels of PLP, SHMT activity and S-adenosylmethionine, respectively, compared to cells cultured in alphaMEM. Cytoplasmic SHMT activity and protein levels, but not mRNA levels, were decreased in cells cultured in vitamin B(6) deficient medium, whereas mitochondrial SHMT activity and protein levels were less sensitive to vitamin B(6) availability. PLP bound to cytoplasmic SHMT with a K(d)=850 nM, a value two orders of magnitude lower than previously reported for the bovine cytoplasmic SHMT isozyme. Collectively, these data indicate that vitamin B(6) restriction decreases the activity and stability of SHMT, and that the cytoplasmic isozyme is more sensitive to vitamin B(6) deficiency than the mitochondrial isozyme in MCF-7 cells.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Experimental study on protection of vitamin B6 on TCDD-induced palatal cleft formation in the mice

BACKGROUND: 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) can cause a high percentage of cleft palate in fetuses when administered during organogenesis in certain strains of mice including the C57BL/6J. In this study, vitamin B₆ (B₆) was tested for antiteratogenic effects on TCDD-induced cleft palate in fetal mice. METHODS: The pregnant C57BL/6J mice were dosed with 24 µg TCDD/kg and/or 5, 10, 20, and 40 mg B₆/kg body weight on gestation day (GD) 10. The control group mice were dosed with 50 ml sesame oil/kg body weight on GD10. The mice were sacrificed on GD12.5, GD13.5, GD14.5, GD15.5, and GD17.5, respectively. The harvested embryos were examined to detect the incidence of cleft palate and the developing palatal shelves in a different phase were investigated morphologically and histologically among different groups. RESULTS: Total frequency of clefts is 55.56% in the TCDD group and 31.81% (5 mg), 44.44% (10 mg), 40.90% (20 mg), and 32.00% (40 mg) in the TCDD+ B₆ groups. There were no statistically significant differences among the TCDD and TCDD+ B₆ groups (p=0.743>0.05). CONCLUSIONS: It was demonstrated in this study that B6 could not antagonize 2, 3, 7, 8-TCDD-indued cleft palate. Birth Defects Res (Part B) 86:357–361, 2009. © 2009 Wiley-Liss, Inc.     

Structural relatedness of mouse lactate dehydrogenase isozymes,A4 (muscle), B4 (heart), and C4 (testis)

Three homotetrameric lactate dehydrogenase isozymes, LDH-M(A₄), LDH-H(B₄), and LDH-X(C₄), from DBA/2J mice have been purified by affinity chromatography. The amino acid compositions of the subunits A, B, and C, based on a molecular weight of 36,000, have been determined. The compositional relatedness of these isozymes indicates that subunits A (muscle) and B (heart) are more closely related to each other than to subunit C (testis). Tryptic peptide maps and amino acid compositions of some active site peptides appear to confirm the compositional relatedness among these isozymes. The sequence of the loop region of mouse C subunit seems to be markedly different from all known A and B sequences, and the structural and functional implications are discussed.     

Malate dehydrogenase isozymes in the longnose dace,Rhinichthys cataractae

The interspecies homology of dace supernatant (A₂, AB, B₂) and mitochondrial (C₂) malate dehydrogenase isozymes has been established through cell fractionation and tissue distribution studies. Isolated supernatant malate dehydrogenase (s-MDH) isozymes show significant differences in Michaelis constants for oxaloacetate and in pH optima. Shifts in s-MDH isozyme pH optima with temperature may result in immediate compensation for increase in ectotherm body pH with decrease in temperature, but duplicate s-MDH isozymes are probably maintained through selection for tissue specific regulation of metabolism.This research was supported in part by NSF Grant SM176-83974 and a grant from the Blakeslee Fund.     

Enhanced levels of vitamin B(6) increase aerial organ size and positively affect stress tolerance in Arabidopsis

Vitamin B₆ is an essential nutrient in the human diet derived primarily from plant sources. While it is well established as a cofactor for numerous metabolic enzymes, more recently, vitamin B₆ has been implicated as a potent antioxidant. The de novo vitamin B₆ biosynthesis pathway in plants has recently been unraveled and involves only two proteins, PDX1 and PDX2. To provide more insight into the effect of the compound on plant development and its role as an antioxidant, we have overexpressed the PDX proteins in Arabidopsis, generating lines with considerably higher levels of the vitamin in comparison with other recent attempts to achieve this goal. Interestingly, it was possible to increase the level of only one of the two catalytically active PDX1 proteins at the protein level, providing insight into the mechanism of vitamin B₆ homeostasis in planta. Vitamin B₆ enhanced lines have considerably larger vegetative and floral organs and although delayed in pre‐reproductive development, do not have an altered overall morphology. The vitamin was observed to accumulate in seeds and the enhancement of its levels was correlated with an increase in their size and weight. This phenotype is predominantly a consequence of embryo enlargement as reflected by larger cells. Furthermore, plants that overaccumulate the vitamin have an increased tolerance to oxidative stress providing in vivo evidence for the antioxidant functionality of vitamin B₆. In particular, the plants show an increased resistance to paraquat and photoinhibition, and they attenuate the cell death response observed in the conditional flu mutant.