The lectin binding sites on the membranes of the nuclear envelope, mitochondria and the cell surface of human lymphoblastoid cells

The membranes of the cell surface, the endoplasmic reticulum, outer and inner mitochondrial leaflet and nuclear envelope were isolated from three human lymphoblastoid cell lines. Membrane components were separated by dodecyl sulfate polyacrylamide gel electrophoresis and the gels incubated with the radioiodinated lectins from lentil, castor bean, scarlet runner bean, gorse seed and Roman snail. After gel slicing and counting, the molecular weights of the lectin binding sites were determined. About 20 glycoproteins were identified as constituents of the plasma membrane, a similar glycoprotein distribution was observed in the endoplasmic reticulum. The outer mitochondrial membrane contained some impurities from the plasma membrane, the inner mitochondrial membrane lacked specific lectin receptors. Two prominent glycoproteins with molecular weights of 70 000 and 60 000 were identified with the castor bean lectin in the nuclear envelope.     

SEPARATION OF MITOCHONDRIAL MEMBRANES OF NEUROSPORA CRASSA : II. Submitochondrial Localization of the Isoleucine-Valine Biosynthetic Pathway

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.     

Protein kinase activity at the inner membrane of mammalian mitochondria.

This paper reports on the discovery of a protein kinase activity associated with the inner membrane of mammalian mitochondria. The enzyme does not respond to addition of cyclic AMP or cyclic GMP and has a preference for whole histone as phosphate acceptor. Some standard assay systems for the cyclic nucleotide-dependent cytosol protein kinases would be unable to pick up this activity of the orthophosphate concentration is higher than 25 mM and the pH or the assay lower than pH 6.5. The enzyme described here has an apparent pH optimum of 8.5. Activity in liver mitochondria is not evident unless the mitochondria are disrupted by either sonication or freezing and thawing. Distribution of kinase activity in centrifugal fractions of both liver and heart mitochondrial sonicates was parallel to that of the two inner membrane marker enzymes succinic dehydrogenase and cytochrome oxidase and quite different from that of the matrix enzyme malic dehydrogenase. Experiments with preparations enriched in outer or inner membranes confirmed the contention that this enzyme is located on the inner membrane. Since disruption of the inner membrane by a freeze-thaw treatment (after the outer membrane had been disrupted by swelling in phosphate) was necessary for full expression of activity by this enzyme, the tentative conclusion was reached that substrate is accepted only from the matrix side of the inner membrane.     

Binding of Ricinus communis agglutinin to the mitochondrial inner membrane as an artifact during preparation.

Endosperm from Ricinus communis was homogenized in the presence of 3H-labelled Ricinus communis agglutinin, with or without addition of lactose. In preparations without the binding-specific sugar the subfraction containing the mitochondrial inner membrane contained sufficient labelled agglutinin to account for the agglutinin reported to be associated with this membrane.     

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.     

Localization of the lactose permease protein(s) in the E. coli envelope.

The amino acid double labeling technique was used to identify and localize membrane-bound lactose operon proteins in $\text{E.}\text{coli}$. Both the “M” protein, thought to be the $\text{y}$ gene product, and a polypeptide of MW ～15,000 appeared in the membrane following $\text{lac}$ operon induction. The amounts of these two proteins were approximately equal.The inner and outer membrane layers of the cell envelope were separated by sucrose density gradient centrifugation or by selective solubilization of inner membranes with the detergent Sarkosyl. When gentle lysis conditions were employed to prepare membrane vesicles, both $\text{lac}$ induced proteins fractionated with the inner membrane. However, the “M” protein was more easily randomized in the envelope structure by sonication than the 15,000 dalton component or an inner membrane marker enzyme.     

Properties and intramitochondrial localization of serine hydroxymethyltransferase in leaves of higher plants

The activity of serine hydroxymethyltransferase in mitochondria isolated from spinach leaves was absolutely dependent on tetrahydrofolate; pyridoxal phosphate has no effect on the activity. The stability of this activity in the isolated mitochondria was dependent on the presence of sulfhydryl compounds. It was apparently more stable at pH 7.0 to 7.5 than at higher pH even though the pH optimum of serine hydroxymethyltransferase was 8.5 for both the mitochondrial and cytoplasmic fractions. Distribution studies have indicated that serine hydroxymethyltransferase was predominantly located in the mitochondria. The activity of serine hydroxymethyltransferase was observed to be co-compartmented with glycine decarboxylation and malate dehydrogenase behind the mitochondrial inner membrane. This activity could be solubilized by KCl from osmotically ruptured mitochondrial membrane fractions but substantial activity (35 to 40%) was still retained with the membrane fractions at 0.3 m KCl. This suggests that the glycine decarboxylation-serine hydroxymethyltransferase complex may be closely bound to the internal surface of the mitochondrial inner membrane.The relationship of this integrated enzyme complex to CO(2) evolution and serine synthesis during photorespiration and the physiological role of the dicarboxylate shuttle were discussed.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase     

Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis

Abstract Highly purified preparations of inner, i.e. cytoplasmic and intracytoplasmic, membranes and outer membranes were isolated from Nitrobacter hamburgensis strain X14 by sucrose density-gradient centrifugation of cell-free extracts. The two membrane fractions differed markedly in morphology, density, and protein composition as determined by polyacrylamide gel electrophoresis. The inner membrane fraction was enriched in NADH oxidase and nitrite oxidase activity. It contained four major protein bands of apparent M _rs of 28 000, 32 000, 70 000, and 116000. The outer membrane fraction was characterized by the presence of 2-keto-3-deoxyoctonate and contained two major proteins of apparent M _rs of 13 000 and 50 000. There was no evidence for differences between cytoplasmic and intracytoplasmic membranes.     

Localization and distribution of Microccus lysodeikticus membrane ATPase determined by ferritin labeling

Antiserum to Ca²⁺-activated ATP phosphohydrolase (EC 3.6.1.3) isolated and purified from membranes of Micrococcus lysodeikicus was prepared in rabbits and guinea pigs. The γ-globulin fractions of these antisera reacted with and inhibited ATPase activity in isolated membranes but failed to absorb to intact protoplasts or purified mesosome fractions. ATPase activity was not detectable in the purified mesosomal preparations and trypsin treatment and sonication failed to release any activity. Ferritin conjugated to the γ-globulin fractions of the antiserum reacted with the ATPase particles on the membrane as visualized in negatively stained preparations examined in the electron microscope. Labeled membranes showed a distribution of ferritin very similar to the patterns observed for ATPase particles on untreated membranes. No significant labeling occurred when the ferritin conjugate was reacted with intact protoplasts or mesosome fractions. Thin sections of ferritin-labeled membranes established the asymmetric disposition of the ATPase, with the conjugate visible on only one side of the membrane. The results indicate that the ATPase protein occurs on the inner face of the membrane. All labeling experiments were verified immunologically. When ferritin-labeled membranes were subjected to the selective release procedure used in releasing the ATPase-like particles from the membranes, a complex of ferritin-conjugate associated with the ATPase particles was released. The selective release of ferritin-antibody-enzyme complexes from the membrane opens up a new way of studying the molecular architecture of cell membranes.     

Subcellular and submitochondrial localization of phospholipid-synthesizing enzymes in Saccharomyces cerevisiae.

Using highly enriched membrane preparations from lactate-grown Saccharomyces cerevisiae cells, the subcellular and submitochondrial location of eight enzymes involved in the biosynthesis of phospholipids was determined. Phosphatidylserine decarboxylase and phosphatidylglycerolphosphate synthase were localized exclusively in the inner mitochondrial membrane, while phosphatidylethanolamine methyltransferase activity was confined to microsomal fractions. The other five enzymes tested in this study were common both to the outer mitochondrial membrane and to microsomes. The transmembrane orientation of the mitochondrial enzymes was investigated by protease digestion of intact mitochondria and of outside-out sealed vesicles of the outer mitochondrial membrane. Glycerolphosphate acyltransferase, phosphatidylinositol synthase, and phosphatidylserine synthase were exposed at the cytosolic surface of the outer mitochondrial membrane. Cholinephosphotransferase was apparently located at the inner aspect or within the outer mitochondrial membrane. Phosphatidate cytidylyltransferase was localized in the endoplasmic reticulum, on the cytoplasmic side of the outer mitochondrial membrane, and in the inner mitochondrial membrane. Inner membrane activity of this enzyme constituted 80% of total mitochondrial activity; inactivation by trypsin digestion was observed only after preincubation of membranes with detergent (0.1% Triton X-100). Total activity of those enzymes that are common to mitochondria and the endoplasmic reticulum was about equally distributed between the two organelles. Data concerning susceptibility to various inhibitors, heat sensitivity, and the pH optima indicate that there is a close similarity of the mitochondrial and microsomal enzymes that catalyze the same reaction.     

Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane

Rat liver mitochondria were isolated by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation, as assessed by marker enzyme analysis, latency of cytochrome-c oxidase, respiratory control index and electron microscopy. Two different methods were compared for the separation of inner and outer membranes. In the swell-shrink-sonicate procedure glycerol was included resulting in the isolation of one outer membrane and two inner membrane fractions of high purity. Using digitonin a highly selective and gradual solubilization of the outer membrane could be accomplished. Analysis of the phospholipid composition of the intact mitochondria and all subfractions showed that the inner membrane was virtually devoid of phosphatidylinositol and -serine, while the outer membrane contained 23% of the total mitochondrial cardiolipin, which did not originate from inner membrane contamination and therefore is a true component of the outer membrane.     

Separation of outer and inner membranes of mitochondria from the brown adipose tissue of infant rats.

Digitonin treatment and the swelling-shrinkage-sonication procedure as used to separate mitochondria membranes were applied to mitochondria from the brown adipose tissue (BAT) of infant rats. Digitonin at a concentration of 0.15 mg/mg mitochondrial protein produced disruption of the outer membrane of BAT mitochondria and a complete release of adenylate kinase. However, fragments of the outer membrane remained firmly attached to the inner membrane-matrix particles (mitoplasts) and sedimented at 10 000 g, as indicated by the activity of monoamine oxidase in the pellet. Only at 0.5 mg digitonin/mg protein did outer membrane become almost entirely separated. Oxidation of external cytochrome c by mitoplasts was only 50% of the total cytochrome oxidase at 0.5 mg digitonin/mg protein, indicating an incomplete exposure of the inner membrane to the external medium. Ultrastructural studies revealed that a large proportion of mitoplasts retained the orthodox configuration under these conditions. Outer membrane fragments obtained by the swelling-shrinkage-sonication procedure were of buoyant density corresponding to 20–30% (weight/vol) sucrose. After a 10 sec sonication of mitochondria, a relatively pure outer membrane fraction could be obtained with a yield not exceeding 20%. Longer sonication increased the yield, but also increased the degree of contamination by inner membrane fragments. Optimum conditions for the separation of outer and inner membranes from brown adipose tissue mitochondria are described.     

Subcellular distribution of rat liver porin

The subcellular distribution of rat liver porin was investigated using the immunoblotting technique and monospecific antisera against the protein isolated from the outer membrane of rat liver mitochondria. Subfractionation of mitochondria into inner membranes, outer membranes and matrix fractions revealed the presence of porin only in the outer membranes. Porin was also not detected in highly purified subcellular fractions, including plasma membranes, nuclear membranes, Golgi I and Golgi II, microsomes and lysosomes. Thus, liver porin is located exclusively in the outer mitochondrial membrane.     

Contact sites from human placental mitochondria: characterization and role in progesterone synthesis

To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate-isocitrate dehydrogenase, porin, and adenosine 5(')-triphosphate-diphosphohydrolase activity showed the capacity to synthesize progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.     

Lactate dehydrogenase activity in the mitochondrial fraction of chicken liver: enzyme binding and kinetic behavior of soluble and bound enzyme

Chicken liver crude mitochondrial fraction showed lactate dehydrogenase activity (6.5% of cytoplasmic enzyme). Most of the mitochondrial lactate dehydrogenase was solubilized by sonication of the mitochondrial fraction in 0.15 M NaCl, pH 6. Total extracted lactate deshydrogenase activity was 3-fold higher than the initial pellet activity. Different isoenzymatic compositions were observed for cytosoluble and mitochondrial extracted lactate dehydrogenase. The pI, values of the 5 lactate dehydrogenase isoenzymes were found to be independent of their origin. The cytosoluble lactate dehydrogenase and the separated H4,H3M and H2M2 isoenzymes were able to bind to the chicken liver mitochondrial fraction in 5 mM sodium phosphate buffered medium, and could be solubilized afterwards with 0.15 M NaCl, pH 6. The enzyme bound to the mitochondrial fraction was less active than the soluble one. Particle saturation by the bound enzyme occurred with all mitochondrial fractions assayed. According to the Langmuir isotherm, the non-sonicated mitochondrial fractions contain a single type of binding sites for lactate dehydrogenase; in contrast, the sonicated mitochondrial fraction should contain different binding sites. Chicken liver crude or sonicated active mitochondrial fractions showed a hyperbolic behavior with respect to NADH and a non-hyperbolic one with respect to pyruvate. This mechanism is different from the bi-bi compulsory order mechanism of the soluble enzyme. With hydroxypyruvate as the substrate, the active mitochondrial fraction fit a sequential mechanism but lost the rapid-equilibrium characteristics of the soluble enzyme.     

Characterization of the iron-sulfur protein of the mitochondrial outer membrane partially purified from beef kidney cortex.

The iron-sulfur protein present in the mitochondrial outer membrane has been partially purified from beef kidney cortex mitochondria by means of selective solubilization followed by DEAE-cellulose chromatography. The EPR spectrum of the iron-sulfur protein with g-values at 2.01, 1.94 and 1.89 was well resolved up to 200 K which is unusual for an iron-sulfur protein. Analyses confirmed a center with two iron and two labile sulfur atoms in the protein. By measuring the effect of oxidation-reduction potential on the EPR signal amplitude, midpoint potentials at pH 7.2 were determined both for the purified iron-sulfur protein, +75 (+/- 5) mV, and in prepared mitochondrial outer membrane, +62 (+/- 6) mV. At pH 8.2 slightly lower values were indicated, +62 and 52 mV, respectively. The oxidation-reduction equilibrium involved a one electron transfer. A functional relationship to the rotenone-insensitive NADH-cytochrome c oxidoreductase in the mitochondrial outer membrane is suggested. Both this activity and the iron-sulfur center were sensitive to acidities slightly below pH 7 in contrast to the iron-sulfur centers of the inner membrane.     

The interaction of yeast citrate synthase with yeast mitochondrial inner membranes

The specific interaction of yeast citrate synthase with yeast mitochondrial inner membranes was characterized with respect to saturability of binding, pH optimum, effect of ionic strength, temperature response, and inhibition by oxalacetate. The binding ability of the inner membranes is inhibited by proteolysis and heat treatment, which implies that the membrane component(s) responsible for binding is a protein. A protein fraction from inner membranes when added to liposomes will bind citrate synthase. In addition, the binding of yeast fumarase, mitochondrial malate dehydrogenase, and cytosolic malate dehydrogenase to yeast inner membranes was examined. For these studies the yeast mitochondrial matrix enzymes, citrate synthase (from two types of yeast), malate dehydrogenase, and fumarase, as well as cytosolic malate dehydrogenase, were purified using rapid new techniques.     

The intracellular localization and properties of carnitine acetyltransferase from ram spermatozoa

Although spermatozoa possess a very active carnitine acetyltransferase, there is no satisfactory explanation for such a high activity. In order to help elucidate possible roles for carnitine acetyltransferase in spermatozoa, we examined the intracellular location and properties of carnitine acetyltransferase from ejaculated ram spermatozoa. The spermatozoa were disrupted by hypotonic treatment with 10 mm phosphate buffer (pH 7.4), followed by mild sonication. The resulting homogenate was separated by sucrose step-gradient centrifugation into soluble, plasma membrane, acrosomal membrane, and mitochondrial fractions. These fractions were characterized by electron microscopy and marker enzyme assays. The particulate fractions were made soluble by treatment with 0.1% deoxycholate and then were assayed for carnitine acetyltransferase activity. Carnitine acetyltransferase activity was found exclusively in the mitochondrial fraction with a specific activity of 0.151 μmol CoASH · min^?1 · mg^?1. The apparent K_m values for acetyl-CoA and l-carnitine were 1.1 × 10^?5 and 1.3 × 10^?4m respectively.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.