Ivan J. Ryrie

Modification of the chloride requirement for photosynthetic O2 evolution: The role of the 23 kDa polypeptide

In thylakoid preparations from spinach and the halophyte Avicennia marina a correlation is observed between functional O2 evolution at low chloride concentrations and the presence of the 23 kDa protein. Addition of spinach 23 kDa protein to polypeptide‐depleted halophyte inside‐out thylakoid vesicles lowers their chloride requirement for optimal O2 evolution activity from 250 to 5 mM. It is suggested that the specific role for the 23 kDa protein is to increase the affinity of the water oxidation site for chloride.

The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant.

1. The subunit compositions of the F1 (oligomycin-insensitive) and F1--F0 (oligomycin-sensitive) mitochondrial ATPase complexes from Saccharomyces cerevisiae have been examined by the highly resolving technique of sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis using a discontinuous buffer system. When isolated in the presence of protease inhibitors, F1 and F1--F0 contained five and twelve bands, respectively; this contrasts with the four- and ten-band patterns seen previously using the less resolving disc gel method. When isolated in the absence of protease inhibitors both F1 and F1--F0 contain spurious polypeptides produced by proteolytic modification. 2. Endogenous protein turnover in S. cerevisiae was impaired in the presence of protease inhibitors. F1--F0 isolated from cells grown in the presence and absence of inhibitors contained an identical polypeptide composition, suggesting that the subunits are not significantly modified by endogenous proteases prior to cell harvesting. 3. Yeast F1--F0 prepared in the presence of protease inhibitors contains a latent, sodium dodecyl sulphate-activated protease contaminant. Sodium dodecyl sulphate-induced proteolysis is largely confined to the 52 000 dalton alpha subunit which degrades into polypeptides of 40 000 and 10 700 daltons. The 40 000 dalton band is apparently equivalent to the polypeptide previously designated subunit 3. 4. Both F1 and F1--F0 were isolated from Torulopsis glabrata, a yeast with considerably shorter mitochondrial DNA than that in S. cerevisiae. F1--F0 catalysed high rates of ATP--32Pi exchange when reconstituted into phospholipid vesicles, thus demonstrating the presence of a complete coupling mechanism. F1--F0 contained approximately twelve subunits and F1 five, like the S. cerevisiae complexes. It therefore appears that the shorter mitochondrial DNA length does not produce a significantly simpler ATPase subunit structure.

Purification and partial characterization of the oligomycin-sensitive ATPase from yeast mitochondria.

Abstract The oligomycin-sensitive ATPase protein has been purified in a properly dispersed form from yeast mitochondrial membranes and has been further characterized, particularly with respect to parameters which affect ATPase activity. The protein is to some degree cold labile, the rate of inactivation being accelerated by chaotropic anions. Essentially complete protection against cold inactivation is afforded by methanol, ethanol, and ADP. A partly latent component of the ATPase activity has been discovered which is activated either by heating in the presence of ATP, or to some extent by trypsin. The heat-activated protein is oligomycin-insensitive and much more susceptible to inactivation by cold. N -ethylmaleimide, and trypsin. It is suggested that like the ATPase from spinach chloroplasts and bovine mitochondria, the yeast protein may contain an ATPase inhibitor polypeptide which is dislodged either by heating or by proteolytic enzymes.

The yeast mitochondrial adenosine triphosphatase complex. Purification, subunit composition, and some effects of protease inhibitors.

Abstract ( i ) The method of preparing the oligomycin-insensitive F 1 -ATPase by chloroform treatment of mitochondrial membranes (Beechey et al. , 1975, Biochem. J. 148 , 533–537) has been modified such that a five-subunit protein is obtained from yeast with an activity of 140 μmol of ATP hydrolyzed/min/mg of protein. Repetition of this procedure in the presence of protease inhibitors (in particular, p -aminobenzamidine) allows isolation of a four-subunit protein with an activity of 243 μmol of ATP hydrolyzed/min/ mg of protein, ( ii ) A modified procedure is described for the preparation of the yeast oligomycin-sensitive F 1 -F 0 ATPase complex, making use of protease inhibitors throughout and solubilization of the ATPase from mitochondrial membranes using Triton X-100 and sodium deoxycholate simultaneously. Two polypeptides Of 42,000 and 29,000 molecular weight are eliminated, the largest corresponding to the missing band of the F 1 sector. The complex retains oligomycin- and uncoupler-sensitive ATP- 32 P i exchange and ATP-driven proton uptake, indicating the retention of a complete coupling mechanism. ( iii ) F 1 -ATPase is released from the F 1 -F 0 complex by brief heating at 50 °C in the presence of ATP. The remaining hydrophobic polypeptides aggregate and are isolated by centrifugation. The F 1 sector can be isolated containing either four or five subunits depending on whether the starting F 1 -F 0 complex contained the 42,000 and 29,000 molecular weight polypeptides. ( iv ) Sensitivity of the F 1 -F 0 ATPase complex to oligomycin and dicyclohexylcarbodiimide varies considerably depending on the activity measured and whether the complex was first reconstituted with phospholipids. The degree of inhibitor sensitivity is considered a poor guide to intactness of the complex.

Reconstitution of ATP-32Pi exchange by phospholipid addition to the purified oligomycin-sensitive ATPase from yeast mitochondria

Abstract A purified preparation of the oligomycin-sensitive ATPase from yeast mitochondria has been shown to elicit ATP-32Pi exchange when combined with phospholipids. The reconstitution was normally carried out by dialysis of an ATPase-phospholipid-bile detergent mixture, but could also be achieved by direct addition of the lipid. Vesicle structures with diameters between 200 and 1500 A were seen by electron microscopy. The ATP-32Pi exchange was independent of electron transport but sensitive to uncouplers and energy-transfer inhibitors. As in mitochondria, ATPase activity in the reconstituted system was stimulated by a range of uncouplers which inhibited ATP-32Pi exchange. Taken together, the results raise the possibility that the terminal coupling mechanism might still be intact within the ATPase complex.

Studies on oxygen evolution of inside-out thylakoid vesicles from mangroves: Chloride requirement, pH dependence and polypeptide composition

Inside-out thylakoid vesicles from the halophyte Avicennia marina were isolated by the aqueous polymer phase partition method. Oxygen-evolution activity measured with ferricyanide and phenyl-p-benzoquinone was absolutely dependent on added chloride, since the vesicles were almost completely depleted of the 23 and 16 kDa polypeptides of the O2-evolving complex. Addition of the spinach 23 kDa protein to the mangrove inside-out vesicles lowered their chloride requirement for O2 evolution at least 50-fold. In the absence of added chloride, the mangrove vesicles were very sensitive to inhibition by light, which could be prevented by high chloride or low chloride plus added purified spinach 23 kDa protein. The preparations were also inactivated by neutral or alkaline pH (greater than 7.2) in the absence of high chloride concentrations. This inactivation was not significantly influenced by addition of spinach 23 kDa protein. Chloride binding and alkaline inhibition may therefore be closely related, either directly via the manganese centers or, more likely, via pKa changes in as yet unidentified proteins.

Energy-linked activities in reconstituted yeast adenosine triphosphatase proteoliposome: Adenosine triphosphate formation coupled with electron flow between ascorbate and ferricyanide

Abstract (1) Conditions are described wherein the yeast oligomycin-sensitive adenosine triphosphatase (ATPase) complex can be reconstituted together with phospholipids to yield extremely high rates of ATP- 32 P j exchange. The vesicles so formed exhibit proton uptake upon addition of Mg 2+ -ATP and a relatively slow decay of the proton gradient. (2) The stimulation of ATP- 32 P i exchange by valinomycin + K + reported previously (Ryrie, I. J. (1975) Arch. Biochem. Biophys . 168, 704–711) is apparently not simply due to a diffusion potential. The findings suggest that an electroimpelled, valinomycin-dependent migration of K + may occur together with the electrogenic movements of protons during ATP hydrolysis and synthesis to establish optimal energized conditions for ATP- 32 P i exchange. (3) An artificial oxidative phosphorylation system in the reconstituted vesicles is described: [ 32 P]ATP formation from ADP and 32 P i is shown to be linked with electron flow between external ascorbate and internal ferricyanide where a permeable proton carrier, such as phenazine methosulfate, is used to establish a proton gradient. That the yeast ATPase is capable of net ATP synthesis has also been demonstrated in a light-dependent reaction using ATPase proteoliposomes reconstituted together with bacteriorhodopsin.

Membrane adhesion in reconstituted proteoliposomes containing the light-harvesting chlorophyll ab-protein complex: The role of charged surface groups

The light-harvesting chlorophyll ab-protein complexes (LHCP) of spinach, pea, and barley thylakoids apparently contain four nonidentical polypeptide subunits of between 29,000 and 23,000 daltons on highly resolving sodium dodecyl sulfate-polyacrylamide gradient gels. Trypsin treatment of the spinach complex degraded at least the two major subunits by approximately 2000 daltons and resulted in a three-subunit pattern on gels. Proteoliposomes reconstituted with LHCP and the chloroplast diacyl lipids aggregated markedly in the presence of cations but vesicles containing LHCP prepared from trypsin-treated thylakoids did not. Amino acid analysis of native- and trypsin-treated LHCP indicated that the fragment(s) released by trypsin, which is essential for cation-induced stacking of thylakoids, contains lysine and arginine, but not aspartate or glutamate, and is thus cationic. Carboxyl groups on the surface of LHCP were charge neutralized using a water-soluble carbodiimide (1-ethyl-(3-dimethylaminopropyl)carbodiimide) plus [14C]glycine ethyl ester. Only two or three sites were labeled per 26,000-dalton polypeptide equivalent and only a minor fraction of this (22–24%) was located in the surface fragment(s) released by trypsin. Both LHCP and LHCP proteoliposomes, after carboxyl modification, aggregated avidly at low salt concentrations. The findings suggest that exposed anionic groups on the surface of LHCP contribute to an electrostatic repulsive force between membranes which must be attenuated, either by cation binding or chemical neutralization, before membrane-membrane adhesion can occur. In line with this the binding of Mn2+ by LHCP (approximately four Mn2+ bound/26,000-dalton polypeptide equivalent) was sharply decreased after carboxyl modification.

Structure and energy-linked activities in reconstituted bacteriorhodopsin-yeast ATPase proteoliposomes

Abstract Bacteriorhodopsin-F1·F0 (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F1·F0 is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F1·F0 by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [32P]ATP formation where pyridine is present during first stage illumination. F1·F0 can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.

Molecular weight determinations of hydrophilic proteins by cationic detergent electrophoresis. Application to membrane proteins.

Abstract 1. 1. A separation of polypeptides is reported based on polyacrylamide-gel electrophoresis in the presence of the cationic detergent cetyltrimethylammonium bromide. Like sodium dodecylsulphate electrophoresis, it shares the advantages of dissociability coupled with a linear relationship between the migration velocity and the logarithm of the molecular weight at least when applied to the simpler hydrophilic proteins. Polypeptide chain molecular weights can thus be estimated with considerable accuracy. 2. 2. More complex membrane proteins generally produced fewer bands with the cationic detergent than with sodium dodecylsulphate. Studies with the oligomycinsensitive ATPase protein indicate that this is due principally to an exclusion from the gel of the subunits with greatest hydrophobicity.