Study on heart mitochondrial creatine kinase using a cross-linking bifunctional reagent. II. The binding sites for the creatine kinase octamer on mitochondrial membranes have different properties

Beef heart mitochondria suspended in 0.25 M sucrose were treated with 0.3% glutaraldehyde (GA). The membranes were disintegrated by ultrasonication in 0.25 M KCl and precipitated by centrifugation. The relative amount of the membrane-bound mitochondrial creatine kinase (CKm) does not depend on the time course of membrane disruption. The enzyme is not removed by repeated washing of the pellet. It is concluded that this part of CKm is cross-linked to mitochondrial membranes. The maximum amount of the enzyme capable of cross-linking to the membrane with an increase in GA concentration or incubation time makes up to about 50% of the total CKm activity present in the mitochondria. It is concluded also that the CKm binding sites differ with respect to their environment.     

Morphology and ATP-ase of isolated mitochondria

Changes in the morphology of rat liver mitochondria brought about by different methods of isolation and the concomitant changes in ATP-ase activity were studied. The morphology was investigated with the electron microscope. It was found that the ATP-ase activity of the isolated mitochondria cannot be readily correlated with the morphology of the mitochondria. The ATP-ase found in these preparations was latent, resembling the enzyme described in mitochondria prepared in 0.25 M sucrose. In confirmation of earlier results the use of 0.88 M sucrose yielded preparations with a higher initial ATP-ase than did other methods. Preparation in 0.25 M sucrose resulted in round, swollen mitochondria of which 30 to 40 per cent appeared to have lost a substantial part of the mitochondrial matrix. Preparations in 0.44 to 0.88 M sucrose contained mainly rod-shaped mitochondria plus a small amount of another type of swollen mitochondria. The matrix of mitochondria isolated in 0.88 M sucrose was highly condensed. By the use of 0.44 M sucrose adjusted to pH 6.2 with citric acid, it was possible to isolate, for the first time, mitochondria closely resembling those in situ and containing latent ATP-ase.     

Interaction of creatine kinase with phosphorylating rabbit heart mitochondria and mitoplasts

This paper demonstrates that the mitochondrial isoenzyme of creatine kinase (CKm) can be solubilized from rabbit heart mitochondria, the outer membrane of which has been removed or at least broken by a digitonin treatment or a short hypotonic exposure, but which has retained an important part of the capacity to phosphorylate ADP. Phosphate, ADP, or ATP, at concentrations which are used to study oxidative phosphorylation and creatine phosphate synthesis, solubilize CKm; the same is true with MgCl2 and KCl. The effect of adenine nucleotides does not seem to be due to their interaction with the adenine nucleotide translocase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that CKm is the main protein released in the described conditions; however, it does not amount to more than 1% of the total protein content of the mitoplasts. When the apparent Km for ATP of CKm was estimated by measuring creatine phosphate synthesis, the values obtained using water-treated mitochondria (0.21 mM) were slightly higher than those of intact mitochondria (0.12 mM) but the difference was not significant. In the former preparation 77% of CKm was in a soluble state. If we can extrapolate these results to intact mitochondria and suppose that in this case a fraction of CKm is also soluble in the intermembrane space, this does not support the theory of functional association between CKm and the adenine nucleotide translocase.     

Extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex. II. ACTH-induced changes in subcellular corticosterone

Zona fasciculata-reticularis subcellular structures were implicated in corticosterone transport and secretion by noting changes in subcellular corticosterone during a 30-min period following ACTH stimulation. Six decapsulated adrenal homogenate subcellular fractions separated by gradient centrifugation were characterized cytochemically and morphologically. Predominant components in each of six fractions were: floating lipid droplets, 0.125 M sucrose (no organelles), cytosol (0.25 M sucrose supernatant with 0.25-1.2 micron electron dense granules), microsomes (interface between 0.5 M and 1.1 M sucrose layers), mitochondria (boundary between 1.1 M and 2.2 M sucrose layers) and nuclei (centrifuge pellet). Whole glands and most subcellular fractions showed peak corticosterone levels 10 to 15, and 30 min after stimulation. Sucrose and cytosolic fractions contained about 75% of the total corticosterone, responded to stimulation most significantly, and were rich in protein. In these two fractions only cytosol contained structures; these consisted of 0.15-1.2 micron electron dense granules.     

A method for isolating intact mitochondria and nuclei from the same homogenate,and the influence of mitochondrial destruction on the properties of cell nuclei

1. An improved type of ground glass homogenizer for soft tissues has been described which brings about a high degree of cell disruption and liberation of nuclei without causing appreciable damage to mitochondria. The gentleness and effectiveness of the new homogenizer in respect to isolation of mitochondria have been ascertained by comparing the ATP-ase activities of mitochondria isolated in 0.25 M sucrose solution without pH adjustment using a previous type of homogenizer with those of mitochondria isolated under the same conditions with the aid of the new homogenizer. In these experiments sucrose of 0.25 molarity without pH adjustment has been used in order to maintain the mitochondria in a rather sensitive state so as to make slightly deleterious effects of homogenization readily apparent. 2. A new method is described for the isolation of morphologically intact mitochondria and cell nuclei from the same homogenate. In this procedure the pH of the homogenate in 0.44 M sucrose is maintained at 6.0-6.2 with citric acid during the homogenization. An alternative method employing 0.44 M sucrose plus 0.005 M CaCl(2) is given for the isolation of nuclei from tumor cells. However, the latter method does not produce unaltered mitochondria. 3. The alpha-ketoglutarate, malate, succinate, and hexanoate oxidases of the "intact" mitochondria isolated in 0.44 M sucrose adjusted to pH 6.0-6.2 with very dilute citric acid as described in this paper have been investigated, and it has been shown that the mitochondria compare favorably to those isolated in 0.25 M sucrose by a previously described method. 4. Mitochondria have been found to contain an enzyme which causes nuclei to lose their ability to form gels in dilute alkali. This enzyme is released from the mitochondria when the latter are disrupted. 5. Some properties of nuclei isolated by the new method have been briefly discussed.     

Interaction of creatine kinase and hexokinase with the mitochondrial membranes,and self-association of creatine kinase: crosslinking studies

Summary Covalent coupling of protein by crosslinking reagents have been used to study the interaction of mitochondrial creatine kinase (CKm) and hexokinase (HK) with the mitochondrial membranes.The effects of crosslinkers were studied either by following the inhibition of solubilization of enzymatic activities or by modification of the electrophoretic patterns of proteins solubilized from mitochondria after treatment with different crosslinkers.Dimethylsuberimidate (DMS) efficiently reduced the amount of HK activity solubilized by various agents but it did not modify solubilization of CKm from mitochondria. The effect of DMS on HK solubilization did not result from non specific crosslinking since it did not impede the solubilization of adenylate kinase.Bissuccinimidyl another class of crosslinker has been tested. Ethyleneglycol bis (succinimidyl succinate)(EGS) efficiently reduced HK solubilization, but in addition it induced osmotic stabilization of mitochondria and thus impeded release of soluble or solubilized proteins from the intermembrane space. Furthermore this agent drastically inhibited CKm activity and thus, in a second set of experiments the effect of crosslinkers have been studied by the disappearance of protein bands in the electrophoretic pattern of soluble fractions obtained from mitochondria, the outer membranes of which have been ruptured to allow free release of soluble proteins. Results of these experiments showed that succinimidyl reagents and Cu⁺⁺-Phenanthroline substantially reduced the amount of CKm released from mitochondria and confirmed that bisimidates were ineffective in inhibiting CKm solubilization.In addition crosslinking reagents have been used to study subunits interactions in purified CKm. Our results showed, in contrast with control experiments with a non oligomeric protein (ovalbumin) which did not give rise to polymers, that in the same conditions electrophoresis of crosslinked CKm resolved a set of species with molecular weights roughly equal to integral multiples of the protomer. These results proved that the polymeric form of CKm was an octamer.Abbreviations AK Adenylate Kinase (EC 2.7.4.3) - CKm Mitochondrial Isoenzyme of Creatine Kinase (EC 2.7.3.2) - DMS Dimethyl Suberimidate - DTT Dithiothreitol - EGS Ethylene Glycol bis (succinimidyl succinate) - EGTA Ethylene Glycol bis (aminoethyl ether) - N,N,N,N Tetraacetic acid - G6P Glucose 6 Phosphate, Hepes - N-2 Hydroxyethyl Piperazine N-2 Ethane Sulfonic Acid - HK Hexokinase (EC 2.7.1.1) - MABI methyl 4-Azido Benzoimidate - NaPi Sodium Phosphate - SANPAH N-Succinimidyl 6(4 azido 2 nitrophenylamino) Hexanoate - SDS Sodium Dodecyl Sulfate (sodium lauryl sulfate) - Tris Tris (hydroxymethyl) Aminomethane     

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.     

Studies on ultrastructure and purification of isolated plant mitochondria

Sweetpotato mitochondria, that showed respiratory control, were studied with respect to ultrastructure. If fixed in media containing sucrose at 0.4 M, the cristae were dilated and the matrix was highly condensed. A more orthodox ultrastructural form was observed when the mitochondria were fixed in a medium containing sucrose at 0.25 M, i.e., the matrix was more expanded, the cristae were less dilated, and peripherally, the inner membrane element lay adjacent to the outer membrane element. These results are discussed in terms of a sucrose-accessible space (space between outer and inner membrane elements including intracristal space), and a space relatively inaccessible to sucrose (matrix). Ultrastructural shifts were not observed with change in metabolic steady state of the mitochondria. High resolution electron micrographs showed that the ultrastructure of sweetpotato mitochondria is very similar to that of animal mitochondria.

Purity and homogeneity of mitochondrial fractions were followed both by phase-contrast and electron microscopy. Preparations from sweetpotato, using older methods, were relatively homogeneous with respect to particle type and size, whereas avocado preparations contained a high proportion of chloroplasts and cellular debris. A method of purification involving sucrose-density-gradient centrifugation was developed. Purified mitochondria exhibited respiratory control and appeared similar to unpurified mitochondria under the electron microscope.

     

Deterioration of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium.

We have investigated the effect of the centrifugation speed on the behavior of rat-liver mitochondria during isopycnic centrifugation in an isoosmotic medium. The gradient was made with a macromolecular compound, glycogen dissolved in 0.25 M aqueous sucrose. The distribution curves of several mitochondrial enzymes change when the centrifugation reaches a certain speed: they are shifted toward regions of lower density. The results are plausibly explained by supposing that the inner mitochondrial membrane becomes permeable to sucrose at high centrifugation speeds, and that the granules swell. The main causal agent of the phenomenon is the hydrostatic pressure the mitochondria are subjected to during centrifugation. Morphological observations show that mitochondria are markedly deteriorated when centrifuged at high speed in the glycogen gradient; they are swollen and the outer membrane is broken; also frequently, a large electron-dense granule is seen in the matrix near the inner mambrane.     

Kinetic properties of the octameric and dimeric forms of mitochondrial creatine kinase and physiological role of the enzyme

It has been found that at pH 7.4 and 2 mg/ml protein, bovine heart mitochondrial creatine kinase (CKm) contains less than 10% of the dimer. The constant for the CKm octamer dissociation into dimers, Kd, in the presence of substrates forming an analog of the complex of the transient state was found to be equal to 4.9 10(-17) M3. Using this value, the experimental conditions were chosen so as to achieve a practically complete dissociation of the octamer into dimers. Evidence has been obtained suggesting that the octamer does not dissociate into dimers during the time course of the kinetic experiments; the corresponding kinetic parameters of the CKm octamer and dimer are as follows: KMgATPm = 82 microM and 42 microM; KCrm = 8.1 mM and 3.4 mM; Vf = 61 and 60 micrograms-equiv. H+ min-1 mg-1; KMgADPm = 43 microM and 17 microM, KCPm = 0.68 mM and 0.23 mM; Vr = 162 and 111 micrograms-equiv. H+ min-1 mg-1. The experimental and calculated data shed additional light on the physiological role of CKm.     

Integrity and Respiration of Red Beet Mitochondria Isolated and Examined in Different Sucrose Concentrations

Respiration experiments with succinate as substrate were made with red beet mitochondria isolated in soluitions containing 0.25 to 1.25 M sucrose. The respiration was measured in reaction media adjusted to be 0.25, 0.50, 0.75 or 1.0 osmolar. With mitochondria isolated in 0.25 or 0.50 M sucrose the rate of succinate oxidation was completely dependent on the osmotic pressure of the reaction medium (decreasing with increasing osmotic pressures). Isolation in 0.75 M sucrose caused a slight after-effect of the osmotic pressure of the isolation medium, and by isolation in 1.0 M or 1.25, M sucrose the after-effect was complete. The rate of oxidation was low and independent of the osmotic pressure of the reaction medium. An electron microscopic examination of the state of the mitochondria before and after the respiration period showed that with the conditions used in the present experiments the structure of the mitochondria remained well preserved regardless of the osmotic pressures used.     

Evidence against direct transfer of the adenine nucleotides by the heart mitochondrial creatine kinase-adenine nucleotide translocase complex

Adenine nucleotide translocase inhibitors, atractyloside and carboxyatractyloside, added to respiration inhibited rat heart mitochondria had no significant effects on the apparent Km's for ATP and ADP and Vmax's of the creatine kinase reaction. The results suggest that there is no sequential, mandatory, direct transfer of the nucleotides between the translocase and the creatine kinase active site. The reaction was run in both the forward and reverse directions in media containing either 0.25 M sucrose or 0.13 M KCl. The apparent Km's, however, were found to be 2–3 times higher in the KCl medium than in sucrose, suggesting the use of the more physiological medium for meaningful kinetic studies.     

Creatine kinase of heart mitochondria. The progressive loss of enzyme activity during in vivo ischemia and its correlation to depressed myocardial function

It is now appreciated that mitochondrial creatine kinase (CKm) may play an important role in heart high-energy phosphate metabolism and that this isozyme is solubilized in vitro by dilute solutions of Pi. Since an increase in cellular Pi is known to occur with even brief periods of myocardial ischemia, we investigated the relationship between CKm activity and myocardial performance in rabbit hearts subjected to total global ischemia. CKm activity is expressed as a ratio to mitochondrial malate dehydrogenase (MDHm), a stable marker enzyme. A significant decline in this ratio was observed after only 10 min of ischemia, a time prior to changes in total homogenate creatine kinase activity. After 60 min of ischemia, the CKm/MDHm ratio was depressed by more than 70%. Since there was no restoration of activity following 30 min of reperfusion, we correlated changes in enzyme activity to contractile dysfunction following variable periods of total ischemia. The data showed a close correlation between the decline in the CKm/MDHm ratio and the reduction in performance, measured as left ventricular developed pressure. No correlation was observed between State 3 respiratory rates and performance. Using KCl arrest at 27 degrees C or hyperthermic ischemia at 40 degrees C, the CKm/MDHm ratio consistently correlated to the degree of postischemic functional depression, independent of the duration of ischemia. Isoenzyme electrophoresis failed to detect soluble CKm activity in the postischemic supernatant. Therefore, CKm activity appears to be altered rapidly and irreversibly by ischemia. The implications of these observations on the integration of myocardial high-energy phosphate metabolism are discussed.     

Production of hypotaurine from l-cysteinesulfinate by rat liver mitochondria

Hypotaurine is the precursor of taurine production from L-cysteinesulfinate. It is recognized that hypotaurine production in the liver occurs in cytosol. In the present study, hypotaurine production from L-cysteinesulfinate in rat liver mitochondria was investigated. The mitochondrial preparation prepared according to the method of Hogeboom and washed repeatedly with 0.25 M sucrose solution was incubated with L-cysteinesulfinate. Products were derivatized with dabsyl chloride and dabsylated amino acids were analyzed by RP-HPLC. Presence of a peak corresponding to dabsyl-hypotaurine was confirmed. The peak of dabsyl-hypotaurine was converted quantitatively to dabsyl-taurine by the treatment with H(2)O(2). Optimum pH of the reaction was shown to be broad between 6.0 and 7.8 and Km for L-cysteinesulfinate was 0.11 mM. Results indicate the presence of L-cysteinesulfinate decarboxylase activity in liver mitochondria. Mitochondrial cysteine metabolism was summarized and possible antioxidant roles of cysteine metabolites including hypotaurine in mitochondria are discussed.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla.

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules (SH (average sedimentation coefficient) = 12 400 S in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin (greater than or approximately 95% of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium. The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Ion Transport in Mitochondria in the Context of Osmotic Concentration of the Buffer

Studies on mitochondria has revealed apparent violation of the laws of osmosis, probably related to ion transport. In this context, we studied changes in the content of potassium, sodium, magnesium, and calcium in mitochondria from wheat (Triticum aestivumL.) roots incubated in the buffer without the substrate that contained 0.3 or 0.5 M sucrose as well as potassium, and phosphate. The most pronounced changes were observed for potassium. Potassium inflow into mitochondria was more active at 0.5 than 0.3 M sucrose. The direction of the cation transport depended on its content in the initial buffer. At the certain threshold level, the inflow was replaced with the outflow; this threshold was lower at 0.3 than 0.5 M sucrose. Magnesium was also released from mitochondria; its stable outflow was primarily observed at its lower initial content in the suspension at 0.3 rather than 0.5 M sucrose. These data showed that the apparent violation of laws of osmosis was false and resulted from ion transport. Passive ion transport as a possible mechanism of adaptation to osmotic properties of the external medium is discussed.     

Effects of different sucrose concentrations on vitrified porcine preantral follicles: Qualitative and quantitative analysis

The aim of the present study was to perform a qualitative and quantitative analysis of the effect of different sucrose concentrations combined with ethylene glycol in the preservation of vitrified porcine preantral follicles. Fragments of ovarian cortex were vitrified in cryotubes containing 200 μl of the vitrification solution (30% Ethylene Glycol; 20% Fetal Bovine Serum; 0 M–0.25 M – 0.75 M or 1 M sucrose) and stored in liquid nitrogen for a week. Histological analysis showed that after vitrification the number of normal follicles decreased compared to the fresh tissue (control). The percentage of normal primordial follicles was sucrose dose dependent. The percentage of normal primary follicles was similar in 0 M or 0.25 M sucrose, while higher concentrations (0.75 M and 1 M) increased significantly the percentage of abnormal follicles (p < 0.05). Morphometric analysis showed a statistically significant reduction in the total area of primordial follicles with 0.75 M sucrose and a significant increase in the cytoplasmic area of primordial follicles with 0 M sucrose (p < 0.05). The qualitative and the quantitative analysis appear to be a complementary tool when choosing a vitrification protocol. For our cryopreservation system - vitrification of ovarian cortex slices in cryotubes-the best vitrification medium was TCM 199-Hepes with 30% de ethylene glycol, 20% of Fetal Bovine Serum and 0 or 0.25 M sucrose. The present study shows that the use of high sucrose concentrations in the vitrification solution has a deleterious effect on the preservation of porcine preantral follicles contained in ovarian tissue. Consequently, its use at 0.75 M or 1 M wouldn't be recommended.     

Oxidation of Various Substrates by Mitochondria as Related to the Concentration of Osmotics in the Medium

Wheat (Triticum aestivumL.) root mitochondria were used to investigate the effect of solute concentration in the medium on the rates of succinate, malate, -ketoglutarate, and glutamate oxidation; the corresponding values of the apparent Michaelis constant (K _M); and the changes in the organelle volumes. The oxidation rates and the K _M values for all substrates were lower in the 0.5 M sucrose solution than in the 0.3 M solution. Under high osmotic concentration, mitochondria did not shrink. On the contrary, in the absence of the substrate, mitochondria swelled more in the 0.5 M sucrose solution than in the 0.3 M solution. This effect was absent when the substrate was added. The authors conclude that the decrease in the oxidation rate imposed by sucrose was not related to the hindered influx of substrates caused by matrix contraction. Rather, the osmotic effect of sucrose is related to the changes in the structure of mitochondrial enzyme complexes.