Interaction of phospholipids with the detergent-solubilized ADP/ATP carrier protein as studied by spin-label electron spin resonance

The interaction of spin-labeled phospholipids with the detergent-solubilized ADP/ATP carrier protein from the inner mitochondrial membrane has been investigated by electron spin resonance spectroscopy. The equilibrium binding of cardiolipin and phosphatidic acid was studied by titration of the protein with spin-labeled phospholipid analogues using a spectral subtraction protocol for the evaluation of the mobile and immobilized lipid portions. This analysis revealed the immobilization of two molecules of spin-labeled cardiolipin per protein dimer. Phosphatidic acid has a similar affinity for the protein surface as cardiolipin. The lipid-protein interaction was less pronounced with the neutral phospholipids and with phosphatidylglycerol. The importance of the electrostatic contribution to the phospholipid-protein interaction shows up with a strong dependence of the lipid binding on salt concentration. Cleavage by phospholipase A2 and spin reduction by ascorbate of the spin-labeled acidic phospholipids in contact with the protein surface suggest that these lipids are located on the outer perimeter of the protein. At reduced detergent concentration, the protein aggregated upon addition of small amounts of cardiolipin but remained solubilized when more cardiolipin was added. This result is discussed with respect to the aggregation state of the protein in the mitochondrial membrane. It is also tentatively concluded that binding of spin-labeled cardiolipin does not displace the tightly bound cardiolipin of mitochondrial origin, which was detected previously by 31P nuclear magnetic resonance spectroscopy.     

Specific binding of mitochondrial protein precursors to liposomes containing cardiolipin

In vitro synthesized precursors of several mitochondrial proteins, including P-450(SCC), adrenodoxin, and malate dehydrogenase, bound to liposomes prepared from mitochondrial phospholipids, but not to those from microsomal phospholipids. When liposomes were prepared from various pure phospholipids, adrenodoxin precursor was bound only to the liposomes that contained cardiolipin. The liposomes containing other phospholipids did not show the binding affinity for the precursor. The binding was observed only with the precursor peptides of adrenodoxin and malate dehydrogenase, and their mature forms were not bound to the liposomes. The binding of the precursors was dependent on the concentration of cardiolipin in the liposomes. Liposomes containing various cardiolipin derivatives with modified polar head groups showed very different binding affinity for adrenodoxin precursor, suggesting the importance of the structure of the polar head of the cardiolipin molecule. Two or three positively charged amino acid residues in the extension peptide of P-450(SCC) precursor were replaced by neutral amino acid residues by site-directed mutagenesis. The mutated P-450(SCC) precursors did not bind to the liposomes containing cardiolipin. The results indicated that mitochondrial protein precursors have specific affinity for cardiolipin, and the affinity was due to the interaction between the extension peptides of the precursors and the polar head of the cardiolipin molecule.     

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4',4'-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatide acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 degrees C. In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of guinea pig liver.     

Binding of cardiolipin/lecithin and monoamine oxidase to lipid-depleted mitochondrial membrane structure.

Lipid-depleted pig liver mitochondrial residues were incubated with different proportions of the acidic phospholipid cardiolipin and the zwitterionic phospholipid lecithin in either separate or mixed liposomes. When cardiolipin and lecithin were present in separate liposomes all of the cardiolipin but no lecithin bound to the residues. When present in the same liposomes, cardiolipin also caused binding of lecithin to the mitochondrial residues. When monoamine oxidase solubilized from pig liver mitochondria by extraction of the phospholipids was included in the incubation, binding of the enzyme to the residues occurred in the presence of cardiolipin. The percentage of enzyme bound followed the same trend as the binding of phospholipids to the mitochondrial residues.     

A spin-label electron spin resonance study of the binding of mitochondrial creatine kinase to cardiolipin

The binding of the mitochondrial creatine kinase to aqueous dispersions of beef heart cardiolipin has been studied via the perturbation of the mobility of spin-labelled cardiolipin, using electron spin resonance (ESR) spectroscopy. In the presence of creatine kinase (1:1 protein/lipid ratio, by mass), the ESR spectra of cardiolipin labelled in a single acyl chain [n-(4,4-dimethyl-oxazolidinyl-N- oxy)stearoylcardiolipin] indicate a restriction of motion both at the C-5 and C-14 positions (n = 5, 14) of the lipid chains. The restriction in mobility was reversed by addition of phosphate or adriamycin, which are thought to inhibit the binding of creatine kinase to the mitochondrial membrane or to displace it from its binding site on the membrane. The effect of the protein on the chain mobility is consistent with surface binding of the protein; no positive evidence was obtained for penetration of the protein into the hydrophobic region of the membrane.     

Two-dimensional 1H nuclear magnetic resonance studies on the gene V-encoded single-stranded DNA-binding protein of the filamentous bacteriophage IKe. II. Characterization of the DNA-binding wing with the aid of spin-labelled oligonucleotides

The DNA-binding domain of the single-stranded DNA-binding protein IKe GVP was studied by means of 1H nuclear magnetic resonance, through use of oligonucleotides of two and three adenyl residues in length, that were spin-labelled at their 3' and/or 5' termini. These spin-labelled ligands were found to cause line broadening of specific protein resonances when bound to the protein, although they were present in small quantities, i.e. of the order of 0.04 molar equivalent and less. The line broadening of protein resonances was made manifest by means of difference one and two-dimensional spectroscopy. Difference one-dimensional experiments revealed line broadening of the same protein resonances upon binding of either 3' or 5' spin-labelled oligonucleotides. Evidence in favour of the existence of a fixed 5' to 3' orientation in the binding of oligonucleotides to the protein surface was therefore not obtained from the spin-labelled oligonucleotide binding studies. Residue-specific assignments of broadened resonances could not, or could only sparsely, be derived from the difference one-dimensional spectra, because of the tremendous overlap in the aliphatic region of the spectrum. In contrast, such assignments were easily obtained from the difference two-dimensional spectra, which were recorded by means of both total correlated spectroscopy and nuclear Overhauser effect spectroscopy. Difference signals were detected for 15 spin systems; ten out of these were assigned to the residues I29, Y27, S20, G18, R16, T28, K22, Q21, V19 and S17 in the amino acid sequence of IKe GVP; the other five spin systems could be assigned to a phenylalanyl residue, an arginyl or lysyl residue, an aspartic acid or asparagyl residue, a glycyl residue and a glutamic acid or glutamyl residue. From the evaluation of the relative difference signals, it was concluded that the direct surroundings of the spin-label group of the labelled oligonucleotide in the bound state is composed of the first five residues in the former group of residues and the five residues in the latter group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective enzymatic radioactive and spin labelling of phospholipids in biological membranes: application to study of temperature-induced changes of microsomal phosphatidylinositols and mitochondrial polyglycerophosphatides.

A new method for the covalent radioactive and spin labelling of lipids within isolated biological membranes has been described in detail and applied to studies of temperature-induced changes of microsomal and mitochondrial membranes. The method is based on the enzymatic use of radioactive substrates carrying covalently bound doxyl derivatives of stearic acid in the biosynthesis of phospholipids in isolated membranes. Radioactive-and spin-labelled lipids bound to the microsomal and mitochondrial membranes were then used as internal probes in monitoring temperature-induced changes of these membranes. Since the analysis of isolated radioactive-and spin-labelled lipids revealed the exact composition of membrane-bound labelled lipids, specific temperature-induced changes were correlated with specific lipids of examined membranes. Phosphatidylinositol of microsomal membranes and polyglycerophosphatides (phosphatidyl-glycerol and cardiolipin) of mitochondrial and inner mitochondrial membranes were found to be involved in the apparent formation of lipid clusters at around 20-30 degrees C. Cardiolipin was found to be involved in the fluidization of inner mitochondrial membranes. These findings are discussed in view of the present state of knowledge of the organization of lipids in biological membranes.     

Magnetic-resonance studies of the geometry of bound substrate, coenzyme and activator on bovine-liver glutamate dehydrogenase.

ADP and ATP with a spin-label linked to the terminal phosphate are activators of glutamate dehydrogenase and bind to the same site as the activator ADP. There is hardly any interaction with the coenzyme site. Glutamate dehydrogenase can be modified with a ketone spin-label at a site in the active centre[Andree and Zantema, (1978) Biochemistry, 17, 778--783]. The spin-labelled activators interact with ketone spin-labelled glutamate dehydrogenase in the same way as with native glutamate dehydrogenase relative to the activator site, but show a stronger binding to the coenzyme site. Upon binding to the coenzyme site a spin-spin interaction between the ketone spin-label and the spin-labelled activators is observed. Nuclear magnetic resonance studies of the linewidth of 2-oxoglutarate and NADP+ bound to their functional sites on glutamate dehydrogenase without and with spin-labels result in distances between the ligand nuclei and the spin-labels. The results show that NADP+ binds in an open conformation consistent with the conformation in other dehydrogenases. The activator ADP binds in the neighbourhood of the active centre, but with very little or no overlap with the coenzyme site.     

Adriamycin, a drug interacting with acidic phospholipids, blocks import of precursor proteins by isolated yeast mitochondria

Acidic phospholipids such as cardiolipin partially unfold an artificial precursor protein which consists of a mitochondrial presequence fused to mouse dihydrofolate reductase (Endo, T., and Schatz, G. (1988) EMBO J. 7, 1153-1158). We now show that import of this precursor protein into isolated yeast mitochondria is blocked by adriamycin, a drug binding to cardiolipin and other acidic phospholipids. This inhibition is lessened if the precursor's dihydrofolate reductase moiety is labilized by point mutations; inhibition is abolished altogether if the "wild-type" precursor is presented to mitochondria in a urea-denatured state. These and other observations suggest that adriamycin interferes with the generation of a translocation-competent, loose structure of the precursor protein. They imply that acidic phospholipids such as cardiolipin participate, directly or indirectly, in the translocation of this fusion protein into isolated mitochondria.     

Spin-label electron paramagnetic resonance studies on the interaction of avidin with dimyristoyl-phosphatidylglycerol membranes

The interaction of avidin--a basic protein from hen egg-white--with dimyristoyl-phosphatidylglycerol membranes was investigated by spin-label electron paramagnetic resonance spectroscopy. Phosphatidylcholines, bearing the nitroxide spin label at different positions along the sn-2 acyl chain of the lipid were used to investigate the effect of protein binding on the lipid chain-melting phase transition and acyl chain dynamics. Binding of the protein at saturating levels results in abolition of the chain-melting phase transition of the lipid and accompanying perturbation of the lipid acyl chain mobility. In the fluid phase region, the outer hyperfine splitting increases for all phosphatidylcholine spin-label positional isomers, indicating that the chain mobility is decreased by binding avidin. However, there was no evidence for direct interaction of the protein with the lipid acyl chains, clearly indicating that the protein does not penetrate the hydrophobic interior of the membrane. Selectivity experiments with different spin-labelled lipid probes indicate that avidin exhibits a preference for negatively charged lipid species, although all spin-labelled lipid species indirectly sense the protein binding. The interaction with negatively charged lipids is relevant to the use of avidin in applications such as the ultrastructural localization of biotinylated lipids in histochemical studies.     

Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin. In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T. (1998) J. Biol. Chem. 273, 28651-28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg(360), Arg(364), and Lys(372)) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo. All mutant proteins maintained activities for DNA replication and ATP binding. A mutant protein in which Lys(372) was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins. Thus, Lys(372) seems to play an important role in the interaction between DnaA protein and acidic phospholipids. Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.     

Lipid-protein interactions in Escherichia coli membranes over-expressing the sugar-H(+) symporter,GalP EPR of spin-labelled lipids

The D-galactose-H(+) symport protein (GalP) of Escherichia coli is a homologue of the human glucose transport protein, GLUT1. After amplified expression of the GalP transporter in E. coli, lipid-protein interactions were studied in gradient-purified inner membranes by using spin-label electron paramagnetic resonance (EPR) spectroscopy. Phosphatidylethanolamine, -glycerol, -choline and -serine, in addition to phosphatidic and stearic acids, were spin-labelled at the 14 C-atom of the sn-2 chain. EPR spectra of these spin labels at probe amounts in GalP membranes consist of two components. One component corresponds to a lipid population whose motion is restricted by direct interaction with the transmembrane sections of the integral protein. The other component corresponds to a lipid population with greater chain mobility, and is similar to the single-component EPR spectrum of the spin-labelled lipids in membranes of E. coli lipid extract. Quantitation of the protein-interacting spin-label component allows determination of the stoichiometry and selectivity of lipid-protein interactions. On average, approximately 20 mol of lipid are motionally restricted per 52 kDa of protein in GalP membranes. At the pH of the transport assay, there is relatively little selectivity between the different phospholipids tested. Only stearic acid displays a stronger preferential interaction with this protein.     

Annexin V binding perturbs the cardiolipin fluidity gradient in isolated mitochondria. Can it affect mitochondrial function?

The phosholipid bilayer fluidity of isolated mitochondria and phospholipid vesicles after calcium-dependent binding of annexin V was studied using EPR spectroscopy. The membranes were probed at different depths by alternatively using cardiolipin, phosphatidylcholine, or phosphatidylethanolamine spin labeled at position C-5 or C-12 or C-16 of the beta acyl chain. Computer-aided spectral titration facilitated observing and quantitating the EPR spectrum from phospholipid spin labels affected by annexin binding, and spectral mobility was calibrated by comparison with standard spectra scanned at various temperatures. In most cases it was found that binding of the protein to the membranes makes the inner bilayer more rigid up to acyl position C-12 than afterward, in agreement with the previously observed effect in SUVs [Megli, F. M., Selvaggi, M., Liemann, S., Quagliariello, E., and Huber, R. (1998) Biochemistry 37, 10540-10546]. Moreover, in isolated mitochondrial membranes, cardiolipin apparently is more readily affected than the other main phospholipids, while in vesicles made from mitochondrial phospholipids, the different species are affected in essentially the same way. This behavior is consistent with the existence of distinct cardiolipin pools in mitochondria, and with the already advanced hypothesis that these domains are the binding site for annexin V to the isolated organelles [Megli, F. M., Selvaggi, M., De Lisi, A., and Quagliariello, E. (1995) Biochim. Biophys. Acta 1236, 273-278]. Keeping in mind the funcional importance of cardiolipin in the mitochondrial membrane, the question is raised as to whether the observed influence of annexin V binding to this phospholipid and its consequent local fluidity alteration might affect the mitochondrial functionality, at least in vitro.     

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.     

How does the Bax-α1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin

A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. ¹H and ³¹P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational changes into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.     

Acidic phospholipids inhibit the DNA-binding activity of DnaA protein,the initiator of chromosomal DNA replication in Escherichia coli

In order to initiate chromosomal DNA replication in Escherichia coli, the DnaA protein must bind to both ATP and the origin of replication (oriC). Acidic phospholipids are known to inhibit DnaA binding to ATP, and here we examine the effects of various phospholipids on DnaA binding to oriC. Among the phospholipids in E. coli membrane, cardiolipin showed the strongest inhibition of DnaA binding to oriC. Synthetic phosphatidylglycerol containing unsaturated fatty acids inhibited binding more potently than did synthetic phosphatidylglycerol containing saturated fatty acids, suggesting that membrane fluidity is important. Thus, acidic phospholipids seem to inhibit DnaA binding to both oriC and adenine nucleotides in the same manner. Adenine nucleotides bound to DnaA did not affect the inhibitory effect of cardiolipin on DnaA binding to oriC. A mobility-shift assay re-vealed that acidic phospholipids inhibited formation of a DnaA-oriC complex containing several DnaA molecules. DNase I footprinting of DnaA binding to oriC showed that two DnaA binding sites (R2 and R3) were more sensitive to cardiolipin than other DnaA binding sites. Based on these in vitro data, the physiological relevance of this inhibitory effect of acidic phospholipids on DnaA binding to oriC is discussed.     

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.     

Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.     

Probing the topography of lectins with site-specific spin-labeled glycosides

Three new spin-labeled glycosides, spin-label I [1-[4-(beta-D-galactopyranosyloxy)phenyl]-3-(2,2,6,6-tetramethyl-1 -oxypiperidin-4-yl)-2-thiourea], spin-label II (2,2,6,6-tetramethyl-1-oxypiperidin-4-yl alpha-D-galactopyranoside), and spin-label III [1-(methyl 2-deoxy-alpha-D-galactopyranosid-2-yl)-3-(2,2,6,6- tetramethyl-1-oxypiperidin-4-yl)-2-thiourea], were investigated as structural probes of Griffonia simplicifolia I isolectins (GS I) A4 and B4, respectively, by electron spin resonance (ESR) and inhibition of guaran isolectin precipitation. The p-aminophenyl beta-galactoside spin-label I was strongly immobilized by the B4 isolectin (Kd = 0.42 mM; 2T parallel = 54.0 +/- 0.3 G), while binding to the A4 isolectin was so weak (KI congruent to 2 mM) that binding was undetectable by ESR. The preference for the B4 isolectin was indicative of a more extended hydrophobic binding locus adjacent to the carbohydrate-specific binding site. The alpha-galactosyl spin-label II bound slightly more strongly to the A4 than to the B4 isolectin, as evidenced in both Kd values and particularly by differences in the degree of immobilization (2T parallel = 53.5 vs. 51.5 G, respectively). The 2-N-substituted methyl galactoside spin-label III was so poor an inhibitor of both isolectins (KI congruent to 1-2 mM) that ESR detection of the bound complex was not feasible. In all cases above, the spin-labels were displaced by specific monosaccharide haptens.     

DNA-bound lipids from eukaryotic (loach spermatozoa, pigeon erythrocytes) and prokaryotic (E. coli B, phage T2) cells

Using thin-layer chromatography, some specific DNA-bound neutral lipids and phospholipids of loach spermatozoa, pigeon erythrocytes, E. coli B and phage T2 cells were studied. These lipids are represented by loosely and firmly bound components. The content of neutral lipids in the above DNAs (per mg of DNA) is 10.6, 4.8, 7.81 and 1.43 micrograms, respectively; that of phospholipids is 4.31, 1.28, 1.14 and 0.54 micrograms, respectively. The eucaryotic DNA-bound lipids are highly deficient of free cholesterol, phosphatidylcholine, phosphatidylinositol and phosphatidylserine but are rich in cardiolipin, phosphatidylethanolamine, cholesterol esters, diglycerides and free fatty acids. The quantitative and qualitative composition of DNA-bound lipids of loach spermatozoa changes during the transition from the superhelical to the relaxed conformation of DNA. Procaryotic DNA-bound neutral lipids are also represented by the free cholesterol, diglyceride and free fatty acid fractions, whereas the DNA-bound phospholipids of procaryotes consist of only two fractions, i.e., cardiolipin and phosphatidylethanolamine. The role of DNA-bound lipids in the structural and functional organization of eucaryotic and procaryotic genomes is discussed.