PROPERTIES OF MEMBRANE-BOUND HEXOKINASE IN RAT BRAIN

Abstract— —-(1) The total hexokinase activity present in the mitochondrial fraction can be solubilized completely by incubation with salt and Triton X-100. This activity cannot be entirely released by washing with sucrose or by freezing and thawing.
(2) A part of the particle bound hexokinase exists in a latent form. The latent form is apparent after incubation with high salt concentrations, detergents or by freezing and thawing.
(3) Solubilization of membrane bound hexokinase is pH-dependent. Incubation in salt solutions increases the specific activity ten-fold. The salt concentration and pH are con-current. At pH 7.0 part of the hexokinase is solubilized. The lower the pH the less salt is required to release the same amount of activity.
(4) Triton X-100 solubilizes particle-bound hexokinase, but to a less extent than salts. The activation of hexokinase is greater with Triton X-100 than with salt.
(5) The possible nature of the bonds between hexokinase and mitochondrial membranes is discussed.     

In Rat Liver Mitochondria All Nucleoside Diphosphate Kinase of the Outer Compartment Is Associated with the Outer Surface of the Outer Membrane

Data on localization of nucleoside diphosphate kinase (NDPK) in the outer mitochondrial compartment are contradictory. We have demonstrated that repeated quintuple wash of a mitochondrial pellet (protein concentration is about 2 mg/ml) solubilized only 60% of total NDPK activity. Since no release of adenylate kinase, the marker enzyme of the intermembrane space, was observed, it was concluded that the solubilized NDPK activity was associated with the outer surface of the outer mitochondrial membrane. Treatment of mitochondria with digitonin solutions in low (sucrose, mannitol) or high (KCl) ionic strength media revealed that solubilization of remaining NDPK activity basically coincided with the solubilization curve of monoamine oxidase, the marker enzyme of the outer mitochondrial membrane, but differed from solubilization behavior of adenylate kinase and malate dehydrogenase. We concluded that the remaining NDPK activity was also associated with the outer mitochondrial membrane and electrostatic interactions were not essential for NDPK binding to mitochondrial membranes. Results of polarographic determination of remaining adenylate kinase and NDPK activities of mitochondria incubated in ice for different time intervals and subjected to subsequent centrifugation suggest that all NDPK activity of the outer compartment of rat liver mitochondria is associated with the outer surface of the outer mitochondrial membrane. We suggest the existence of at least three NDPK fractions. They represent 70, 15, and 15% of total NDPK activity of the outer compartment and differ by tightness of membrane binding.     

Studies on the nature of adenosine diphosphatase activity from rat liver mitochondria

Adenosine diphosphatase (ADPase) activity was studied in rat liver with [beta-32P]ADP as a substrate. Mitochondria and outer mitochondrial membrane fractions were isolated and assayed for ADPase and various marker enzymes. ADPase activity was strikingly reduced when the outer membranes were removed from the mitochondria whether by digitonin treatment or osmotic shock. Addition of the inter-membrane space subfraction to the purified outer membranes resulted in enhanced ADPase activity. Addition of the inter-mitochondrial membrane enzyme adenylate kinase to outer membranes also produced a large stimulation of activity. The ADPase activity could also be reconstituted in vitro with adenylate kinase and either mitoplast ATPase or ouabain-sensitive (Na+ + K+ + Mg2+)-ATPase. Chloroform-released ATPase, however, was not capable of producing an ADPase activity when combined with adenylate kinase. Gel permeation chromatography of Triton-solubilised outer mitochondrial membranes was unable to resolve ADPase activity from contaminating ATPase. These results suggest that the majority of ADPase activity in rat liver mitochondria consists of the coupled activity of adenylate kinase and ATPase.     

Cellular and Subcellular Localization of Hexokinase, Glutamate Dehydrogenase, and Alanine Aminotransferase in the Honeybee Drone Retina

Abstract: Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[³H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.     

Solute effects on mitochondria from an elasmobranch (Raja erinacea) and a teleost (Pseudopleuronectes americanus)

Varying osmolarity with sucrose/KCl media resulted in similar effects on the oxidation of glutamate by mitochondria isolated from the livers of an elasmobranch, Raja erinacea, and a teleost, Pseudopleuronectes americanus. In both species trimethylamine oxide (TMAO) inhibited mitochondrial oxidation of glutamate. Urea penetrated the inner mitochondrial membrane of both species and equilibrated with a ratio ureai/ureao of unity. Urea had little effect on the oxidation of glutamate in both species at concentrations as high as 760 mM. Addition of urea (urea/TMAO, 2:1) did not overcome the detrimental effects of TMAO in the mitochondria of either species. In the case of the elasmobranch, the osmolarity of the urea/TMAO media giving the optimal rate of respiration was hypoosmotic with respect to the intracellular osmolarity. The rate of glutamate oxidation steadily declined as osmolarity increased above this value. Assuming the osmotic profile obtained with the urea/TMAO (2:1) medium resembled most closely the in vivo situation, higher rates of oxidation or organic solutes at low osmolarity would help deplete the cell of these solutes and could contribute to cell volume regulation during hypoosmotic stress. It is suggested that two broad classes of intracellular solutes can be defined based on their effects on mitochondrial respiration. Solutes such as K+, C1-, and TMAO penetrate the inner mitochondrial membrane slowly or not at all. Increasing concentrations of these solutes result in lower rates of oxidation. This capacity may be important in regulating intracellular levels of organic solutes during osmotic stress. Solutes such as urea rapidly penetrate the cell and inner mitochondrial membrane reducing the mitochondrial volume changes associated with osmotic stress. The known detrimental effects of urea on protein structure may prevent its exclusive use as an intracellular osmotic effector.     

Colligative and non-colligative freezing damage to thylakoid membranes

When spinach thylakoid membranes were frozen in vitro in solutions containing constant molar ratios of cryotoxic to cryoprotective solute, maintenance of functional integrity strongly depended on initial osmolarities. Optimum cryopreservation of cyclic photophosphorylation was observed when the membranes were suspended in solutions of intermediate osmolarities (approx. 50–100 mM NaCl, 75–150 mM sucrose). Both higher and lower initial osmolarities were found to result in decreased cryopreservation. In the absence of added salt, more than 100 mM sucrose were needed for full cryopreservation of the membranes. When thylakoids were frozen in solutions containing low concentrations of NaCl (2 mM), the ratio of sucrose to salt necessary to give full protection was high (up to 50). When the salt concentration was about 60 mM, ratios as low as 1.5 were sufficient for maintaining membrane integrity. This ratio increased again, as the initial NaCl concentration was increased beyond 60 mM. During freezing, proteins dissociated from the membranes, and the amount of the released proteins was correlated linearly with inactivation of photophosphorylation. The gel electrophoretic pattern of proteins released at low initial osmolarities differed from that of proteins released at high initial osmolarities. Cryopreservation was also found to depend on membrane concentration. Concentrated membrane suspensions suffered less inactivation than dilute suspensions. The protective effect of high membrane concentrations was particularly pronounced at high initial solute concentrations. It is proposed that damage at low initial osmolarities is caused predominantly by mechanical stress and by osmotic contraction/expansion. Damage at high initial osmolarities is thought to be caused mainly by solute effects. Under these conditions, both the final volume of the unfrozen solution in coexistence with ice and the membrane concentration affect membrane survival by influencing the extent of the loss of membrane components through dissociation reactions. Membrane protection by sugars is caused by colligative action under these circumstances.     

Mitochondrial boundary membrane contact sites in brain: points of hexokinase and creatine kinase location, and control of Ca2+ transport

The location of hexokinase at the surface of brain mitochondria was investigated by electron microscopy using immuno-gold labelling techniques. The enzyme was located where the two mitochondrial limiting membranes were opposed and contact sites were possible. Disruption of the outer membrane by digitonin did not remove bound hexokinase and creatine kinase from brain mitochondria, although the activity of outer membrane markers and adenylate kinase decreased, suggesting a preferential location of both enzymes in the contact sites. In agreement with that, a membrane fraction was isolated from osmotically lysed rat brain mitochondria in which hexokinase and creatine kinase were concentrated. The density of this kinase-rich fraction was specifically increased by immuno-gold labelling of hexokinase, allowing a further purification by density gradient centrifugation. The fraction was composed of inner and outer limiting membrane components as shown by the specific marker enzymes, succinate dehydrogenase and NADH-cytochrome-c-oxidase (rotenone insensitive). As reported earlier for the enriched contact site fraction of liver mitochondria the fraction from brain mitochondria contained a high activity of glutathione transferase and a low cholesterol concentration. Moreover, the contacts showed a higher Ca2+ binding capacity in comparison to outer and inner membrane fractions. This finding may have regulatory implications because glucose phosphorylation via hexokinase activated the active Ca2+ uptake system and inhibited the passive efflux, resulting in an increase of intramitochondrial Ca2+.     

Further characterization of contact sites from mitochondria of different tissues: topology of peripheral kinases

A membrane fraction of intermediate density between inner and outer membrane was isolated by density gradient centrifugation from osmotically disrupted mitochondria of rat liver, brain, and kidney. The fraction was hexokinase rich and could therefore be further purified using specific antibodies against hexokinase and immunogold labelling techniques. In agreement with recent findings the gradient fraction which cosedimented with hexokinase contained the boundary membrane contact sites because it was composed of outer and inner membrane components and beside hexokinase, was enriched also by activity of creatine kinase and nucleoside diphosphate kinase. In contrast the activity of adenylate kinase appeared to be concentrated beyond the contact sites in the outer membrane fraction. By employing surface proteolysis analysis and specific blockers of the outer membrane pore we observed that the location of the kinases relative to the membrane components in the contact fraction resembled that of intact mitochondria. This specific organization of some peripheral kinases in the contact sites suggested an important role of the voltage dependence of the outer membrane pore, in that the pore may become limiting in anion exchange because of influence of the inner membrane potential on the closely attached outer membrane. Such control of anion exchange would lead to a dynamic compartmentation at the mitochondrial surface by the formation of contact sites, which may explain the preferential utilization of cytosolic creatine by the mitochondrial creatine kinase, as postulated in the phosphocreatine shuttle.     

Mechanical and chemical injury to thylakoid membranes during freezing in vitro

The relative contributions of membrane rupture due to osmotic stress and of chemical membrane damage due to the accumulation of cryotoxic solutes to cryoinjury was investigated using thylakoid membranes as a model system. When thylakoid suspensions were subjected to a freeze-thaw cycle in the presence of different molar ratios of NaCl as the cryotoxic solute and sucrose as the cryoprotective solute, membrane survival first increased linearly with the osmolality of the solutions used to suspend the membranes, regardless of the molar ratio of salt to sucrose. It subsequently decreased when the ratio of sucrose to salt was not sufficiently high for complete cryopreservation by sucrose. There was an optimum of cryopreservation at intermediate osmolalities (approx. 0.1 osmol/kg). This optimum of cryopreservation at a given sucrose concentration could be shifted to lower solute concentration, if mixtures of NaCl and NaBr were used instead of NaCl alone. At suboptimal initial osmolalities, damage is attributed mainly to membrane rupture. Under these conditions, cryopreservation is not influenced by the chaotropicity of the suspending medium. At supraoptimal initial solute concentrations, solute (i.e., chemical) effects determine membrane survival. Under these conditions, increased ratios of sugar to salt increased cryoprotection. In mixtures of NaCl and NaBr at constant molar ratios of salt to sucrose, chemical membrane damage was quantitatively related to the lyotropic properties of the ions used. The degree of chemical damage becomes more pronounced with rising osmolalities of the suspending media. With NaF as the cryotoxic solute, damage was more severe than should be expected from its lyotropic properties. This may reflect a specific interaction of fluoride with the membranes. Protein release from the membranes during freezing in the presence of different anions was qualitatively comparable at identical ratios of sugar to salt. However, the total amount of protein released was correlated linearly with membrane inactivation, even when different anions acted on the membranes. Gel electrophoretic analysis of proteins released from thylakoid membranes during freezing revealed discrete bands indicative of mechanical and chemical damage, respectively.     

NaCl对小麦苗叶片脯氨酸氧化酶活性和游离脯氨酸累积的影响

在含NaCl营养液中培养的小麦幼苗较之无NaCl营养液中的幼苗。其脯氨酸氧化酶活性降低,而游离脯氨酸含量则升高;培养液的渗透势越低,培养时间越长,则脯氨酸氧化酶的活性越低,且游离脯氨酸的含量越高。去除胁迫后酶活性恢复,脯氯酸含量下降。不同渗透剂对氧化酶活性抑制强弱顺序为MgCl_2>NaCl>甘露醇,引起脯氨酸累积效应的强度顺序为MgCl_2>NaCl>甘露醇。超微结构显示,高NaCl浓度下部分线粒体结构受损伤,膜和嵴部分消失。     

Hyperosmotic relaxation lysis of chromaffin granules is caused by interactions between the granular membrane and intragranular vesicles

Bovine chromaffin granules undergo irreversible structural changes during osmotic shrinkage in hypertonic sucrose and salt solutions, such that, on reexposure to isoosmotic conditions they do not regain their original morphology, but undergo lysis ('hyperosmotic relaxation lysis'). Irreversible alterations of granules were induced by hypertonic incubations lasting for as little as 1 min. Fluorescence and EPR membrane labelling experiments showed that hypertonicity did not induce membrane loss for instance by inwardly or outwardly directed pinching off of membrane material. The mean sizes of chromaffin granules as a function of increasing and subsequently decreasing osmotic pressure were measured by photon correlation spectroscopy; there was no significant difference in sizes of hyperosmotically pretreated granules as compared with controls. Freeze-fracture electron micrographs showed the formation of 'twins' and 'triplets' under hypertonic conditions. They also revealed intragranular vesicles of 50-200 nm in diameter in both hypertonically and isotonically suspended granules. 'Twin' and 'triplet' granules were formed by the attachment of intragranular vesicles to the granule membranes. We suggest that hyperosmotic relaxation lysis is caused by the fact that this adhesion partly prevents the granule membrane from reexpanding, thus, leading to its rupture.     

Hexokinase of rat brain mitochondria: relative importance of adenylate kinase and oxidative phosphorylation as sources of substrate ATP, and interaction with intramitochondrial compartments of ATP and ADP

Interactions between intramitochondrial ATP-generating, ADP-requiring processes and ATP-requiring, ADP-generating phosphorylation of glucose by mitochondrially bound hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) have been investigated using well-coupled mitochondria isolated from rat brain. ADP generated by mitochondrially bound hexokinase was more effective at stimulating respiration than was ADP generated by hexokinase dissociated from the mitochondria, and pyruvate kinase was less effective as a scavenger of ADP generated by the mitochondrially bound hexokinase than was the case with ADP generated by the dissociated enzyme. These results indicate that ADP generated by the mitochondrially bound enzyme is at least partially sequestered and directed toward the mitochondrial oxidative phosphorylation apparatus. Under the conditions of these experiments, the maximum rate of ATP production by oxidative phosphorylation was approximately 10-fold greater than the maximum rate of ATP generation by the adenylate kinase reaction. Moreover, during periods of active oxidative phosphorylation, adenylate kinase made no detectable contribution to ATP production. Thus, adenylate kinase does not represent a major source of ATP for hexokinase bound to actively phosphorylating brain mitochondria. With adenylate kinase as the sole source of ATP, a steady state was attained in which ATP formation was balanced by utilization in the hexokinase reaction. In contrast, when oxidative phosphorylation was the source of ATP, a steady state rate of Glc phosphorylation was attained, but it was equivalent to only about 40-50% of the rate of ATP production and thus there was a continued net increase in ATP concentration in the system. Rates of Glc phosphorylation with ATP generated by oxidative phosphorylation exceeded those seen with equivalent levels of exogenously added ATP. Moreover, at total ATP concentrations greater than approximately 0.2 mM, hexokinase bound to actively phosphorylating mitochondria was unresponsive to continued slow increases in ATP levels; acute increase in ATP (by addition of exogenous nucleotide) did, however, result in increased hexokinase activity. The relative insensitivity of mitochondrially bound hexokinase to extramitochondrial ATP suggested dependence on an intramitochondrial pool (or pools) of ATP during active oxidative phosphorylation. Two intramitochondrial compartments of ATP were identified based on their selective release by inhibitors of electron transport or oxidative phosphorylation. These compartments were distinguished by their sensitivity to inhibitors and the kinetics with which they were filled with ATP generated by oxidative phosphorylation. Exogenous glycerol kinase competed effectively with mitochondrially bound hexokinase for extramitochondrial ATP, with relatively low levels of glycerol kinase completely inhibiting phosphorylation of Glc.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the mechanism of oligomycin inhibition of Ca-induced mitochondrial respiration

The addition of oligomycin in the presence of Ca²⁺ increased the ADP pool in mitochondrial suspension. It is suggested that oligomycin inhibition of Ca²⁺-induced mitochondrial respiratory activation is the function of the increased endogenous ADP pool. Low ADP concentrations (5–20 μM) produce the same inhibitory effect as oligomycin. The increase of ADP levels in the presence of glucose plus hexokinase resulted in the inhibition of Ca²⁺-induced respiration, while the addition of phosphoenol pyruvate plus pyruvate kinase followed by a reduction in ADP levels, reversed the oligomycin inhibitory effect. One of the essential stages of ADP accumulation in mitochondrial suspensions in the presence of oligomycin and Ca²⁺ is proposed to be the formation of ADP from AMP and ATP, effected by adenylate kinase.     

The effect of salt or various cryoprotective agents on frog sciatic nerves

Desheathed frog sciatic nerves were tested for membrane damage by noting changes in their conduction velocity, externally recorded action potential, and absolute refractory period. Nerves were exposed for 45–60 min depending on the treatment to 1.5 osmolar environments consisting of concentrated Ringer's solution or Ringer's solution plus glycerol, sucrose, or dimethyl sulfoxide (DMSO). The nerves were exposed to these hypertonic solutions in one of four ways: (1) Gradual exposure plus gradual de-exposure, (2) Gradual exposure (plus direct de-exposure), (3) (direct exposure plus) plus gradual de-exposure, (4) Direct exposure (plus direct de-exposure). After treatment the nerves were allowed to recover for 45–60 min depending on the type of treatment in normal Ringer's solution. The results suggest the following: (1) Concentrated salt solutions damage nerve axons due to increased ion concentration rather than increased tonicity. (2) The effect of osmotic shock on sciatic nerves is relatively minor and completely reversible. (3) Axon membranes behave as simple permeability barriers with respect to sucrose and DMSO, but they appear to behave as dynamic entities which alter their metastable states with respect to concentrated salt or concentrated glycerol in Ringer's solution. (4) Freeze-thaw damage to sciatic nerve axon membranes—and perhaps to any cell membrane—is probably due to increased ionic concentration as well as increased osmolarity.     

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 µM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed. (Mol Cell Biochem 174: 43–51, 1997)     

The cationically selective state of the mitochondrial outer membrane pore: a study with intact mitochondria and reconstituted mitochondrial porin.

The outer mitochondrial membrane pore at a voltage above 20 to 30 mV can adopt a state of low conductance which may restrict free permeability of mitochondrial substrates. In order to obtain insight into the physiological meaning of this property we took advantage of the fact that the low conductance pore state could be induced by a polyanion in lipid bilayer membranes as well as in intact mitochondria. Upon reconstitution in artificial bilayers the pore in this substate became exclusively cation selective when the polarity of the applied voltage was negative on the cis-side. This behaviour of the pore would explain why induction of the low conductance pore state in intact mitochondria led to a complete inhibition of mitochondrial intermembranous kinases, such as creatine kinase and adenylate kinase, but not of peripheral kinases, for example hexokinase, when utilizing external ATP. The possibility that the inner membrane potential might be transduced to the outer membrane in the contact sites, suggests the existence of cation selective pores in these sites. This aspect may be important in the regulation of peripheral kinases like creatine kinase, nucleoside diphosphate kinase and adenylate kinase which are located behind the outer mitochondrial membrane.     

The purification and characterization of plasma membranes and the subcellular distribution of adenylate cyclase in mouse parotid gland.

1. Plasma membranes have been purified 17-fold from mouse parotid gland homogenates prepared in hypertonic sucrose media using differential centrifugation. The method is fast and simple. The membranes were characterised by electron microscopy, enzyme composition and chemical composition. Further purification was achieved by isopycnic centrifugation in discontinuous sucrose gradients. 2. The purified membranes contain an adenylate cyclase activity which is stimulated by isoproterenol and fluoride. Only 50% of the total adenylate cyclase activity sedimented in the plasma membrane fraction. The rest of the activity resided in the crude nuclear and mitochondrial pellets. However, this adenylate cyclase activity was not associated with these organelles but with membrane fragments in the pellets. Purified nuclei did not contain adenylate cyclase activity. 3. Adenylate cyclase activity was also localised by electron microscopic cytochemistry. Besides being found at the plasma membrane, large amounts of adenylate cyclase were found in a small proportion of the vesicles within the acinar cells, which appeared to be secondary lysosomes. 4. Adenylate cyclase activities, under standard assay conditions, are proportional to the time of incubation and the concentration of enzyme. The enzyme requires both Mg-2+ and CA-2+ for activity. Isoproterenol increased activity 2-fold and this increase is abolished by beta-adrenergic blocking agents.     

Micromolar concentrations of Al3+ induce phase separation, aggregation and dye release in phosphatidylserine-containing lipid vesicles

It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed.     

Theoretical and experimental studies on freezing point depression and vapor pressure deficit as methods to measure osmotic pressure of aqueous polyethylene glycol and bovine serum albumin solutions

For survival in adverse environments where there is drought, high salt concentration or low temperature, some plants seem to be able to synthesize biochemical compounds, including proteins, in response to changes in water activity or osmotic pressure. Measurement of the water activity or osmotic pressure of simple aqueous solutions has been based on freezing point depression or vapor pressure deficit. Measurement of the osmotic pressure of plants under water stress has been mainly based on vapor pressure deficit. However, differences have been noted for osmotic pressure values of aqueous polyethylene glycol (PEG) solutions measured by freezing point depression and vapor pressure deficit. For this paper, the physicochemical basis of freezing point depression and vapor pressure deficit were first examined theoretically and then, the osmotic pressure of aqueous ethylene glycol and of PEG solutions were measured by both freezing point depression and vapor pressure deficit in comparison with other aqueous solutions such as NaCl, KCl, CaCl(2), glucose, sucrose, raffinose, and bovine serum albumin (BSA) solutions. The results showed that: (1) freezing point depression and vapor pressure deficit share theoretically the same physicochemical basis; (2) theoretically, they are proportional to the molal concentration of the aqueous solutions to be measured; (3) in practice, the osmotic pressure levels of aqueous NaCl, KCl, CaCl(2), glucose, sucrose, and raffinose solutions increase in proportion to their molal concentrations and there is little inconsistency between those measured by freezing point depression and vapor pressure deficit; (4) the osmotic pressure levels of aqueous ethylene glycol and PEG solutions measured by freezing point depression differed from the values measured by vapor pressure deficit; (5) the osmotic pressure of aqueous BSA solution measured by freezing point depression differed slightly from that measured by vapor pressure deficit.     

Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities

The estuarine crab Neohelice granulata was exposed (96h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2ppt salinity, 1mg Cu/L; isosmotic crabs: 30ppt salinity, 5mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes observed suggest no impact in the overall tissue ATP production. Also, findings suggest that copper exposure would stimulate the pentose phosphate pathway to support the antioxidant system requirements. Although N. granulata is very tolerant to copper, acute exposure to this metal can disrupt the energy balance by affecting biochemical systems involved in carbohydrate metabolism.