Factors influencing the activity of succinate dehydrogenase in membrane preparations from Micrococcus lysodeikticus

1. Some properties of succinate dehydrogenase [succinate-(acceptor) oxidoreductase, EC 1.3.99.1] in membrane preparations from Micrococcus lysodeikticus (N.C.T.C. 2665) were investigated. 2. In the spectrophotometric assay system adopted the reaction velocity was shown to be proportional to the amount of membrane added. Dichlorophenol-indophenol, reduced photochemically in the presence of phenazine methosulphate, or enzymically by the membrane-bound enzyme, was shown to undergo reoxidation in the dark. 3. The membrane-bound enzyme was found to be inactivated at temperatures above 10 degrees C. 4. The specific activity of membrane-bound succinate dehydrogenase was found to increase between two- and three-fold in diluted membrane preparations equilibrated at 0 degrees C for 6h. Membranes treated with sodium deoxycholate showed no enzyme activation on dilution but displayed maximal activity, all activity being sedimentable at 103000g. The increase in specific activity observed on dilution could be partially inhibited by fixation with glutaraldehyde, or by the presence of bovine serum albumin. 5. The addition of Mg(2+) or Ca(2+) ions to membrane suspensions caused an overall depression of enzyme activity. 6. The results suggest the presence of an ;inhibitor' that affects the expression of membrane bound succinate dehydrogenase activity.     

Evidence for the existence of enzymatic variants of beta-hydroxybutyrate dehydrogenase from rat liver and brain mitochondria.

A comparison of rat brain and liver β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has revealed that significant differences exist between the enzymes with regard to their kinetic and physical properties. In contrast to the liver enzyme, brain β-hydroxybutyrate dehydrogenase is rapidly inactivated at 46° and is unstable when stored at ?20°. The brain dehydrogenase was found to have a larger K_m (apparent) for the 3-acetylpyridine analog of NAD⁺, and a greater energy of activation in the direction of β-hydroxybutyrate oxidation than the liver enzyme. In the reverse direction, the brain and liver dehydrogenase exhibit substrate inhibition by NADH (0.22 mM and 0.36 mM, respectively). The brain and liver β-hydroxybutyrate dehydrogenase did not differ significantly with regard to the Michaelis-Menten constants measured for NAD⁺ and β-hydroxybutyrate. The K_m constants of brain β-hydroxybutyrate dehydrogenase for acetoacetate (0.39 mM) and NADH (0.05 mM) were lower than those determined for the liver enzyme, acetoacetate (0.73 mM) and NADH (0.35 mM) respectively. These results suggest that the β-hydroxybutyrate dehydrogenase from rat brain and liver are isozymic variants.     

Analysis of the mechanisms of mitochondrial NADH regulation in cardiac trabeculae.

We have previously shown that increased cardiac work initially caused a rapid Ca(2+)-independent fall of mitochondrial [NADH] ([NADH](m)) to a minimum level, and this was followed by a slow Ca(2+)-dependent recovery toward control level (Brandes and Bers, Biophys. J. 71:1024-1035, 1996; Brandes and Bers, Circ. Res. 80:82-87, 1997). The purpose of this study is to improve our understanding of the factors that control [NADH](m) during increased work. [NADH](m) was monitored using fluorescence spectroscopy in intact rat trabeculae isolated from the right ventricular wall. Work was increased by increasing sarcomere length, pacing frequency, external [Ca(2+)], or by decreased temperature. The results were: 1) The initial fall of [NADH](m) during increased pacing frequency depends independently on increased myofilament work and on increased Ca(2+)-transport ATPase activity. 2) The [NADH](m) recovery process depends on average cytosolic [Ca(2+)] (Av[Ca(2+)](c)), but not on absolute work level. 3) The initial fall of [NADH](m) and the [NADH](m) recovery are similar whether increased work is associated with low frequency and high Ca(2+)-transient amplitude or vice versa (at the same myofilament work level and Av[Ca(2+)](c)). 4) The mechanisms associated with the smaller fall and recovery of [NADH](m) at 37 degrees C versus 27 degrees C, may be explained by lowered Av[Ca(2+)](c) and myofilament work. The NADH control mechanisms that operate at lower temperature are thus qualitatively similar at more physiological temperatures.     

Development of a commercial amperometric biosensor electrode for the ketone D-3-hydroxybutyrate

Representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue (MB) 3, 4-methyl-1,2-benzoquinone (4-MBQ) 4, 1-methoxy phenazine methosulphate (1-MeO-PMS) 5 and 2,6-dichloroindophenol (DCIP) 6, are shown to inhibit the NAD-dependent enzyme D-3-hydroxybutyrate dehydrogenase (HBDH), severely limiting their utility in the construction of a stable biosensor electrode for the ketone body D-3-hydroxybutyrate (3-OHB). It is proposed that these mediators bind covalently to important thiol groups in the enzyme. This mode of inhibition is overcome through the use of mediators such as 1,10-phenanthroline quinone (1,10-PQ) 7, which avoid 1,4-nucleophilic addition with enzyme amino acid residues such as Cys. As a result, 1,10-PQ 7 was selected for incorporation in a biosensor electrode for 3-OHB. The resulting MediSense Optiumtrade mark beta-Ketone electrode is stable (相似文献   

Ca2+-surfactant interactions affect enzyme stability in detergent solutions

Detergent proteases and amylases generally bind Ca(2+) ions. These bound ions enhance enzyme stability, reducing the rates of degradative reactions such as unfolding and proteolysis. Thus, surfactant aggregates, such as micelles, affect protease and amylase stability indirectly, by competing with the enzymes for Ca(2+) ions. Dissociation constants for Ca(2+) interactions with anionic surfactant micelles are in the 10(-3) to 10(-2) M range. These interactions are weak relative to enzyme-Ca(2+) interactions (K(d) of order 10(-6) M). However, surfactant is typically present at much higher concentration than enzyme, and it is the Ca(2+)-micelle equilibrium that largely determines the amount of free Ca(2+) available for binding to enzymes. The problem of surfactant-mediated Ca(2+) removal from enzymes can be avoided by adding calcium to a detergent formulation in an amount such that the concentration of free Ca(2+) is around 10(-5)M.     

Microbial metabolism of amino ketones. Aminoacetone formation from 1-aminopropan-2-ol by a dehydrogenase in Escherichia coli

1. Washed-cell suspensions of Escherichia coli, incubated at the optimum pH of 6.4 and with a saturating substrate concentration of approx. 10mm, convert dl-1-aminopropan-2-ol into aminoacetone at a rate of approx. 4.0mmumoles/mg. dry wt. of cells/min. at 30 degrees . 2. Mg(2+), Mn(2+), Co(2+), Zn(2+), Ca(2+), K(+) and NH(4) (+), as sulphates, and EDTA have no effect on this rate, although Cu(2+) inhibits and Fe(2+) activates to some extent. 3. Conditions of growth markedly affect the rate of aminoacetone production by cell suspensions. 4. Dialysed cell-free extracts of E. coli exhibit 1-aminopropan-2-ol-dehydrogenase activity, the enzyme having optimum activity at pH7.0, a requirement for NAD(+) and K(+), and a K(m) for the amino alcohol substrate of 0.8mm, calculated for a single enantiomorph. 5. Under optimum conditions 1-aminopropan-2-ol dehydrogenase forms aminoacetone at rate of approx. 3.0mmumoles/mg. of protein/min. at 37 degrees . The enzyme is only slightly inhibited by dl-3-hydroxybutyrate and dl-2-hydroxy-2-phenylethyl-amine. 6. l-Threonine-dehydrogenase activity is exhibited by both whole cells and cell-free extracts. Whole cells produce aminoacetone from l-threonine more slowly than they do from dl-1-aminopropan-2-ol, whereas the situation is reversed in cell-free extracts. Both kinetic evidence, and the fact that synthesis of 1-aminopropan-2-ol dehydrogenase, but not of threonine dehydrogenase, is repressed by compounds such as glucose and pyruvate, provide evidence that the amino alcohol is oxidized by a specific enyme. 7. The metabolic role of 1-aminopropan-2-ol dehydrogenase is discussed.     

Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm(3+), whose ionic radius is closest to that of Ca(2+), is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100mum in the presence of 5mm-Ca(2+). Er(3+) and Lu(3+), which both have ionic radii smaller than either Ca(2+) or Sm(3+), inhibit less efficiently, and La(3+), which is slightly larger than Ca(2+) or Sm(3+), inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca(2+)-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca(2+) [ionic radius 0.099nm (0.99 A)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca(2+), requires lanthanide concentrations 50-100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50mum, Sm(3+) lowers the apparent K(m) for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent V(max.) from 0.29 Wünsch-Heidrich unit to 0.018 unit. Thus Sm(3+) enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme-Sm(3+)-substrate complex. Inhibition by Sm(3+) is competitive with regard to Ca(2+). The apparent dissociation constant, K(d), of Ca(2+) is 0.27mm, where the K(i) for Sm(3+) is 12mum. Clostridiopeptidase is more thermolabile in the absence of Ca(2+). With Sm(3+), thermoinactivation of the enzyme at 53 degrees C or 60 degrees C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca(2+) at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.     

The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca(2+) transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5'-nucleotidase and Ruthenium Red-sensitive Ca(2+) transport. Initial rates of Ruthenium Red-insensitive Ca(2+) transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca(2+) transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5'-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca(2+) transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca(2+) transport activity, glucose 6-phosphatase and 5'-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca(2+) transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3-4 days, but then rapidly decreases to approach adult values. Maximal activity of 5'-nucleotidase in the microsomal and nuclear fractions is seen about 4-6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca(2+) transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2-3-fold the initial rate of Ruthenium Red-insensitive Ca(2+) transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1mug of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca(2+) transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca(2+) transport are an integral part of the physiological network in the liver cell.     

Calcium activation of heart mitochondrial oxidative phosphorylation: rapid kinetics of mVO2, NADH, AND light scattering

Parallel activation of heart mitochondria NADH and ATP production by Ca(2+) has been shown to involve the Ca(2+)-sensitive dehydrogenases and the F(0)F(1)-ATPase. In the current study we hypothesize that the response time of Ca(2+)-activated ATP production is rapid enough to support step changes in myocardial workload ( approximately 100 ms). To test this hypothesis, the rapid kinetics of Ca(2+) activation of mV(O(2)), [NADH], and light scattering were evaluated in isolated porcine heart mitochondria at 37 degrees C using a variety of optical techniques. The addition of Ca(2+) was associated with an initial response time (IRT) of mV(O(2)) that was dose-dependent with a minimum IRT of 0.27 +/- 0.02 s (n = 41) at 535 nm Ca(2+). The IRTs for NADH fluorescence and light scattering in response to Ca(2+) additions were similar to mV(O(2)). The Ca(2+) IRT for mV(O(2)) was significantly shorter than 1.6 mm ADP (2.36 +/- 0.47 s; p < or = 0.001, n = 13), 2.2 mm P(i) (2.32 +/- 0.29, p < or = 0.001, n = 13), or 10 mm creatine (15.6.+/-1.18 s, p < or = 0.001, n = 18) under similar experimental conditions. Calcium effects were inhibited with 8 microm ruthenium red (2.4 +/- 0.31 s; p < or = 0.001, n = 16) and reversed with EGTA (1.6 +/- 0.44; p < or = 0.01, n = 6). Estimates of Ca(2+) uptake into mitochondria using optical Ca(2+) indicators trapped in the matrix revealed a sufficiently rapid uptake to cause the metabolic effects observed. These data are consistent with the notion that extramitochondrial Ca(2+) can modify ATP production, via an increase in matrix Ca(2+) content, rapidly enough to support cardiac work transitions in vivo.     

Effect of micromolar concentrations of free Ca2+ ions on pyruvate dehydrogenase interconversion in intact rat heart mitochondria.

1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.     

Interaction of group IA phospholipase A2 with metal ions and phospholipid vesicles probed with deuterium exchange mass spectrometry

Deuterium exchange mass spectrometric evaluation of the cobra venom (Naja naja naja) group IA phospholipase A 2 (GIA PLA 2) was carried out in the presence of metal ions Ca (2+) and Ba (2+) and phospholipid vesicles. Novel conditions for digesting highly disulfide bonded proteins and a methodology for studying protein-lipid interactions using deuterium exchange have been developed. The enzyme exhibits unexpectedly slow rates of exchange in the two large alpha-helices of residues 43-53 and 89-101, which suggests that these alpha-helices are highly rigidified by the four disulfide bonds in this region. The binding of Ca (2+) or Ba (2+) ions decreased the deuterium exchange rates for five regions of the protein (residues 24-27, 29-40, 43-53, 103-110, and 111-114). The magnitude of the changes was the same for both ions with the exception of regions of residues 24-27 and 103-110 which showed greater changes for Ca (2+). The crystal structure of the N. naja naja GIA PLA 2 contains a single Ca (2+) bound in the catalytic site, but the crystal structures of related PLA 2s contain a second Ca (2+) binding site. The deuterium exchange studies reported here clearly show that in solution the GIA PLA 2 does in fact bind two Ca (2+) ions. With dimyristoylphosphatidylcholine (DMPC) phospholipid vesicles with 100 microM Ca (2+) present at 0 degrees C, significant areas on the i-face of the enzyme showed decreases in the rate of exchange. These areas included regions of residues 3-8, 18-21, and 56-64 which include Tyr-3, Trp-61, Tyr-63, and Phe-64 proposed to penetrate the membrane surface. These regions also contained Phe-5 and Trp-19, proposed to bind the fatty acyl tails of substrate.     

Conformational stability and integrity of alpha-amylase from mung beans: evidence of kinetic intermediate in GdmCl-induced unfolding

alpha-Amylase from mung beans (Vigna radiata) being one of the few plant alpha-amylases purified so far was studied with respect to its conformational stability by CD and fluorescence spectroscopy. The enzyme was shown to bind 3-4 Ca(2+) ions, which all are important for its activity. In contrast to other alpha-amylases no inhibition was observed at high Ca(2+) concentrations (100 mM). Depletion of calcium decreased the transition temperature from 87 to 48 degrees C. Kinetic stopped-flow fluorescence measurements allowed detecting two unfolding phases at >6 M GdmCl, whereas only one phase was observed at <5 M GdmCl. These results suggest that the first (reversible) step of unfolding is slower than the second (irreversible) step at low GdmCl concentrations, whereas the rates of these two steps are opposite at high GdmCl concentrations.     

Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5''-phosphate.

1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.     

Inhibition of lipogenesis by halothane in isolated rat liver cells.

1. Halothane at clinically effective concentrations [2.5 and 4% (v/v) of the gas phase of the incubation flask] was found to inhibit significantly lipogenesis from endogenous substrates, e.g., glycogen, or from added lactate plus pyruvate. This was accompanied by a decrease in the ratio of the free [NAD+]/[NADH] of the mitochondrion and the cytoplasm, as shown by the [3-hydroxybutyrate]/[acetoacetate] ratio and the [lactate]/[pyruvate] ratio. 2. Acetoacetate or pyruvate decreased the inhibitory effect of halothane and restored lipogenesis to control rates. They were reduced rapidly by 3-hydroxybutyrate dehydrogenase or lactate dehydrogenase respectively, with the concomitant oxidation of NADH and the generation of NAD+. 3. These results suggest that the mechanism by which halothane inhibits lipogenesis from glycogen or lactate is by inhibition of the oxidation of NADH; this results in inhibition of flux of carbon through pyruvate dehydrogenase and a shortage of acetyl-CoA for fatty acid synthesis. Thus when NADH acceptors are added in the presence of halothane, the concentration of mitochondrial NAD+ is raised so that the flux of carbon through pyruvate dehydrogenase increases and lipogenesis is restored.     

Reversible thermal inactivation of the quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. Ca2+ ions are necessary for re-activation.

The soluble form of the homogeneous quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus is reversibly inactivated at temperatures above 35 degrees C. An equilibrium is established between active and denatured enzyme, this depending on the protein concentration and the inactivation temperature used. Upon thermal inactivation the enzyme dissociates into the prosthetic group pyrroloquinoline quinone and the apo form of glucose dehydrogenase. After inactivation at 50 degrees C active enzyme is re-formed again at 25 degrees C. Ca2+ ions are necessary for the re-activation process. The velocity of re-activation depends on the protein concentration, the concentration of the prosthetic group pyrroloquinoline quinone and the Ca2+ concentration. The apo form of glucose dehydrogenase can be isolated, and in the presence of pyrroloquinoline quinone and Ca2+ active holoenzyme is formed. Even though native glucose dehydrogenase is not inactivated in the presence of EDTA or trans-1,2-diaminocyclohexane-NNN'NH-tetra-acetic acid, Ca2+ stabilizes the enzyme against thermal inactivation. Two Ca2+ ions are found per subunit of glucose dehydrogenase. The data suggest that pyrroloquinoline quinone is bound at the active site via a Ca2+ bridge. Mn2+ and Cd2+ can replace Ca2+ in the re-activation mixture.     

Decreased rate of ketone-body oxidation and decreased activity of D-3-hydroxybutyrate dehydrogenase and succinyl-CoA:3-oxo-acid CoA-transferase in heart mitochondria of diabetic rats.

Heart mitochondria from chronically diabetic rats ('diabetic mitochondria'), in metabolic State 3, oxidized 3-hydroxybutyrate and acetoacetate at a relatively slow rate, as compared with mitochondria from normal rats ('normal mitochondria'). No significant differences were observed, however, with pyruvate or L-glutamate plus L-malate as substrates. Diabetic mitochondria also showed decreased 3-hydroxybutyrate dehydrogenase and succinyl-CoA: 3-oxoacid CoA-transferase activities, but cytochrome content and NADH-dehydrogenase, succinate dehydrogenase, cytochrome oxidase and acetoacetyl-CoA thiolase activities proved normal. The decrease of 3-hydroxybutyrate dehydrogenase activity was observed in diabetic mitochondria subjected to different disruption procedures, namely freeze-thawing, sonication or hypoosmotic treatment, between pH 7.5 and 8.5, at temperatures in the range 6-36 degrees C, and in the presence of L-cysteine. Determination of the kinetic parameters of the enzyme reaction in diabetic mitochondria revealed diminution of maximal velocity (Vmax) as its outstanding feature. The decrease in 3-hydroxybutyrate dehydrogenase in diabetic mitochondria was a slow-developing effect, which reached full expression 2-3 months after the onset of diabetes; 1 week after onset, no significant difference between enzyme activity in diabetic and normal mitochondria could be established. Insulin administration to chronically diabetic rats for 2 weeks resulted in limited recovery of enzyme activity. G.l.c. analysis of fatty acid composition and measurement of diphenylhexatriene fluorescence anisotropy failed to reveal significant differences between diabetic and normal mitochondria. The Arrhenius-plot characteristics for 3-hydroxybutyrate dehydrogenase in membranes of diabetic and normal mitochondria were similar. It is assumed that the variation of the assayed enzymes in diabetic mitochondria results from a slow adaptation to the metabolic conditions resulting from diabetes, rather than to insulin deficiency itself.     

Electrochemical evaluation of lactate dehydrogenase immobilized in polyacrylamide gels

Lactate dehydrogenase (EC 1.1.1.27) has been immobilized in polyacrylamide gels over a platinum grid matrix. The immobilized enzyme is used to oxidize L-lactate in the presence of nicotinamide adenine dinucleotide (NAD+) and ferricyanide. The NADH produced is then chemically oxidized back to NAD+ by ferricyanide. The coupled reduction of ferricyanide ions to ferrocyanide ions results in a measurable electrochemical potential. This measurable zero-current potential is found to be Nernstian in nature and directly proportional to the logarithm values of L-lactate concentration over the range of 2 X 10(-5) to 5 X 10(-2)M. The results indicate that immobilized lactate dehydrogenase can be incorporated into a system to detect L-lactate acid in aqueous solutions.     

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.