Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Voltage-dependent orientation of membrane proteins

In order to study the influence of electrostatic forces on the disposition of proteins in membranes, we have examined the interaction of a receptor protein and of a membrane-active peptide with black lipid membranes. In the first study we show that the hepatic asialoglycoprotein receptor can insert spontaneously into lipid bilayers from the aqueous medium. Under the influence of a trans-positive membrane potential, the receptor, a negatively charged protein, appears to change its disposition with respect to the membrane. In the second study we consider melittin, an amphipathic peptide containing a generally hydrophobic stretch of 19 amino acids followed by a cluster of four positively charged residues at the carboxy terminus. The hydrophobic region contains two positively charged residues. In response to trans-negative electrical potential, melittin appears to assume a transbilayer position. These findings indicate that electrostatic forces can influence the disposition, and perhaps the orientation, of membrane proteins. Given the inside-negative potential of most or all cells, we would expect transmembrane proteins to have clusters of positively charged residues adjacent to the cytoplasmic ends of their hydrophobic transmembrane segments, and clusters of negatively charged residues just to the extracytoplasmic side. This expectation has been borne out by examination of the few transmembrane proteins for which there is sufficient information on both sequence and orientation. Surface and dipole potentials may similarly affect the orientation of membrane proteins.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

Long-lived primary radical pair state detected by time-resolved fluorescence and absorption spectroscopy in an isolated Photosystem two core

A Photosystem two (PS II) core preparation containing the chlorophyll a binding proteins CP 47, CP 43, D1 and D2, and the non-chlorophyll binding cytochrome-b559 and 33 kDA polypeptides, has been isolated from PS II-enriched membranes of peas using the non-ionic detergent heptylthioglucopyranoside and elevated ionic strengths. The primary radical pair state, P680⁺Pheo^-, was studied by time-resolved absorption and fluorescence spectroscopy, under conditions where quinone reduction and water-splitting activities were inhibited. Charge recombination of the primary radical pair in PS II cores was found to have lifetimes of 17.5 ns measured by fluorescence and 21 ns measured by transient decay kinetics under anaerobic conditions. Transient absorption spectroscopy demonstrated that the activity of the particles, based on primary radical pair formation, was in excess of 70% (depending on the choice of kinetic model), while time-resolved fluorescence spectroscopy indicated that the particles were 91% active. These estimates of activity were further supported by steady-state measurements which quantified the amount of photoreducible pheophytin. It is concluded that the PS II core preparation we have isolated is ideal for studying primary radical pair formation and recombination as demonstrated by the correlation of our absorption and fluorescence transient data, which is the first of its kind to be reported in the literature for isolated PS II core complexes from higher plants.Abbreviations CP 43 and CP 47 chlorophyll binding proteins of PS II having apparent molecular weights on SDS-PAGE of 43 kDa and 47 kDa, respectively - D1 and D2 polypeptides PS II reaction centre polypeptides encoded by the psbA and psbD genes, respectively - HPLC high performance liquid chromatography - PS II Photosystem two - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - P680 primary electron donor of PS II - Pheo phenophytin a - SPC single photon counting - PBQ phenyl-p-benzoquinone - DPC 1,5-diphenylcarbazide AFRC Photosynthesis Research Group, Department of Biochemistry     

Energy status of cells lacking dystrophin: an in vivo/in vitro study of mdx mouse skeletal muscle

Although great strides have been made in understanding the genetics of Duchenne muscular dystrophy (DMD), uncertainty still remains as to the metabolic changes which are associated with the disease. We have used the recently discovered animal model of DMD, the mdx mouse, to study aspects of high energy phosphate metabolism and metabiolic control indices in dystrophic muscle. This model of DMD has the dual advantage of having a genetic defect which is homologous to that in human DMD, and it lacks the fatty infiltration and ncecrosis which makes biochemical analysis of DMD so difficult. We have used nuclear magnetic resonance sperctroscopy (NMR) to monitor developmental changes in high energy phosphates and pH. No differences were observed between young (< 40–50 days old) control and mdx mice. The pH increase and alterations in phosphate ratios (i.e., decline in PCr/ATP) observed in adult mdx vs. control mice are quantilatively similar to those observed in humans. Biochemical analysis showed a small decline in ATP and PCr content and a decline in some indices of energy status in adult mdx mice. As young mdx mice appeared to be normal, the lack of dystrophin does not correlate with metabolic changes. The changes which were observed were small enough that alterations in fibre composition could be the major contributory factor.     

Early electrochemical events in lectin-lymphocyte interaction

Concanavalin A, at extremely low concentrations, will produce significant increases in the electrophoretic mobility of murine splenic T lymphocytes. It has been established that the alteration in cellular surface charge is mediated by a factor produced by those lymphocytes that have reacted directly with con A. We originally conjectured that the mobility change might be the consequence of an alteration in the distribution of the charged moieties of membrane glycoproteins. The results of experiments conducted at low temperature raise some questions about this mechanism. Further experiments have been performed to establish the nature of the physicochemical alterations in the peripheral zone of the factor-stimulated lymphocytes that are manifest as changes in cellular surface charge. The results of these studies indicate that, subsequent to the interaction of T lymphocytes with con A, there is a reduction in the number of positively charged amino groups effective at the electrophoretic surface of the cells.