The effect of trypsin treatment on rabbit muscle phosphofructokinase

The consequences of trypsin treatment of rabbit muscle phosphofructokinase, in terms of the physical and kinetic properties of the enzyme, have been investigated. At 1% trypsin (w/w) and 25 °C, no activity is lost over a period of 60 min. The complex sedimentation behavior at pH 8 (three peaks) is unchanged by this treatment as is the extent of dissociation of the enzyme when the pH is lowered from 8 to 6 or reassociation when the pH is raised back to 8. However, the trypsin-treated enzyme shows a subunit molecular weight, determined in the guanidine HCl or 0.5 m acetic acid, of 35,000–40,000 compared to the subunit molecular weight of the untreated enzyme at 75,000–80,000. Similarly, SDS gels give only a single species of about 80,000 for the native enzyme but two species, 42,000 and 48,000, for the trypsin-treated enzyme. Kinetic studies showed no differences in the regulatory properties of the enzyme including fructose 6-phosphate cooperativity, ATP inhibition, NH_4⁺ activation, and cAMP activation. Small differences in stability and inhibition by citrate and creatine phosphate were observed.     

Factors defining the functional coupling of bacteriorhodopsin and ATP synthase in liposomes

Optimal conditions for the co-reconstitution of bacteriorhodopsin and yeast mitochondria ATP synthase were determined. Reconstitution was achieved with a quick two-step procedure. Preparations obtained by this method displayed in optimal cases 2–3-times higher activities (up to 500 nmol ATP/min per mg protein) compared with maximal values reported in the literature, when light-driven ATP synthesis was measured under similar conditions. The final activities depended on the purification method used for the ATP synthase, and it is shown that the oligomycin-sensitive ATP hydrolysis activity was not a good measure for the ability of the ATP synthase preparations to perform ATP synthesis after co-reconstitution. Light-driven ATP synthesis activities depended also on the type of phospholipid used, soybean phospholipid giving the best results. A close relation to the bacteriorhodopsin proton pump activity was found. Using different phospholipids, different $\text{H}{}^{\mathrm{+}}\text{ATP}$ ratios were found, calculated from ATP synthesis activities and initial and steady-state light-driven proton pump activities. From this, together with the findings that the ATP synthase displayed the same ATP hydrolysis and ATP-³²P_i exchange activities with these different phospholipids used, it is concluded that the protein distribution for the two proteins among the liposomes is different relative to each other for the different phospholipids. The light-driven ATP synthesis activity did not correlate with the variation in leakiness of the membrane for protons when different phospholipids were used. An explanation is given by the finding that at high light intensities, the ATP synthesis became independent of the presence of protonophore.     

In vivo and in vitro incorporation of endogenous nucleotides by the energy-transducing ATPase of Streptococcus faecalis

The soluble ATPase isolated from Streptococcus faecalis membranes containing tightly bound endogenous nucleotides do not exchange in the presence of ATP and Mg+2 added during the purification of the enzyme. In this paper the stoichiometry of endogenous nucleotides in the soluble ATPase obtained from (a) growing cells, (b) nongrowing glycolyzing cells, and (c) isolated cell membranes has been defined. The time course of incorporation was also studied in nongrowing, glycolyzing cells and isolated cell membranes. In all cases, 1-2 mol of nucleotide was bound per mol of enzyme. Maximal incorporation required approximately 1 h at 38 degrees C. Incorporation of cytoplasmic nucleotide into the enzyme occurred by a process of slow exchange for bound nucleotide. N,N'-dicyclohexylcarbodiimide, which inhibits the membrane-bound ATPase and prevents generation of the protonmotive force, had no effect on incorporation of endogenous nucleotides in glycolyzing cells. Treatment of glycolyzing cells with gramicidin D plus K+, which dissipates the protonmotive force but has no effect on ATPase activity, did not inhibit incorporation of nucleotide. These results support the view that the slow exchange-incorporation of endogenous nucleotide(s) is independent of ATP hydrolysis and a protonmotive force. An in vitro system for the study of nucleotide binding at endogenous sites is described.     

Dynamic properties of bacteriorhodopsin in purple membranes upon heat treatment

Reversible temperature-dependent conformational changes in bacteriorhodopsin of the purple membranes from Halobacterium halobium have been studied by the method of deuterium exchange. A noticeable increase in the mobility of structured peptide groups in bacteriorhodopsin was revealed upon reorganization of the supermolecular structure at about 60 degrees C. In the supermolecular structure formed, bacteriorhodopsin molecules have no contacts with external medium at 75-80 degrees C. Membrane destruction results in a drastic increase in molecular mobility within the narrow temperature range 100-110 degrees C. The effects observed are induced by predenaturation changes in the bacteriorhodopsin structure and rearrangements in the structure of a protein-lipid complex. The temperature dependence of the number of peptide groups involved in reversible conformational rearrangements is in good agreement with the microcalorimetry data.     

The incorporation of solubilized choline-transport activity into liposomes.

The choline-transport system has been solubilized from synaptic plasma membrane by using either sodium cholate or Triton X-100, and re-incorporated into unilamellar liposomes by using the technique of cholate dialysis. The criteria of choline-transport activity were saturability by excess choline, inhibition by hemicholinium-3, and trans-activation (i.e. stimulation of the uptake of [3H]choline into liposomes by preloading them with non-radioactive choline). Liposomes prepared from detergent extracts of synaptic plasma membrane and added lipid showed uptake of [3H]choline fulfilling these three criteria. Data on choline-transport activity of liposomes at various choline concentrations could be interpreted as implying that the transport system has two apparent Km values (2-5 microM and 50-100 microM), or alternatively that the system is composed of two or more negatively co-operating subunits (or units). It was shown by t.l.c. that the transported radioactivity was choline and that it was not significantly acetylated. Replacing Na+ by K+ on the outside of these liposomes partially inhibited uptake, and the formation of a potential gradient (inside negative) with valinomycin increased the total but not the saturable components of uptake when liposomes were prepared in a K+ medium, and transferred to an Na+ medium.     

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5-7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple-blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H(+). At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.     

The effect of the glycosaminoglycan chain removal on some properties of the human urinary trypsin inhibitor

The major urinary trypsin inhibitor UTI I is a proteoglycan. UTI c (Mr 26,000), produced by chrondroitin lyase digestion of UTI I, was isolated and characterized. About 90% of the glycosaminoglycan chain was removed by this treatment without proteolytic modification, as assessed by amino-acid composition and N-terminal sequence of UTI c. Its electrophoretic mobilities on alkaline and SDS-PAGE are identical with those of UTI II which occurs in urine during storage. To study the role of the glycosaminoglycan chain on the inhibitory properties of UTI I, UTI I and UTI c were compared using different proteinases as target enzymes. The inhibitory activity towards bovine trypsin and chymotrypsin as well as human granulocytic cathepsin G did not differ significantly. However, towards human granulocytic elastase, the equilibrium dissociation constant (Ki) is 5 times higher for UTI c than for UTI I. Weak inhibitory activities were measured on human plasmin, UTI c being more efficient than UTI I. The acid-stability of UTI I is not modified after chrondroitin lyase treatment. UTI I and UTI c are equally sensitive to trypsinolysis indicating that the covalently bound glycosaminoglycan chain does not play an important role for the stability of UTI I.     

The incorporation of tritiated retinyl moiety into the active-site lysine residue of bacteriorhodopsin.

Purple membranes were isolated from Halobacterium halobium bleached and regenerated with all-trans-[15-3H]retinal. The incorporation of label was 1.2 mol of retinal/mol of bacterio-opsin. The [3H]retinyl-bacterio-opsin obtained from regeneration was hydrolysed to give tritiated retinyl-lysine, which, on hydrogenation to N-epsilon-perhydro[3H]retinyl-lysine and reaction with 1-fluoro-2,4-dinitrobenzene, gave bis-(2,4-dinitrophenyl)-N-epsilon-perhydro[3H]retinyl-lysine. This result confirmed that the retinyl moiety of the chromophore is attached to an epsilon-amino group of lysine.     

Effect of sterol incorporation on head group separation in liposomes

Electrophoretic mobilities of multilamellar liposomes of varying composition have been measured to determine the effect of incorporated sterols on surface charge density. Liposomes made from mixtures of zwitterionic egg phosphatidylcholine (PC) and anionic egg phosphatidylglycerol (PG) in varying proportions were shown to have electrophoretic mobilities consistent with the anticipated surface charge density. Incorporation of cholesterol up to 50 mole per cent in the bilayer produced no detectable change in surface charge density. Similar results were obtained for lanosterol and epicoprostanol. These results are interpreted to mean that incorporation of the sterols into the bilayers produced no detectable change (less than 3%) in the spacing of charged phospholipids. It is inferred that sterols are incorporated among the fatty acyl chains of these phospholipid bilayers with little or no displacement of the head groups at the surface.     

The effect of chain length and lipid phase transitions on the selective permeability properties of liposomes.

This paper describes experiments showing the importance of the fatty acid chain length on the barrier properties of liposomal bilayers, prepared from saturated lecithins, under conditions of lateral phase separation. 1. Above the gel to liquid crystalline phase transition temperature, liposomes prepared from saturated lecithins with 14 or more carbon atoms per acyl chain exist as stable bilayers, which are practically impermeable to ions. 2. At temperatures well above the transition temperature dilauroyl phosphatidylcholine liposomes exhibited osmotic shrinkage, which was dependent on the ionic size of the solute used to bring about the osmotic gradient, indicating that the permeation through these less stable bilayers takes place mainly via individual diffusion of the permeating ions. 3. An enhanced release of trapped potassium from liposomes was demonstrated in the vicinity of the transition temperature. The extent of the increase, however, depended strongly on the length of the paraffin chain. 4. From measurements of the shrinkage behaviour of liposomes in the vicinity of the transition temperature it is concluded that the increased permeability decreases with increasing diameter of the permeating ion. This finding implies that the increased permeability at the transition temperature cannot be ascribed to "macroscopic" rupture of the liposomal membrane. The maximum permeability in the vicinity of the Tc is discussed in terms of probability and size distribution of statistical pore formation at the boundaries of liquid and solid domains.     

The effect of pH on proton transport by bacteriorhodopsin

The pH-dependence of proton motion during the photocycle was investigated by measuring the photoelectric signals due to charge displacement inside bacteriorhodopsin molecules. Measurements were performed on purple membranes oriented in suspension and the kinetics of flash excited electric and light absorption signals was compared. It was found that in the pH range 4.5–8 the photocycle and the successive proton movements have identical kinetics, and do not depend on pH. In the pH range 8–10 both kinetics change, though differently; the charge motion decouples from the photocycle and the photocycle seems to split up into two parallel paths, the photoelectric signal becomes faster. However, the net proton transfer remains the same as at lower pH values. Above pH ≈ 10, the photocycle behaves differently and cannot be described by the parallel pathway model and the net proton displacement drops. The results are explained by the successive titration of two groups (probably tyrosine) participating in proton translocation.     

Counterion collapse and the effect of diamines on bacteriorhodopsin

A recent report of electrical measurements on oriented bacteriorhodopsin in gels [(1986) FEBS Lett. 195, 164 168] concluded that low concentrations of diamines reversed the direction of the proton pump. Calculations are presented which show that in low diamine concentrations, charge displacements of the counterion atmosphere in the direction opposite to proton pumping are expected following H+ ejection. It is also shown that the effect will be sharply reduced by raising the diamine concentration or by adding excess salt, as was observed. Hence it is not necessary to conclude that diamines reverse the direction of the proton pump itself.     

Thermodynamical characteristics and volume compressibility of dipalmitoylphosphatidylcholine liposomes containing bacteriorhodopsin.

The effects of bacteriorhodopsin (BR) interaction with large dipalmitoylphosphatidylcholine (DPPC) liposomes (approx. 100 nm in diameter) were examined at various BR/DPPC ratios, using differential scanning calorimetry (DSC) and ultrasonic velocimetry (USV). On DSC, the lipid phase transition temperature, Tc, and the half-width of the phase transition peak, delta T1/2, showed significant non-monotonic changes with the increasing BR concentration. Two exponential segments could be distinguished in the dependence of the transition enthalpy change per mol of lipid (delta H/nL) on the BR/DPPC ratio: one corresponding to ratios between 0:1 and 1:64, and another corresponding to ratios between 1:44 and 1:16. A maximal value of delta H/nL was observed for BR/DPPC ratio 1:44, probably corresponding to maximal BR-lipid ordering with each BR molecule being surrounded by two layers of lipid molecules. The nonmonotonic changes of thermodynamical parameters suggest long-distance interactions between regions of altered bilayer structure which form around each BR molecule. The results obtained with USV provided support for the above conclusions. The dependence of ultrasound velocity increment A on BR concentration supplies information on relative changes of membrane volume compressibility. Decreasing volume compressibility is reflected in increasing values of parameter A. Within T less than Tc, the values of A increased with the increasing BR concentration; saturation was observed at BR/DPPC ratio 1:500 (A = A(BR/DPPC]. No significant BR-concentration dependent changes of A were observed at T greater than Tc. From these values the average diameter of the distorted region of lipid bilayer was estimated to be approximately 20 nm.