Gabriella Sandri

Isolation of a soluble Ca2+ binding glycoprotein from ox liver mitochondria

Abstract A soluble glycoprotein has been extracted from ox liver mitochondria and purified to a very considerable degree by polyacrylamide gel electrophoresis. Its protein moiety has a molecular weight of about 42,000. It contains about 5 per cent total carbohydrates, including some sialic acid, and up to 30 per cent phospholipids. It binds Ca 2+ to 2 classes of sites having different affinity. The binding of Ca 2+ is sensitive to some of the inhibitors of Ca 2+ binding and transport in mitochondria, among them La 3+ and ruthenium red.

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+.

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.

Hydrogen peroxide production by monoamine oxidase in isolated rat-brain mitochondria: its effect on glutathione levels and Ca2+ efflux

H2O2 production and accumulation during incubation of isolated rat-brain mitochondria with substrates of monoamine oxidase A and B were investigated. All substrates gave rise to an accumulation of H2O2 which was inhibited by malate + pyruvate or isocitrate, consistent with a need for mitochondrial NADPH to maintain glutathione in the reduced state. However, in the absence of these additions the level of reduced glutathione decreased only by about 30%, indicating that only a fraction of the mitochondrial glutathione pool was accessible to the glutathione peroxidase and glutathione reductase activities responsible for the continuous removal of H2O2 generated by monoamine oxidase. The H2O2 accumulation was also inhibited by externally added reduced glutathione or NADPH but not NADH. External NADPH was oxidized by added oxidized glutathione but not alpha-ketoglutarate + NH4+. These results suggest that the removal of H2O2 generated by monoamine oxidase proceeds by way of special fractions of glutathione peroxidase and glutathione reductase that are located in the intermembrane space of mitochondria in such a way that they can react with both intra- and extra-mitochondrial glutathione and NADPH, possibly at the contact sites between the inner and outer mitochondrial membranes. Evidence is also presented that H2O2 generated by monoamine oxidase enhances Ca2+ release from mitochondria and may thus function as a regulator of mitochondrial Ca2+ efflux.

Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.

Distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat testis mitochondria

The distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat testis mitochondria was investigated, using a reverse sucrose density gradient centrifugation procedure for the separation of the inner and outer membranes and the contact sites between the two membranes. The results indicate that PHGPx is largely localized in the contact sites fraction. This finding might therefore suggest that the enzyme has more than just an antioxidant function.

Reconstitution in vitro of sulfobromophthalein transport by bilitranslocase

Liposomes containing 150 mM KCl and 0.48 mM sulfobromophthalein have been prepared. The internal pH was set at 6.5, a value at which sulfobromopthalein is colorless. When brought to alkaline pH a certain amount of the dye is deprotonated and can be read spectrophotometrically as external sulfobromophthalein. Upon addition of Triton X-100 the membrane is dissolved and all sulfobromophthalein present in the preparation may be measured. Addition of bilitranslocase to such a preparation of liposomes causes the internal sulfobromophthalein to leave the internal compartment. The rate of this phenomenon may be followed directly and shown to be greatly accelerated by the addition of valinomycin. The latter finding indicates that sulfobromophthalein transport occurs in response to a membrane diffusion potential created by permeabilisation to K+ of liposomes brought about by valinomycin (uniport). The permeability change induced by bilitranslocase is specific and does not reflect an alteration of the normal impermeability of liposomes to small ions such as protons or Ca2+.

A glycoprotein located in the intermembrane space of rat liver mitochondria

Recently much has been focused on the glycoprotein content of biological membranes and in particular of mitochondria [ I1 1 ] . In a previous paper on this subject [ 121, we have reported the distribution of glycoprotein components between inner and outer mitochondrial membranes. An interesting observation in that paper was that a large portion of sialic acid and hexosamine-containing material was released during the separation of the two membranes. No information was however available about the original intramitochondrial location of the released glycoprotein. We found therefore of interest to compare the supernatant obtained from mitochondria upon swelling and contraction with that obtained by sonication from pre-swollen and contracted mitochondria. In this paper we report an electrophoretic study of the proteins released in the soluble fraction by different procedures. A technique is also described for the isolation of the glycoprotein, released by swelling, in an electrophoretically homogenous form.

The calcium-binding glycoprotein and mitochondrial calcium movements

Abstract The calcium-binding glycoprotein isolated from mitochondria can be shown to move from one mitochondrial compartment to another as a function of calcium and magnesium presence as well as calcium transport. The movement is reversible in vitro and the possibility is therefore considered that the glycoprotein may behave as a mobile calcium-carrier. In the presence of acetate and phosphate, calcium-pre-loaded mitochondria release the cation upon addition of uncoupling concentrations of pentachlorophenol. The rate of calcium efflux can be modulated either by changing pentachlorophenol or phosphate concentrations. Simultaneously a release of calcium-binding glycoprotein can be detected and a negative linear relation has been found between amount of glycoprotein released and rate of calcium passive efflux. The data are interpreted to indicate that calcium efflux occurs only when the glycoprotein is bound to the mitochondrial membranes.

Differential Splicing of the Phospholipid Hydroperoxide Glutathione Peroxidase Gene in Diploid and Haploid Male Germ Cells in the Rat

Abstract Phospholipid hydroperoxide glutathione peroxidase (PHGPx, 20 kDa) and sperm nuclei glutathione peroxidase (snGPx, 34 kDa) are two selenoproteins present in mammalian testis and epididymal spermatozoa. They originate from the differential splicing of the PHGPx gene and appear to play important roles in sperm physiology. To determine the stages of spermatogenesis in which they are present, we compared the expression pattern of these two enzymes in highly purified populations of germ cells during specific phases of differentiation. In Northern and Western blotting experiments, both PHGPx transcript and protein were markedly expressed in pachytene spermatocytes and round spermatids. In contrast, the testis-specific snGPx was detected at both the mRNA and protein level only in haploid round spermatids. Accordingly, the developmental analysis of testicular RNAs from rats of different ages first revealed the appearance of PHGPx and snGPx transcripts at Day 20 and Day 30, respectively. Furthermore, both meiotic and postmeiotic cells contained catalytically active PHGPx/snGPx, with higher activity in the haploid cells. The intracellular distribution of PHGPx in mitochondria and nuclei of meiotic cells was demonstrated by immunocytochemical electron microscopy and Western blotting. These findings provide evidence that the PHGPx gene is differentially spliced during the meiotic prophase and haploid cell phases of spermatogenesis.