Fluorescence studies on the interaction of myoglobin with mitochondria

To determine the nature and characteristic parameters of the myoglobin-mitochondrion interaction during oxymyoglobin (MbO₂) deoxygenation in the cell, we studied the quenching of the intrinsic mitochondrial flavin and tryptophan fluorescence by different liganded myoglobins in the pH range of 6–8, as well as the quenching of the fluorescence of the membrane probes 1,8-ANS and merocyanine 540 (M 540) embedded into the mitochondrial membrane. Physiologically active MbO₂ and oxidized metmyoglobin (metMb), which are unable to bind oxygen, were used as the quenchers. The absence of quenching of flavin and tryptophan fluorescence implies that myoglobin does not form quenching complexes with either electron transport chain proteins of the inner mitochondrial membrane or with outer membrane proteins. We found, however, that MbO₂ and metMb effectively quench 1,8-ANS and M 540 fluorescence in the pH range of 6–8. Characteristic parameters of 1,8-ANS and M 540 fluorescence quenching by the myoglobins (extent of quenching and quencher binding constant, K _m) are very similar, indicating that both probes are localized in phospholipid sites of the mitochondrial membrane, and myoglobin is complexed with these sites. The dependence of K _m on ionic strength proves the important role of coulombic interactions in the formation of the quenching complex. Since the overall charge of myoglobin is shown not to influence the K _m values, the ionic strength dependence must be due to local electrostatic interactions in which polar groups of some part of the myoglobin molecule participate. The most likely candidates to interact with anionic groups of mitochondrial phospholipids are invariant lysine and arginine residues in the environment of the myoglobin heme cavity, which do not change their ionization state in the pH range investigated.     

Biochemical and ultrastructural properties of osmotically lysed rat-liver mitochondria

Isolated rat-liver mitochondria were osmotically lysed by suspension and washing 3 times in cold, distilled water. Pellets obtained by centrifugation at 105,000 g for 30 min were resuspended, fixed with glutaraldehyde and OsO₄, and embedded in Epon 812. Thin sections show the presence of two distinct membranous populations, each of which is relatively homogeneous in size and appearance. Swollen mitochondria (∼1.5 µ in diameter), which have been stripped of their outer membranes, are largely devoid of matrix and normal matrix granules and are referred to as "ghosts." The smaller (0.2 to 0.4 µ in diameter), empty appearing, vesicular elements, derived primarily from the outer mitochondrial membrane, can be differentiated from the ghosts on the basis of their smaller size and complete absence of internal structures, especially cristae. Each membranous element is enclosed by a single, continuous membrane; the "double membrane" organization typical of intact mitochondria is not observed. These findings indicate that the outer membrane of rat-liver mitochondria is spatially dissociated from the inner mitochondrial membrane by osmotic lysis of the mitochondria in distilled water. Three parameters of structural and functional significance in freshly isolated rat-liver mitochondria have been correlated with the structural alterations observed: (a) chemical composition (total protein, lipid phosphate and total phosphate), (b) specific and total activities of marker enzymes for mitochondrial matrix and membranes (malate dehydrogenase (MDH), D-β-hydroxybutyrate dehydrogenase (BDH) and cytochromes), and (c) integrated multienzyme functions (respiration, phosphorylation, and contraction). The data presented indicate that all mitochondrial membranes are completely conserved in the crude ghost preparation and that, in addition, about ⅓ of the matrix proteins (estimated by assays for MDH activity and protein) are retained. The study of integrated mitochondrial functions shows that a number of physiologically important multienzyme activities also are preserved in the water-washed preparation. The respiratory rate of ghosts per milligram of protein is 1.5 to 2.0 times that of intact mitochondria, which shows that the respiratory chain in the ghosts is functionally intact. The rate of phosphorylation is reduced, however, to about 25% of that measured in freshly isolated mitochondria and accounts for lowered P:O ratios using succinate as substrate (P:O ranges from 0.4 to 0.9). The phosphorylation of ADP to ATP is the only biochemical function, so far investigated, that is greatly affected by osmotic lysis. In addition, two lines of evidence suggest that the ghosts undergo an energy-dependent transformation resulting in contraction: (a) suspensions of the crude ghost preparation in 0.02 M Tris-0.125 M KCl medium show a marked increase in optical density upon the addition of ATP, and (b) ghost preparations incubated in ion-uptake medium in the absence of added calcium but in the presence of added ATP contain a large number of highly condensed ghosts (about 50% of the total profiles) when viewed as thin sections in the electron microscope. The correlated biochemical and morphological study presented here shows that the outer membrane of rat-liver mitochondria can be removed by controlled osmotic lysis without greatly impairing a number of integrated biochemical functions associated with the inner membrane.     

Tritrichomonas foetus: ultrastructural localization of basic proteins and carbohydrates

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver proteinate technique was used to localize carbohydrates. With the AS technique reaction product was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa, in the hydrogenosomes, and at the region of the recurrent flagellum's adhesion to the cell body. Previous acetylation of the cells under conditions that block most free amino groups abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex, and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA techniques to detect basic proteins is based on observations made with T. foetus as well as with other cell types.     

Biochemical studies on sub-fractions of rat liver mitochondria

Highly purified mitochondria from rat liver were separated into six sub-fractions by differential centrifugation. The sub-fractions represent a spectrum from “heavy” to “very light” mitochondria. Enzymes representative of mitochondrial compartments were assayed to see whether functional differences occurred among the various mitochondrial sub-fractions. Respiratory control and NADH oxidase activity, both of which are indicators of mitochondrial structural integrity, were also measured. An enzyme marker for endoplasmic reticulum (glucose-6-phosphatase, G-6-Pase) was also assayed. Specific activities for monoamine oxidase (outer membrane marker), cytochrome oxidase (inner membrane marker) and malate-cytochrome c reductase did not vary within experimental error in all sub-fractions; similarly, for respiratory control and NADH oxidase activity. Malate dehydrogenase, a component of malate-cytochrome c reductase is located within the matrix surrounded by the inner membrane. Specific activity of adenylate kinase (located between the outer and inner membrane) decreased markedly from the “heavy” mitochondria to the “very light” fractions. Specific activity for G-6-Pase, very low in the “heavy” fractions, increased markedly in the “light” to “very light” fractions. Isopycnic density centrifugation on a linear sucrose density gradient of each of the fractions indicated that the correlation coefficient for the sucrose concentrations at which cytochrome oxidase and G-6-Pase activities peaked was 0.995. Thus the “light” to “very light” mitochondria may represent mitochondria whose outer membrane is still contiguous with the endoplasmic reticulum. Microsomes containing the endoplasmic reticulum peaked on the gradient at a significantly lower sucrose concentration than any of the mitochondrial sub-fractions. A buoyant effect of endoplasmic reticulum still attached to any of the mitochondrial sub-fractions would be expected to lower the density of attached mitochondria and thus give rise to “light” and “very light” mitochondria.     

Cholesterol and the interaction of proteins with membrane domains

Cholesterol is not uniformly distributed in biological membranes. One of the factors influencing the formation of cholesterol-rich domains in membranes is the unequal lateral distribution of proteins in membranes. Certain proteins are found in cholesterol-rich domains. In some of these cases, it is as a consequence of the proteins interacting directly with cholesterol. There are several structural features of a protein that result in the protein preferentially associating with cholesterol-rich domains. One of the best documented of these is certain types of lipidations. In addition, however, there are segments of a protein that can preferentially sequester cholesterol. We discuss two examples of these cholesterol-recognition elements: the cholesterol recognition/interaction amino acid consensus (CRAC) domain and the sterol-sensing domain (SSD). The requirements for a CRAC motif are quite flexible and predict that a large number of sequences could recognize cholesterol. There are, however, certain proteins that are known to interact with cholesterol-rich domains of cell membranes that have CRAC motifs, and synthetic peptides corresponding to these segments also promote the formation of cholesterol-rich domains. Modeling studies have provided a rationale for certain requirements of the CRAC motif. The SSD is a larger protein segment comprising five transmembrane domains. The amino acid sequence YIYF is found in several SSD and in certain other proteins for which there is evidence that they interact with cholesterol-rich domains. The CRAC sequences as well as YIYF are generally found adjacent to a transmembrane helical segment. These regions appear to have a strong influence of the localization of certain proteins into domains in biological membranes. In addition to the SSD, there is also a domain found in soluble proteins, the START domain, that binds lipids. Certain proteins with START domains specifically bind cholesterol and are believed to function in intracellular cholesterol transport. One of these proteins is StAR-D1, that also has a mitochondrial targeting sequence and plays an important role in delivering cholesterol to the mitochondria of steroidogenic cells.     

Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin

The aim of this study was to correlate the supramolecular organization of conglutinin (BK) with its primary and tertiary structure and to gain more knowledge of functionally important regions of the molecule. BK analyzed by SDS-PAGE under standard reducing conditions (40 mM DTT) showed a major band at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6-50 mM L-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O-linked glycans of GalNAc and alpha (2-3) linked sialic acid type, whereas no N-linked glycans were demonstrated. Binding experiments with GlcNAc-gold suggested that multivalency is required for carbohydrate binding to BK. Electron microscopy showed mostly tetramers, 96 nm in diameter, but also mono-, di-, and trimers were seen. The tetramers consisted of 40-nm strands, each with a peripheral globular head composed of subunits and connected to a common central lobe built from four ring-formed structures. The strands occasionally showed two bends, one close to the central lobe and another 25 nm from the lobe. These bends most likely correspond to the interrupted Gly-Xaa-Yaa repeats at residues 38 and 123.     

Comparative X-ray studies on the interaction of carotenoids with a model phosphatidylcholine membrane

The interaction of structurally different carotenoids with a membrane molecular model was examined by X-ray diffraction. The selected compounds were beta-carotene, lycopene, lutein, violaxanthin, zeaxanthin, and additionally carotane, a fully saturated derivative of beta-carotene. They present similarities and differences in their rigidity, the presence of terminal ionone rings and hydroxy and epoxy groups bound to the rings. The membrane models were multibilayers of dipalmitoylphosphatidylcholine (DPPC), chosen for this investigation because the 3 nm thickness of the hydrophobic core of its bilayer coincides with the thickness of the hydrophobic core of thylakoid membranes and the length of the carotenoid molecules. Results indicate that the six compounds induced different types and degrees of structural perturbations to DPPC bilayers in aqueous media. They were interpreted in terms of the molecular characteristics of DPPC and the carotenoids. Lycopene and violaxanthin induced the highest structural damage to the acyl chain and polar headgroup regions of DPPC bilayers, respectively.     

Calorimetric studies on the interaction of gangliosides with phospholipids and myelin basic protein

Differential scanning calorimetry was employed to investigate the interaction of GM1 gangliosides with phospholipids (phosphatidylethanolamine, phosphatidylserine or phosphatidylcholine). It was found that GM1 is completely miscible with phosphatidylethanolamine; however, the interaction with phosphatidylserine is minimal. Addition of excess Ca2+ to the interaction products of GM1 with phosphatidylcholine or phosphatidylethanolamine did not induce phase separation. The influence of myelin basic protein on the thermotropic behaviour of GM1 was also studied. It was found that basic protein has a very strong perturbing effect on GM1 micelles.     

Biochemical studies of the excitable membrane of paramecium tetraurelia. IX. Antibodies against ciliary membrane proteins

The excitable ciliary membrane of Paramecium regulates the direction of the ciliary beat, and thereby the swimming behavior of this organism. One approach to the problem of identifying the molecular components of the excitable membrane is to use antibodies as probes of function. We produced rabbit antisera against isolated ciliary membranes and against partially purified immobilization antigens derived from three serotypes (A, B, and H), and used these antisera as reagents to explore the role of specific membrane proteins in the immobilization reaction and in behavior. The immobilization characteristics and serotype cross- reactivities of the antisera were examined. We identified the antigens recognized by these sera using immunodiffusion and immunoprecipitation with 35S-labeled ciliary membranes. The major antigen recognized in homologous combinations of antigen-antiserum is the immobilization antigen (i-antigen), approximately 250,000 mol wt. Several secondary antigens, including a family of polypeptides of 42,000-45,000 mol wt, are common to the membranes of serotypes A, B, and H, and antibodies against these secondary antigens can apparently immobilize cells. This characterization of antiserum specificity has provided the basis for our studies on the effects of the antibodies on electrophysiological properties of cells and electron microscopic localization studies, which are reported in the accompanying paper. We have also used these antibodies to study the mechanism of cell immobilization by antibodies against the i-antigen. Monovalent fragments (Fab) against purified i- antigens bound to, but did not immobilize, living cells. Subsequent addition of goat anti-Fab antibodies caused immediate immobilization, presumably by cross-linking Fab fragments already bound to the surface. We conclude that antigen-antibody interaction per se is not sufficient for immobilization, and that antibody bivalency, which allows antigen cross-linking, is essential.     

Biochemical and ultrastructural evidence for the association of basic fibroblast growth factor with cardiac gap junctions

Basic fibroblast growth factor (bFGF) is a ubiquitous and multifunctional polypeptide that is believed to have a role in tissue repair and to act as a morphogen in embryonic development. Here, we have used immunohistochemical and biochemical methods with antibodies directed against the amino-terminal domain of bFGF, designated IS2, which recognize native and denatured bFGF, to demonstrate that in addition to its known intracellular and extracellular localization in heart, bFGF is also associated with cardiomyocyte gap junctions. In tissue sections, IS2 labeled regions of intercalated discs, producing an immunofluorescence pattern virtually indistinguishable from that obtained with antibodies against the heart gap junction protein connexin-43. By electron microscopy, gap junctions but not other regions of plasma membrane were heavily immunolabeled with this antibody. By solid phase immunoassay, bFGF was found to be more concentrated in a fraction enriched in cardiac gap junctions than in whole sarcolemmal preparations. Finally, an 18-kDa protein was recognized by several different antibodies specific for bFGF on Western blots of heart subcellular fractions enriched in gap junctions. We suggest that bFGF-like peptides are either an integral part of, or exist in close association with, cardiac gap junctions and thus may play a role in modulating gap junctional intercellular communication.     

NMR studies on interaction of lauryl maltoside with cytochrome c oxidase: a model for surfactant interaction with the membrane protein

Interaction of lauryl maltoside (LM) surfactant with bovine heart cytochrome c oxidase (CcO) has been studied by NMR techniques. Detailed 2-D (1)H and (13)C NMR techniques were used to assign the NMR signals of the surfactant nuclei. Paramagnetic dipolar shift of the surfactant (13)C NMR signals were used to identify the atoms close to the enzyme. The diamagnetic carbon monoxide complex of CcO did not cause any shift in the surfactant NMR spectra suggesting that the paramagnetic centres of the native CcO cause the shifts by dipolar interactions. The results showed that the polar head groups of the surfactant comprised of two maltoside rings are more affected, while the hydrophobic tail groups did not show any significant change on binding of the surfactant to the enzyme. This indicated that surfactant head groups possibly bind to the enzyme surface and the hydrophobic tail of the surfactant forms micelles and remains away from the enzyme. Based on the results, we propose that the membrane bound enzyme is possibly stabilised in aqueous solution by association with the micelles of the neutral surfactant so that the polar heads of the micelles bind to the polar surface of the enzyme. These micelles might form a 'belt like' structure around the enzyme helping it to remain monodispersed in the active form.     

Stereologic studies on mitochondrial configuration in mitochondria of immobilized mice muscle

The effect of long-term (3 weeks) immobilization on mice skeletal (m. gastrocnemius) and heart muscles was investigated. Morphometric determinations were carried out for analysis surface and volume parameters in mitochondria. The ultrastructural visual evaluation of mitochondria showed that there are no difference between control and experimental groups. Morphometric data obtained from stereological analysis were shown that mitochondria in immobilized skeletal and heart muscles represent tendency of transformation to low-energy states (condensed state). Based upon data obtained in this study we recommend that these changes in mitochondria there are reversible.     

Direct evidence for the interaction of platelet surface membrane proteins GPIIb and III with cytoskeletal components: protein crosslinking studies

When intact platelets are incubated at 37 degrees C with Concanavalin A (ConA), the two major surface membrane proteins GPIIb and III become associated with the Triton-insoluble cytoskeleton. Preincubation of platelets with a variety of metabolic inhibitors, including cytochalasin B, 2-deoxy-D-glucose, and antimycin A or lidocaine, had no effect on the ability of ConA to produce this effect. These results suggested that the ConA-induced anchorage of GPIIb and III to the Triton-insoluble cytoskeleton is a passive process requiring clustering of GPIIb-III molecules but not requiring the metabolic energy of an intact cell. This was supported by experiments that showed that ConA binding to plasma membrane-rich fractions at 37 degrees C could induce association of GPIIb and III with a sedimentable actin-rich, Triton-insoluble membrane matrix. Similar results were obtained when membranes were first isolated from ConA-treated cells. Adding DNAse I, an actin depolymerizing agent, into the Triton extraction buffer inhibited the ConA-induced sedimentation of GPIIb-III and actin by 50% in the presence of Mg2+-ATP. Treatment of ConA-treated membranes with dimethyl-3,3'-dithiobispropiomidate, a bifunctional, reducible protein crosslinking agent, produced Triton-insoluble crosslinked species of discrete molecular weights. When these cross-linked species were analyzed by SDS-PAGE in the presence of beta-mercaptoethanol, they were found to be composed of a 180-200 K dalton protein, GPIIb, GPIII, and actin. Crosslinking of these components was equally effective after Triton treatment and indicated as well that the species crosslinked in the intact membrane was stable after Triton extraction. Addition of crosslinker to membranes not treated with ConA produced similar crosslinked species. Analysis of their composition on reduced gels revealed that the amounts of GPIIb and III were reduced greatly (less than 10% of the total input GPIIb and III) but that the 180-200 k dalton protein and actin content were similar to that seen with ConA-treated membranes. These results are consistent with the notion that ConA clusters mobile, unanchored molecules of GPIIb-III (approximately 90-95% of the total) around a small fraction of IIb-III that is associated with a submembranous cytoskeleton.     

Biochemical studies of the actions of ethanol on acetylcholinesterase activity: ethanol-enzyme-solvent interaction

1. Biochemical studies of the actions of ethanol on the activity of acetylcholinesterase (AChE), isolated from electric eel (Electrophorus electricus) and purified by affinity chromatography, were performed to elucidate ethanol-enzyme-solvent interactions. 2. Ethanol at a low concentration [( EtOH] = 2.7-200 mM) was found to enhance AChE activity slightly and systematically. 3. This observation was consistent with the result from enzyme-kinetic studies that ethanol might noncompetitively activate AChE activity at this lower concentration range. 4. If ethanol alters the hydrophobic site interaction on the enzyme and subsequently induces a favorable conformation for the active center of the enzyme, then a slight increase in the AChE activity in the presence of a low concentration of ethanol will be observed. 5. This speculation was supported by the finding of ethanol's ability to perturb the inhibition of AChE activity by tetrabutylammonium bromide and to affect hydrophobic interaction between this salt and AChE, as investigated by enzyme activity and microcalorimetric measurements. 6. The ethanol effect on the activity of this soluble AChE was found to be distinguishable from that on a membrane-bound AChE. 7. Furthermore, to elucidate the effect of ethanol-solvent interaction on AChE activity, enzyme activity in the presence of much higher concentrations of ethanol was also examined. 8. At [EtOH] greater than 800 mM, ethanol can perturb the structure of water around hydrophobic areas of AChE, causing an instability in the enzyme conformation and subsequently decreasing AChE activity.     

On the mechanism of sphingomyelin interaction with solubilized membrane proteins

Studies on the high-affinity receptor for IgE from rat basophilic leukemia cells (RBL-2H3) have shown that the phospholipid sphingomyelin remains attached to the protein complex during washing of the affinity immobilized complex under solubilizing conditions. Here we extended these findings and compared the species distribution patterns in sphingomyelin and phosphatidylcholine of the receptor-bound lipids to those of the plasma membrane lipids. FC epsilon-receptor-bound sphingomyelin but not phosphatidylcholine was enriched in long-chain fatty acids. We then examined other membrane proteins with respect to sphingomyelin enrichment. RBL-2H3 cell surface proteins, immobilized on concanavalin A-Sepharose and washed under solubilizing conditions, also showed a two- to six-fold enrichment in the associated sphingomyelin. Similar observations were also derived from other cell types, such as the mouse fibroblast cell line A-9 and the pig kidney epithelial cell line PK-1. Since this has been observed in all the three cell sources, it was suggested that sphingomyelin enrichment in FC epsilon-receptor preparations, although reproducible, was not specific for this protein. That this phenomenon was not specific for a particular protein might also be concluded from experiments that have shown nonhomogenous distribution of sphingomyelin in protein-free lipid-detergent mixtures. These results are compatible with a model whereby the interaction between sphingomyelin and soluble membrane proteins results from preference to nonmicellar phases or to structures with extended hydrophobic domains, probably due to the imperfect fitness of the detergent micelles to properly accomodate these lipids. This feature makes long-chain sphingomyelin a plausible candidate for the lipid responsible for the stabilizing effect that crude lipid preparations exert on the structural and functional properties of some membrane protein, e.g., FC epsilon R.     

Biochemical and structural studies of the interaction of Cdc37 with Hsp90

The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors. In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones. Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases. These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others. Here, the biochemical details of the interaction of human Hsp90 beta and Cdc37 have been characterised. Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37. The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.     

Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins

Interest in amyloidogenesis has exploded in recent years, as scientists recognize the role of amyloid protein aggregates in degenerative diseases such as Alzheimer's and Parkinson's disease. Assembly of proteins or peptides into mature amyloid fibrils is a multistep process initiated by conformational changes, during which intermediate aggregation states such as oligomers, protofibrils, and filaments are sampled. Although once it was assumed that the mature fibril was the biologically toxic species, more recently it has been widely speculated that soluble intermediates are the most damaging. Because of its relevance to mechanism of disease, the paths traversed during fibrillogenesis, and the kinetics of the process, are of considerable interest. In this review we discuss various kinetic models used to describe amyloidogenesis. Although significant advances have been made, construction of rigorous, detailed, and experimentally validated quantitative models remains a work in progress. We briefly review recent literature that illustrates the interplay between kinetics and amyloid-membrane interactions: how do different intermediates interact with lipid bilayers, and how does the lipid bilayer affect kinetics of amyloidogenesis?