Isolation and characterization of a laminin-binding protein from rat and chick muscle

A major laminin-binding protein (LBP), distinct from previously described LBPs, has been isolated from chick and rat skeletal muscle (Mr 56,000 and 66,000, respectively). The purified LBPs from the two species were shown to be related antigenically and to have similar NH2-terminal amino acid sequences and total amino acid compositions. Protein blots using laminin and laminin fragments provided evidence that this LBP interacts with the major heparin-binding domain, E3, of laminin. Studies on the association of this LBP with muscle membrane fractions and reconstituted lipid vesicles indicate that this protein can interact with lipid bilayers and has properties of a peripheral, not an integral membrane protein. These properties are consistent with its amino acid sequence, determined from cDNAs (Clegg et al., 1988). Examination by light and electron microscopy of the LBP antigen distribution in skeletal muscle indicated that the protein is localized primarily extracellularly, near the extracellular matrix and myotube plasmalemma. While a form of this LBP has been identified in heart muscle, it is present at low or undetectable levels in other tissues examined by immunocytochemistry indicating that it is probably a muscle-specific protein. As this protein is localized extracellularly and can bind to both membranes and laminin, it may mediate myotube interactions with the extracellular matrix.     

Isolation and characterization of smooth muscle cell membranes

A method for the isolation of guinea pig ileum smooth muscle cell membranes is described. The plasma membrane fraction possessed a (Na⁺, K⁺)-ATPase which was inhibitied by ouabain. The Mg²⁺-dependent ATPase of the membrane fraction was stimulated by 1 μM Ca²⁺. A basal ATPase, not dependent on Mg²⁺, was directly stimulated by Ca²⁺ in the range of 1 μM to 1 mM.The isolated membranes contracted in response to the following substances: ATP, angiotensin II and some of its analogs, bradykinin, acetylcholine and histamine. The contractility was inhibited by ouabain and chlorambucil-angiotensin II, but not by cytochalasin B. No contraction was produced by AMP, angiotensin I and adrenaline.     

Isolation of sarcolemmal membranes from cardiac muscle

• 1.1. Sarcolemmal membranes from cardiac tissue were isolated to a high degree of purity by brief extraction of homogenate with KC1 followed by 2 successive discontinuous sucrose density gradients of extracted particles.
• 2.2. Sarcolemmal membranes contained 40% adenylate cyclase, 22% guanylate cyclase and 20% ouabain-sensitive (Na⁺−K⁺) ATPase: contamination of sarcolemma by other intracellular membranes was negligible.
• 3.3. Electron microscopic examination of membranes showed presence of relatively empty vesicles of various shapes and sizes while electrophoretic analysis revealed about 20 protein bands of which 6 were prominent.

     

Isolation of an anorexigenic protein from membranes

Satiety means an internal state that leads to termination of eating. We have isolated an anorexigenic glycoprotein (MW 50,000 dalton) from human and rat erythrocyte membrane and from rat liver plasma membrane. The substance isolated from all these membrane sources has almost same onset and offset anorectic effect in rats deprived of food for 96 h as well as in normally fed rats without any rebound. Similar properties of membrane anorectic substance and plasma satietin indicated that it has membrane origin. The results also suggest that the loss of appetite in chronic diseases involving damage or turn-over of cell membranes could be due to release of a glycopeptide from cellular membranes into the circulation.     

Isolation of cell membranes from Saccharomyces cerevisiae for evaluation of the protein composition

We describe a simple method for the isolation of membrane fractions from Saccharomyces cerevisiae yeasts, containing the complex of plasma membranes and cell walls. The method is based on cell disruption on an INBI flow disintegrator. This device spares subcellular structures, which simplifies the isolation of cell membranes. The membrane fraction obtained by this method was suitable for studies of protein composition of these structures by means of two-dimensional electrophoresis.     

Isolation and characterization of plasma membranes from rabbit skeletal muscle

An improved procedure was developed for the isolation of skeletal muscle plasma membranes. This method includes a DNAse treatment of the homogenate prior to the isolation of membranes by differential and sucrose gradient centrifugation techniques. We obtained two light fractions which were highly enriched in many biochemical and chemical plasma membrane markers. These fractions were shown to be mostly inside-out vesicles containing a Ca2+-ATPase activity. These results suggested that this enzyme could participate in the extrusion of calcium ions from the muscle cells.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F-actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.     

Isolation and characterization of cell membranes

The steps outlined above should provide a framework for developing and evaluating a scheme for isolating a particular membrane system of interest. Further characterization often focuses on the specific research interests of an investigator. Most subsequent techniques will probably involve biochemical analyses of protein and lipid components and biophysical analyses of membrane properties. The techniques to be used, such as SDS gel electrophoresis, enzyme kinetics, receptor binding, protein purification, etc. are not unique to membrane systems and will not be described in detail. However, it is likely that the functional relevance and success of subsequent analyses will be aided if the starting membranes are as homogeneous and well-characterized as possible. Even if some of the fractionation steps are bypassed, such as might occur if a specific membrane protein is detergent-extracted, purified, and reconstituted directly from a crude homogenate, a full understanding of membrane protein function will probably require complementary studies on intact cells and isolated membranes.     

Mapping the laminin-binding and adhesive domain of the cell surface-associated Hlp/LBP protein from Mycobacterium leprae

Binding of Mycobacterium leprae to and invasion of Schwann cells (SC) represent a crucial step that initiates nerve damage in leprosy. We and others have described that M. leprae colonization of the peripheral nerve system may be mediated in part by a surface-exposed histone-like protein (Hlp), characterized as a laminin-binding protein (LBP). Hlp/LBP has also been shown to play a role in the binding of mycobacteria to alveolar epithelial cells and macrophages. In the present study we report that M. leprae expresses Hlp/LBP protein during the course of human infection. Additionally, we analyzed the interaction of Hlp/LBP with the extracellular matrix and host cell surface. We show that Hlp/LBP, besides laminin, also binds heparin and heparan sulfate. Testing truncated recombinant Hlp molecules corresponding to the N-terminal (rHlp-N) and the C-terminal (rHlp-C) domains of the protein, we established that interaction of Hlp/LBP with laminin-2 and heparin is mainly mediated by the C-terminal domain of the protein. Moreover, the same domain was found to be involved in Hlp/LBP-mediating bacterial binding to human SC. Finally, evidence is shown suggesting that M. leprae produces a post-translationally modified Hlp/LBP containing methyllysine residues. Methylation of the lysine residues, however, seems not to affect the adhesive properties of Hlp/LBP. Taken together, our observations reinforce the involvement of Hlp/LBP as an adhesin in mycobacterial infections and define its highly positive C-terminal region as the major adhesive domain of this protein.     

Isolation of a Zn-binding protein mediating cell adhesion from common carp

Extraordinarily high concentrations of Zn (300-500 microg/[g fresh tissue]) are often found in the digestive tract tissue of common carp Cyprinus carpio, and most of the Zn is bound to membrane protein located on plasma membranes that are attached to basal laminae. To isolate the Zn-binding protein, the basolateral plasma membranes were separated from the extracellular matrix by treating the nuclei/cell debris fraction of the tissue with collagenase type IV and Arg-Gly-Asp (RGD) peptide. The Zn-binding protein was isolated from the separated plasma membranes by immobilized metal affinity chromatography and affinity chromatography on laminin-Sepharose. A 43 kDa protein was bound by the laminin-Sepharose and specifically eluted with tirofiban (a mimic of RGD). Affinity chromatography on wheat germ agglutinin and concanavalin A-Sepharose showed that the 43 kDa protein is a glycoprotein. The 43 kDa protein was labelled with 65Zn and became incorporated into liposomes at a high efficiency. Liposomes containing this protein were bound to laminin-Sepharose or reconstituted basement membrane. We propose that the Zn-binding protein is a cell surface receptor involved in the adhesion of cells to laminin.     

Isolation and characterization of a new 40-kilodalton protein from bovine cardiac muscle

A new protein having a subunit weight of 40,000 has been purified from myosin-extracted bovine cardiac myofibrils. Its amino acid composition and isoelectric point are distinct from actin, eu-actinin, and a variety of sarcoplasmic proteins of similar size. Affinity-purified antibodies made to this protein only react with a single 40-kDa protein band from cardiac myofibrils on immunoblots. The anti-40-kDa protein also shows cross-reactivities with cardiac myofibrils from rabbits, rats, and chickens. Immunofluorescence studies demonstrate that the 40-kDa protein is localized at the Z-bands of cardiac myofibrils and at the intercalated discs. The antibody did not react with skeletal muscle myofibrils by immunofluorescence or immunoblotting. It appears that the 40-kDa protein may play a role in the strong attachments between adjacent myofibrils in cardiac muscle.