Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate

Pyruvate dehydrogenase complex activity (PDHC) measured by CO2 release isotopic assay has generally been much lower than activity measured by the spectrophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [1-14C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units +/- 12.3 SD (n = 22) was observed at 4 mM pyruvate (1 unit = 1 nmol pyruvate decarboxylated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units +/- 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 mM oxaloacetate, and without oxaloacetate activity fell to 15.4 units +/- 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.     

ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning from Vicia faba mesophyll and epidermis: Characterization and influence of abscisic acid and fusicoccin

Plasmalemma-rich microsomal vesicles were prepared from whole leaf and acid-washed epidermal tissue of Vicia faba L. cv. Osnabrücker Markt by aqueous two-phase partitioning in dextran T-500 and polyethylenglycol 1350 aqueous phases. These vesicles were tightly sealed and predominantly right-side out, and contained a K⁺ -stimulated, mg²⁺-dependent and vanadate-sensitive ATPase. The enzyme from both tissues exhibited nearly identical properties: pH optimum 6.4, K_m for ATP 0.60 mM(whole leaf) and 0.67 mM (epidermis). V_max -480 nmol (mg protein)¹ min¹ (whole leaf) and 510 nmol (mg protein)¹ min¹ (epidermis), I₅₀ (Na₃,VO₄) 7.5 μM (whole leaf) and 15 μM (epidermis). The enzyme was not inhibited by NO₃(50 mM)or sodium azide (I mM). DCCD (20 μM) reduced enzyme activity to 50% (whole leaf) and 58% (epidermis), gramicidin S (20 μM) to 36% (whole leaf) and 41%(epidermis). Ca²⁺ inhibited the ATPase [I₅₀, C²⁺: 0.5 mM(whole leaf) and 0.8 mM(epidermis)]. Ca²⁺ inhibited the ATPase [I₅₀, C²⁺ 0.5 mM(whole leaf) und 0.8 (epidermis)]. The vanadate-sensitive ATPase from whole leaf and epidermal tissue was slightly but significantly stimulated by fusicoccin (FC) at a concentration (0.13 μM) promoting stomatal opening. The stimulation was not seen in the solubilized ATPase. Stomata of the cultivar used here were insensitive lo (±)ABA up to 2 μM level which is effective in most other cultivars and species. Likewise, at this concentration no effect of ABA on the activity of the epidermal ATPase was observed. The data are discussed with respect to the interaction of FC and ABA with the ATPase.     

Response of aromatic-pathway enzymes to changing physiological states of growth in suspension cultures of Nicotiana silvestris

The activity levels of enzymes of aromatic amino acid biosynthesis respond to changing physiological states of growth, as illustrated by results obtained from suspension-cultured cells of Nicotiana silvestris Speg. et Comes line ANS 1 (2N=24). The experimental system provides a foundation for interpretations about overall regulation of enzyme levels in relationship to growth physiology. Levels of activity for shikimate dehydrogenase (EC 1.1.1.25), prephenate aminotransferase and arogenate dehydrogenase were followed throughout a growth cycle obtained by a conventional subculture protocol. Enzyme date were also obtained from cell cultures maintained in continuous exponential growth for greater than 10 generations (EE cells). Both shikimate dehydrogenase and prephenate aminotransferase exhibited elevated stationary-phase levels of enzyme, much of which was carried over into a subsequent subculture. At least 4 generations of exponential growth were required before diminution of the latter two enzymes to the levels characteristic of truly exponential-phase growth (EE cells) occurred. This is reminiscent of the overall behavior of 3-deoxy-D- arabino -heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15), specifically attributed to the properties of the cytosolic isozyme species (DAHP synthase-Co). Elevation of arogenate dehydrogenase also occurred in stationary-phase cells, but diminished rapidly during lag phase to reach the level characteristic of EE cells.     

Fermentation of starch hydrolysates byLactobacillus plantarum

Summary Lactic acid production by an isolated ofLactobacillus plantarum was standardised on enzyme-hydrolysed tapioca (Manihot esculenta) flour, tapioca starch and soluble starch. Calculated yields of lactic acid (g from 100 g reducing sugars used) in nutrient media containing the abovementioned hydrolysates (10% reducing sugars) were 21.8%, 16.2% and 16.2%, respectively. Higher yields (29–34%) were obtained in media containing 5% reducing sugars. A conversion efficiency of 80–99% was achieved when the acid produced in the broth was neutralised periodically. One hundred milliliters of the medium (5% sugars) yielded 4.0–4.5 g of calcium lactate. These results indicate that unrefined starchy material can be successfully employed for the economic production of lactic acid. The same substrate can also be utilised for biomass production, as viable lactobacilli are being used for therapy in medicine.     

Myocardial cell vulnerability to exogenous phospholipase attack

Myocardial cell vulnerability to phospholipase C (PC-PLC) attack was investigated in three different preparations of rat myocardial cells: triacylglycerol (TG)-loaded, hypothermic/rewarmed and energy depleted myocytes. The attack by PC-PLC was evaluated as PC-PLC induced glycerol output due to the combined action of phospholipase C and intracellular lipases. PC-PLC induced glycerol output was significantly higher (p < 0.05) in all three myocyte preparations, compared to their respective controls. Cell morphology (% rod shaped myocytes) of TG-loaded or hypothermic/rewarmed myocytes was not different from their controls, whereas energy depleted myocytes almost exclusively were rounded up, due to hypercontraction of the myofilaments. Hypothermic/rewarmed and energy depleted myocytes showed a significantly higher release of lactate dehydrogenase (LDH), compared to their controls although the difference was much more pronounced in the latter. Finally, the cellular contents of ATP were maintained both in TG-loaded and hypothermic rewarmed myocytes, while energy depleted myocytes contained only about 25% of the normal ATP level. These results demonstrate that attack from exogenously added phospholipases can occur, not only in seriously damaged cardiac myocytes, but in myocytes with a more subtle damage as well. (Mol Cell Biochem 116: 47–52, 1992)     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Genetic,biochemical and immunological evidence for the involvement of two alcohol dehydrogenases in the metabolism of propane by Rhodococcus rhodochrous PNKb1

NAD⁺-dependent propan-1-ol and propan-2-ol dehydrogenase activities were detected in cell-free extracts of Rhodococcus rhodochrous PNKb1 grown on propane and potential intermediates of propane oxidation. However, it was unclear whether this activity was mediated by one or more enzymes. The isolation of mutants unable to utilize propan-1-ol (alcA^-) or propan-2-ol (alcB^-) as sole carbon and energy sources demonstrated that these substrates are metabolized by different alcohol dehydrogenases. These mutants were also unable to utilize propane as a growth substrate indicating that both alcohols are intermediates of propane metabolism. Therefore, propane is metabolized by terminal and sub-terminal oxidation pathways. Westernblot analysis demonstrated that a previously purified NAD⁺-dependent propan-2-ol dehydrogenase (Ashraf and Murrell 1990) was only synthesized after growth on propane and sub-terminal oxidation intermediates (but not acetone), and not propan-1-ol or terminal oxidation intermediates. Therefore, our evidence suggest that another dehydrogenase is involved in the metabolism of propan-1-ol and this agrees with the isolation of the alcA^- and alcB^- phenotypes. The previously characterized NAD⁺-dependent propan-2-ol dehydrogenase from R. rhodochrous PNKb1 is highly conserved amongst members of the propane-utilizing Rhodococcus-Nocardia complex.     

Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels.

An algorithm is presented for the accurate and rapid generation of multiple protein sequence alignments from tertiary structure comparisons. A preliminary multiple sequence alignment is performed using sequence information, which then determines an initial superposition of the structures. A structure comparison algorithm is applied to all pairs of proteins in the superimposed set and a similarity tree calculated. Multiple sequence alignments are then generated by following the tree from the branches to the root. At each branchpoint of the tree, a structure-based sequence alignment and coordinate transformations are output, with the multiple alignment of all structures output at the root. The algorithm encoded in STAMP (STructural Alignment of Multiple Proteins) is shown to give alignments in good agreement with published structural accounts within the dehydrogenase fold domains, globins, and serine proteinases. In order to reduce the need for visual verification, two similarity indices are introduced to determine the quality of each generated structural alignment. Sc quantifies the global structural similarity between pairs or groups of proteins, whereas Pij' provides a normalized measure of the confidence in the alignment of each residue. STAMP alignments have the quality of each alignment characterized by Sc and Pij' values and thus provide a reproducible resource for studies of residue conservation within structural motifs.     

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.