G.L. Sottocasa

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.

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.

Further studies on bilitranslocase, a plasma membrane protein involved in hepatic organic anion uptake

Bilitranslocase, a plasma membrane protein involved in bilirubin and other organic anion uptake by the liver, exhibits a high molecular weight (170 000) when isolated in the presence of deoxycholate. This value is decreased to approx. 100 000 if deoxycholate is not included in the isolation medium. Both preparations can be resolved into two kinds of subunit, alpha and beta , of 37 000 and 35 500, respectively, by reduction with 2-mercaptoethanol and addition of sodium dodecyl sulfate. Under these conditions the two subunits are still capable of high-affinity sulfobromophthalein binding and, despite the presence of the detergent, may be isolated by preparative polyacrylamide gel electrophoresis still associated with the dye. It may be suggested that the physiological subunit composition of bilitranslocase is alpha 2-beta.

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.

Lysosomes in heart tissue

Abstract 1. 1. The acid hydrolase (β-glucuronidase, EC 3.2.1.31; cathepsin; β-galactosidase, EC 3.2.1.23; acid ribonuclease, EC 2.7.7.17 and acid deoxyribonuclease, EC 3.1.4.6) of beef-heart muscle are associated with cytoplasmic particles and are poorly reactive towards external substrates; however, their activity is enhanced or even fully displayed by injuring the particles with a variety of treatments (osmotic shock, prolonged homogenization, alternate freezing and thawing, addition of Triton X-100). 2. 2. By gradually increasing the concentration of Triton X-100 in the enzyme assays, the acid hydrolases are gradually liberated from the particles, although not in equal proportions. 3. 3. When particles in suspension are submitted to the action of various “protein” or “lipid” reagents, it appears that both types of reagent are able to increase the availability of the hydrolases for their substrates, but that a high solubilization of the enzymes is achieved only by attacking the membrane phospholipids. 4. 4. Preincubation either in hypo- or hypertonic sucrose solutions produces an increase in the availability of the hydrolases; under the same conditions, a partial solubilization, albeit to a different extent for each enzyme, is also observed. 5. 5. Each of the enzymes considered displays a peculiar intracellular distribution pattern, but none of them is strictly the same as that of succinoxidase. By isopicnic centrifugation of a mitochondrial fraction it is possible to isolate a particulate fraction ( d = 1.174) showing a high concentration of latent hydrolytic enzymes and a comparatively low concentration of cytochrome c oxidase. 6. 6. The acid hydrolases of beef heart do not seem, therefore, to belong to mitochondria but to exhibit the main features of lysosomal enzymes. A comparison between the properties of beef-heart and rat-liver lysosomes is also made and the heterogeneity of the former is discussed.

Glycoprotein components, sialic acid and hexosamines, bound to inner and outer mitochondrial membranes.

Much attention has been focused in the past on protein and phospholipid constituents of biological membranes. More recently, however, carbohydratecontaining materials have become subject of interest as important components of plasma membrane. These macromolecules have been recognized as responsibte for a number of phenomena including contact-inhibition, transport and antigenic properties (for review see [ 11). Intracellular membrane structures have also been shown to possess a glycoprotein complement [2-81 and an active synthesis of such components has been reported to occur in vitro in isolated mitochondria and microsomes [9111. The availability of a technique for the separation of inner and outer membranes [ 121 from isolated mitochondri~i makes it possible to investigate the intramitochondrial distribution of glycoproteins. In this paper we report the distribution between mitochondrial membranes of two typical constituents of glycoproteins such as sialic acid and hexosamines.

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono- and poly-clonal antibodies

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane.

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria.

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca2+-binding glycoprotein and its Ca2+-transporting activity may be as low as 10--20% of the starting value. Both the rate of Ca2+ uptake and the capacity to maintain a high Ca2+ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca2+-transporting ability. The active component in the supernatant is the Ca2+-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 microM Mg2+, the glycoprotein alone may restore fully the Ca2+-transporting ability of the particles. The maximal velocity is already reached at 0.1 microgram glycoprotein/mg mitochondrial protein.

Measurement of sulfobromophthalein uptake in isolated rat hepatocytes by a direct spectrophotometric method

A spectrophotometric technique is described for the continuous recording of sulfobromophthalein uptake by isolated hepatocytes. The technique is based on the principle that sulfobromophthalein behaves as a pH-indicator and may be followed photometrically when moving from the medium at pH 7.8 into the interior of the cell. Data show that upon addition of cells to a sulfobromophthalein solution, an absorbance change can be recorded. The kinetics of the process is biphasic and the initial rate is linearly related to the amount of cells added. By this technique it was confirmed that the substrate dependence of the initial velocity of transport is a compound function including a saturable portion with an apparent Km in the mu molar region. Experiments carried out either in the presence of valinomycin or of high concentrations of potassium chloride indicate that the presence of a membrane potential opposes the entry of sulfobromophthalein into isolated hepatocytes. This finding is in agreement with previous observations in isolated plasma membrane vesicles and in liposomes reconstituted with purified bilitranslocase which indicate a rheogenic type of transport for the dye. Low concentrations of nicotinate (1.6 microM) efficiently inhibit the saturable transport. It is suggested, in addition, that the sensitivity of the transport to valinomycin could be used as an early indication of the functional integrity of cell preparations.

THE EFFECT OF X-IRRADIATION ON THE ACTIVITIES OF SOME LYSOSOMAL HYDROLASES OF HEART TISSUE.

The location of the initial lesions produced by ionizing radiations in living systems has been investigated by many radiobiologists. One result of radiation injury is the increase in enzymic activities frequently observed in irradiated organisms. Bacq and Alexander (1) suggested that this effect could be explained on the basis that radiations break down internal barriers by altering the cellular and intracellular membranes with consequent release of enzymes. Recently it has been shown (2) that membrane-bound organelles containing hydrolytic enzymes are found in the cytoplasm of cells in many tissues: these particles have been named lysosomes. According to the enzyme release theory, if an ionizing radiation produces damage to the intracellular membranes, and therefore also to the membranes of these lysosomes, a release of the lysosomal enzymes should be expected after irradiation. This hypothesis seems to be supported by some experimental evidence. An increase of acid DNase (deoxyribonucleate 3-nucleotidohydrolase, E.C. 3.1.4.6) (lysosomal enzyme) has been reported, for instance, a few hours after irradiation in the serum and in the urine of animals treated with X-rays (3, 4). Furthermore, this nuclease and the acid RNase (polyribonucleotide 2-oligonucleotide-transferase (cyclizing), E.C. 2.7.7.16), which is also a lysosomal enzyme, appear to be increased in the soluble fraction of homogenates of tissues derived from animals treated with X-rays (5, 6). Zeman et al. (7) have also demonstrated, by histochemical reactions, that, in mice brain irradiated with microbeams of accelerated deuterons, lysosomal enzymes such as acid phosphatase (o-phosphoric monoester phosphohydrolase, E.C. 3.1.3.2), B3-glucuronidase (3-D-glucuronide glucuronohydrolase, E.C. 3.2.1.31), and cathepsin c (E.C. 3.4.4.9) become evident. The positive histochemical reaction appears along the path of the ionizing particles.