2 functions of proteoglycans in erythrocyte aggregation and adhesion

It has been shown that rabbit red cells treated with formalin form aggregates in the presence of hyaluronic acid (HUA) soluble protein-chondroitin-keratan sulfate (PCKS) and cartilage proteoglycan aggregates (PA) but to a lesser degree than normal red cells. It is suggested that the proteoglycans under consideration can specifically interact with red cells. Aggregation of red cells in the presence of HUA, PCKS and PA is the result of the combined action of these two factors.     

The role of different proteoglycan salts as factors in steric exclusion

It has been shown that the capacity of Ca2+ salts of hyaluronic acid (HA) and nonaggregating protein-chondroitin-keratan-sulfate (PCKS) to divide in erythrocyte-saline suspension into liquid and cell phases was stronger than the analogous capacity of K+ salts. It was suggested that this is connected with a tendency to form different three-dimensional structures in solutions, which was more expressed in HA and PCKS Ca2+ salts than in K+ salts of these proteoglycans.     

Joint action of protein-chondroitin-4-keratan-sulfate and hyaluronic acid on erythrocyte aggregation and adhesion]

It was shown that the rate and the degree of erythrocytes aggregation brought about by a mixture of protein-chondroitin-4-keratan sulfate (PCKS) and hyaluronic acid (HA) was greater than the sum of the values of the corresponding indices observed during separate independent action of these proteoglycans on the aggregation of the mentioned cells concentrations as in the mixtures. It may be supposed that such phenomenon is connected with formation in the mixture of a hybrid PCKS-HA complex which is more active in respect to the erythrocyte aggregation than its components separately.     

The location of cytochrome c on the surface of ultrathin lipid multilayer films using x-ray diffraction.

X-ray diffraction and spectroscopic techniques were used to characterize ultrathin fatty acid multilayers having a bound surface layer of cytochrome c. Three to six monolayers of arachidic acid were deposited onto an alkylated glass surface, using the Langmuir-Blodgett method. These fatty acid multilayer films were stored either in a 1 mM NaHCO3 pH 7.5 solution or a buffered 10 microM cytochrome c solution, pH 7.5. After washing extensively with buffer, these multilayer films were assayed for bound cytochrome c by optical spectroscopy. It was found that the cytochrome c bound only to the odd-numbered monolayer films (which have hydrophilic surfaces). The theoretical number of cytochrome c molecules bound to the ultrathin multilayer films having three or five monolayers was calculated as N = 1.2 x 10(13)/cm2 (assuming a hexagonally close-packed monolayer of protein), which would produce an optical density of 0.002 at a wavelength of 550 nm; for a three or five monolayer ultrathin film that was incubated with cytochrome c, OD550 approximately equal to 0.002. The protein was released from the film when treated with greater than 100 mM KCl solution, as would be expected for an electrostatic interaction. Meridional x-ray diffraction data were collected from the arachidic acid films with and without a bound cytochrome c layer. A box refinement technique, previously shown to be effective in deriving the profile structures of nonperiodic ultrathin films, was used to determine the multilayer electron density profiles. The electron density profiles and their autocorrelation functions showed that bound cytochrome c resulted in an additional electron dense feature on the multilayer surface, consistent with a bound cytochrome c monolayer. The position of the bound protein relative to the multilayer surface was independent of the number of fatty acid monolayers in the multilayer. Future studies will use these methods to investigate the structures of membrane protein complexes bound directly to the surface of multilayer films.     

Studies on the filtration properties of isolated renal basement membranes.

1. The filtration properties of films of renal basement membrane were studied in vitro using pressure filtration chambers. 2. Retention of cytochrome c by the films was found to be dependent upon the filtration pressure indicating that it was transferred across the films by convective as well as diffusive flow. In contrast, serum albumin was transferred by diffusive movement only. 3. When solutions containing both cytochrome c and IgG were filtered it was found that increasing the filtration pressure reduced the flux of cytochrome c across the films. A similar phenomenon occurred when serum was filtered, less protein passed through the films at high filtration pressures. These phenomena are explained by concentration-polarisation effects. 4. The flux of cytochrome c through the films was found to decrease in a non-linear manner as the films thickness was increased. With thin films, the flux of cytochrome c increased in a non-linear manner as the concentration of the protein in the overstanding solution was increased. With thicker films the flux was linearly dependent on concentration. These findings are interpreted as supporting the view that movement of cytochrome c occurs, at least in part, by convective flow.     

Structural investigation of the covalent and electrostatic binding of yeast cytochrome c to the surface of various ultrathin lipid multilayers using x-ray diffraction.

X-Ray diffraction was used to characterize the profile structures of ultrathin lipid multilayers having a bound surface layer of cytochrome c. The lipid multilayers were formed on an alkylated glass surface, using the Langmuir-Blodgett method. The ultrathin lipid multilayers of this study were: five monolayers of arachidic acid, four monolayers of arachidic acid with a surface monolayer of dimyristoyl phosphatidylserine, and four monolayers of arachidic acid acid with a surface monolayer of thioethyl stearate. Both the phosphatidylserine and the thioethyl stearate surfaces were found previously to covalently bind yeast cytochrome c, while the arachidic acid surface electrostatically binds yeast cytochrome c. Meridional x-ray diffraction data were collected from these lipid multilayer films with and without a bound yeast cytochrome c surface layer. A box refinement technique, previously shown to be effective in deriving the profile structures of ultrathin multilayer lipid films with and without electrostatically bound cytochrome c, was used to determine the multilayer electron density profiles. The surface monolayer of bound cytochrome c was readily apparent upon comparison of the multilayer electron density profiles for the various pairs of ultrathin multilayer films plus/minus cytochrome c for all cases. In addition, cytochrome c binding to the multilayer surface significantly perturbs the underlying lipid monolayers.     

Bioelectronic device consisting of cytochrome c/poly-L-aspartic acid adsorbed hetero-Langmuir-Blodgett films

A bioelectronic device consisting of protein-adsorbed hetero-Langmuir-Blodgett (LB) films was investigated. Four kinds of functional molecules, cytochrome c, viologen, flavin, and ferrocene, were used as a secondary electron acceptor (A2), a first electron acceptor (A1), a sensitizer (S), and an electron donor (D), respectively. To fabricate the cytochrome c adsorbed hetero-LB film, poly-L-aspartic acid was used as the bridging molecule. The hetero-LB film was fabricated by subsequently depositing ferrocene, flavin, and viologen onto the pretreated ITO glass. Cytochrome c-adsorbed hetero-LB films were prepared by the adsorption of cytochrome c onto the poly-L-aspartic acid treated-LB films by intermolecular electrostatic attraction. Finally, the MIM (metal/insulator/metal) structured molecular device was constructed by depositing aluminum onto the surface of the cytochrome c-adsorbed hetero-LB films. Hetero-LB films were analyzed by Atomic Force Microscopy (AFM), and cytochrome c adsorption onto the films confirmed. The photoswitching function was achieved and the photoinduced unidirectional flow was in accordance with the rectifying characteristics of the molecular device. The direction of energy flow was in accordance with the energy level profile across molecular films. Based on the measurement of the transient photocurrent of the molecular device efficient directional flow of photocurrent through the redox potential difference was observed. The photodiode characteristics of the proposed bio-electronic device were verified and the proposed molecular array mimicking the photosynthetic reaction center could be usefully applied as a model system for the development of the bio-molecular photodiode.     

Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.     

Spin-label study of the relation between enzymatic activity and lipid-protein organization in reconstituted cytochrome c oxidase.

Lipid-depleted cytochrome c oxidase (EC 1.9.3.1) containing less than 20 microgram lipids per milligram protein was reconstituted with pure phospholipids of well-defined chemical structure and fatty acid composition without using detergents and (or) sonication. For the maximal restoration of electron transport activity, lipid-depleted cytochrome c oxidase required acidic phospholipds such as phosphatidylglycerol or phosphatidylserine or lysophospholipids such as lysophosphatidylcholine or lysophosphatidic acid, but no specific phospholipid fatty acid composition was necessary. The organization of the lipid environment of the reconstituted cytochrome c oxidase, having a well-defined lipid composition, morphology, and a high specific activity, was examined by electron spin resonance spectroscopy using 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (16-doxyl stearic acid) and 16-doxyl stearic acid - containing phosphatidylglycerol. The presence of boundary lipid was established in both lamellar and micellar organizations of reconstituted cytochrome c oxidase and was not necessarily related to the enzymatic activity of the complex. Our results have established that aside from structural considerations, the boundary lipid, at least in the reconstituted cytochrome c oxidase, is a necessary but not sufficient condition for the enzymatic expression of cytochrome c oxidase.     

Complex formation between chlorophyll a and cytochrome c: surface properties at the air-water interface. Absorbance, fluorescence and fluorescence-lifetime in Langmuir-Blodgett films.

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (II), surface potential (delta V) and [14C]cytochrome c surface-concentration (gamma) isotherms were measured versus molecular area (sigma) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN.m-1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy-transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.     

Orientation and lateral mobility of cytochrome c on the surface of ultrathin lipid multilayer films.

We have previously shown that cytochrome c can be electrostatically bound to an ultrathin multilayer film having a negatively charged hydrophilic surface; furthermore, x-ray diffraction and absorption spectroscopy techniques indicated that the cytochrome c was bound to the surface of these ultrathin multilayer films as a molecular monolayer. The ultrathin fatty acid multilayers were formed on alkylated glass, using the Langmuir-Blodgett method. In this study, optical linear dichroism was used to determine the average orientation of the heme group within cytochrome c relative to the multilayer surface plane. The cytochrome c was either electrostatically or covalently bound to the surface of an ultrathin multilayer film. Horse heart cytochrome c was electrostatically bound to the hydrophilic surface of fatty acid multilayer films having an odd number of monolayers. Ultrathin multilayer films having an even number of monolayers would not bind cytochrome c, as expected for such hydrophobic surfaces. Yeast cytochrome c was covalently bound to the surface of a multilayer film having an even number of fatty acid monolayers plus a surface monolayer of thioethyl stearate. After washing extensively with buffer, the multilayer films with either electrostatically or covalently bound cytochrome c were analyzed for bound protein by optical absorption spectroscopy; the orientation of the cytochrome c heme was then investigated via optical linear dichroism. Polarized optical absorption spectra were measured from 450 to 600 nm at angles of 0 degrees, 30 degrees, and 45 degrees between the incident light beam and the normal to the surface plane of the multilayer. The dichroic ratio for the heme alpha-band at 550 nm as a function of incidence angle indicated that the heme of the electrostatically-bound monolayer of cytochrome c lies, on average, nearly parallel to the surface plane of the ultrathin multilayer. Similar results were obtained for the covalently-bound yeast cytochrome c. Furthermore, fluorescence recovery after photobleaching (FRAP) was used to characterize the lateral mobility of the electrostatically bound cytochrome c over the monolayer plane. The optical linear dichroism and these initial FRAP studies have indicated that cytochrome c electrostatically bound to a lipid surface maintains a well-defined orientation relative to the membrane surface while exhibiting measurable, but highly restricted, lateral motion in the plane of the surface.     

Study of different proteoglycan salts

The authors have devised the methods for preparing free hyaluronic acid (HA) and non-aggregating fraction of protein-chondroitin-keratan sulfate (PCKS), as well as those for preparing their Na+, K+, Ca2+ and Mg2+ salts (acid and neutral). Infrared spectroscopy has demonstrated the presence of intermolecular hydrogen bonds, formed by hydroxyl groups, in HA and PCKS macrocomplexes and in PCKS acid salts. HA salts appeared not to form macrocomplexes at the expense of intermolecular hydrogen bonds.     

Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged nylon 66

We have examined the detection by a 125I-labeled basic protein, cationized cytochrome c, of selected proteoglycans (PGs) and standard preparations of glycosaminoglycans (GAGs) immobilized on Nylon 66 and also on positively charged Nylon 66. Immobilization on Nylon 66 appears to allow a relative freedom of interaction between PGs or GAGs and 125I-cationized cytochrome c, but a more restricted reaction was observed when PGs and GAGs were immobilized to positively charged Nylon 66. On this support PGs with large numbers of GAG side chains reacted well with 125I-cationized cytochrome c, but GAGs were minimally reactive. By taking advantage of some of the properties of large-pore agarose-acrylamide gels, rapid partial characterization of some PGs can be accomplished in the 10-ng range, and therefore at a sensitivity equal to PGs with internal biosynthetic labels.     

Lipid-protein interaction in reconstituted cytochrome c oxidase/phospholipid membranes.

Deuterium and 31P nuclear magnetic resonance have been employed in an investigation of the effect of cytochrome c oxidase (EC 1.9.3.1) on the structure of lecithin bilayers. Cytochrome c oxidase was isolated from beef heart mitochondria in lipid-free form and reconstituted as a functional enzyme in bilayers composed of synthetic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Two separate reconstitution experiments were performed in which the lipid was selectively deuterated either at the C-5' or at the C-14' segment of the palmitic acyl chain. The phospholipid-to-protein ratio of both reconstituted complexes was 0.74 (mg/mg), corresponding to about 200 molecules lipid per molecule cytochrome c oxidase. The deuterium quadrupole splitting deltanuQ, and the phosphorus chemical shielding anisotropy, deltasigma, of the cytochrome c oxidase-phospholipid recombinants were measured as a function of temperature and compared to the results obtained for the pure lipid membrane without protein for the pure lipid membrane without protein. deltanuQ and deltasigma are highly sensitive to the structural organization of the lipid membrane and these measurements demonstrate that the incorporation of cytochrome c oxidase into phosphatidylcholine bilayers leads to a more disordered conformational state of the lipids. This result can be explained by a rapid exchange between lipids in direct contact with hydrophobic protein and those further away from it (exchange rate greater than 10(4) Hz). The irregular protein surface is sensed by all lipid molecules and induces a more disordered bilayer structure. In contrast to previous interpretations, our measurements do not suggest a special type of boundary lipid.     

High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo

Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA) affects insulin resistance in cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS) production. Western blot analysis was used to examine the levels of insulin receptor (IR), phosphorylated insulin receptor substrate 1 (IRS1, Ser307) and phospho-Akt (Ser473). We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307) and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307) response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307) and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.     

Interactions of cytochrome c and [14C]-carboxymethylated cytochrome c with monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin

1. The interactions between cytochrome c (native and [(14)C]carboxymethylated) and monolayers of phosphatidylcholine, phosphatidic acid and cardiolipin at the air/water interface was investigated by measurements of surface radioactivity, pressure and potential. 2. On a subphase of 10mm-or m-sodium chloride, penetration of cytochrome c into egg phosphatidylcholine monolayers, as measured by an increase of surface pressure, and the number of molecules penetrating, as judged by surface radioactivity, were inversely proportional to the initial pressure of the monolayer and became zero at 20dynes/cm. The constant of proportionality was increased when the cytochrome c was carboxymethylated or decreased when the phospholipid was hydrogenated, but the cut-off point remained at 20dynes/cm. 3. Penetrated cytochrome c could be removed almost entirely by compression of the phosphatidylcholine monolayer above 20dynes/cm. 4. With phosphatidic acid and cardiolipin monolayers on 10mm-sodium chloride the binding of cytochrome c was much stronger and cytochrome c penetrated into films nearing the collapse pressure (>40dynes/cm.). The penetration was partly electrostatically facilitated, since it was decreased by carrying out the reaction on a subphase of m-sodium chloride, and the relationship between the surface pressure increment and the initial film pressure moved nearer to that observed with phosphatidylcholine. 5. Surface radioactivity determinations showed that [(14)C]carboxymethylated cytochrome c was still adsorbed on phosphatidic acid and cardiolipin monolayers after the cessation of penetration. This adsorption was primarily electrostatic in nature because it could be prevented and substantially reversed by adding m-sodium chloride to the subphase and there was no similar adsorption on phosphatidylcholine films. 6. The penetration into and adsorption on the three phospholipid monolayers was examined as a function of the pH of the subphase and compared with the state of ionization of both the phospholipid and the protein, and the area occupied by the latter at an air/water interface. 7. It is concluded that the binding of cytochrome c to phospholipids can only be partially understood by a consideration of the ionic interaction between the components and that subtle conformational changes in the protein must affect the magnitude and stability of the complex. 8. If cytochrome c is associated with a phospholipid in mitochondria then cardiolipin would fulfil the characteristics of the binding most adequately.     

The interaction of cytochrome c with monolayers of phosphatidylethanolamine

1. The interaction between [(14)C]carboxymethylated cytochrome c and monolayers of egg phosphatidylethanolamine at the air/water interface has been investigated by measurements of surface radioactivity, pressure and potential. 2. On adding (14)C-labelled cytochrome c to the subphase under monolayers with a surface pressure below 24dynes/cm. there was an initial surface pressure increment as the protein penetrated, followed by an adsorption that could be detected only by a continued increase in the surface radioactivity. 3. Above film pressures of 24dynes/cm. only adsorption was observed, i.e. an increment in surface radioactivity with none in surface pressure. 4. The changes in surface parameters with penetration of cytochrome c added to the subphase were indirectly proportional to the initial pressure of the monolayer. With hydrogenated phosphatidylethanolamine the constant of proportionality was increased but penetration again ceased at 24dynes/cm. 5. On compressing a phosphatidylethanolamine film containing penetrated cytochrome c to 40dynes/cm. only a proportion of the protein was ejected on a subphase of 10mm-sodium chloride, whereas on a subphase of m-sodium chloride nearly all the protein was lost. 6. With both penetration and adsorption only a small proportion of the added cytochrome c interacted with the phospholipid films, and initially the amount bound was proportional to the added protein concentration. There was no evidence of a stoicheiometric relationship between the protein and phospholipid or the build-up of multilayers. The bonded protein was not released by removing cytochrome c from the subphase. 7. The addition of m-sodium chloride to the subphase delays the rate of protein penetration into low-pressure films, but the final surface-pressure increment is not appreciably decreased. In contrast, m-sodium chloride almost completely stops adsorption on to films at all pressures. 8. When sodium chloride is added to the subphase below cytochrome c adsorbed to monolayers at high pressures, so that the final concentration is 1m, only a proportion of the protein is desorbed and this decreases as the time of the interaction increases. This indicates that adsorption is initially electrostatic, followed by the formation of non-ionic bonds. 9. Alteration of the subphase pH under a high-pressure film leads to a steady increase in adsorption from pH3 to 8.5 followed by a rapid fall to zero adsorption at pH11. 10. The penetration into phospholipid monolayers at 10dynes/cm. shows a rate that is consistent with the relative electrostatic status of the two components of the interaction as the subphase pH is varied between 3 and 10.5. The final equilibrium penetration shows a pronounced peak in the increments of surface pressure at pH9.0 although a similar peak is not observed in the surface radioactivity. This indicates that more residues of the protein are penetrating into the film at about this pH. 11. Determinations were made of the electrophoretic mobilities of phosphatidylethanolamine particles both alone and after interaction with cytochrome c. 12. The electrophoretic mobilities of cytochrome c adsorbed on lipid particles showed an isoelectric point below that of cytochrome c. This and the observations on the monolayers suggest that, with cytochrome c, protein-protein interactions are weak compared with other proteins.     

Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis.

The genes coding for the photosynthetic reaction center cytochrome c subunit (pufC) and the soluble cytochrome c2 (cycA) from the purple non-sulfur bacterium Rhodopseudomonas viridis were expressed in Escherichia coli. Biosynthesis of the reaction center cytochrome without a signal peptide resulted in the formation of inclusion bodies in the cytoplasm amounting to 14% of the total cellular protein. A series of plasmids coding for the cytochrome subunit with varying N-terminal signal peptides was constructed in attempts to achieve translocation across the E. coli cytoplasmic membrane and heme attachment. However, the two major recombinant proteins with N-termini corresponding to the signal peptide and the cytochrome were synthesized in E. coli as non-specific aggregates without heme incorporation. An increased ratio of precursor as compared to 'processed' apo-cytochrome was obtained when expression was carried out in a proteinase-deficient strain. Cytochrome c2 from R. viridis was synthesized in E. coli as a precursor associated with the cytoplasmic membrane. An expression plasmid was designed encoding the N-terminal part of the 33 kDa precursor protein of the oxygen-evolving complex of Photosystem II from spinach followed by cytochrome c2. Two recombinant proteins without heme were found to aggregate as inclusion bodies with N-termini corresponding to the signal peptide and the mature 33 kDa protein.     

The intra-mitochondrial cytochrome c distribution varies correlated to the formation of a complex between VDAC and the adenine nucleotide translocase: this affects Bax-dependent cytochrome c release

The mechanism of Bax-dependent cytochrome c release is still controversial and may also depend on the actual localisation of cytochrome C: (i) we studied the distribution of cytochrome c in sub-fractions of rat kidney mitochondria and found that 10-20% of the total cytochrome c was associated at the peripheral inner membrane and to some extent organised in the contact sites. (ii) Cytochrome c concentrations in the contact site fractions varied related to surface bound hexokinase activity. It decreased upon reduction of contact sites by glycerol or specific dissociation of the VDAC-ANT complexes by bongkrekate, whereas it increased upon induction of contacts by dextran or association of VDAC-ANT complexes by atractyloside. (iii) The outer membrane pore (VDAC) acquires high capacity for hexokinase binding by interacting with the ANT. Thus, surface-attached hexokinase protein indicated the frequency of VDAC-ANT complexes and the correlation between hexokinase activity and cytochrome c suggested association of the latter to the complexes. (iv) Substances affecting exclusively the structure of either hexokinase (glucose-6P) or cytochrome c (borate) led to a decrease only of the effected protein without changing the concentration of other contact site constituents. (v) Hexokinase was furthermore used as a tool to isolate the contact site forming complex of outer membrane VDAC and inner membrane ANT from Triton-dissolved membranes. Cytochrome c remained attached to the hexokinase VDAC-ANT complexes that were reconstituted in phospholipid vesicles. (vi) The vesicles were loaded with malate and BaxDeltaC released the endogenous cytochrome c from the reconstituted complexes without forming unspecific pores for malate. BaxDeltaC targeted a cytochrome c fraction associated at the VDAC-ANT complex. The cytochrome c organisation was dependent on the actual structure of VDAC and ANT. Thus, the BaxDeltaC effect was suppressed either by hexokinase utilising glucose and ATP or by bongkrekic acid both influencing the pore and ANT structure.     

Collagen-associated sulphated proteoglycans. Ultrastructure after formaldehyde-cetylpyridinium chloride fixation.

In the course of an ultrastructural cytochemical study of intracellular sulphated proteoglycans involving the addition of cetylpyridinium chloride in the primary aldehyde fixative, a remarkable ultrastructural preservation of the collagen-associated sulphated proteoglycans was observed. Together with the preservation of their localization among the collagen fibrils (with, for some of them, a 50 nm periodic association with d-bands) and of their native elongated shape, previously observed under similar technical conditions, these stick-shaped and chondroitinase ABC-sensitive proteoglycans exhibited a typical pattern with several dense longitudinal parallel tracks (periodicity: 3-4 nm) not described as yet. Readily observable without high iron diamine-staining, the morphology of these cetylpyridinium chloride-precipitated and collagen-associated polyanions was particularly enhanced after incubation in the diamine solution which ascertained their sulphate content. Such a common ultrastructural organization with parallel tracks for both intracellular (i.e., in eosinophilic polymorphonuclear cells and Kurloff cells) and extracellular CPC-precipitated sulphated proteoglycans could correspond to intrinsic properties of the complexed molecules and could be related to 'double track' proteoglycans observed under other technical conditions in basement membranes.