Ward E. Harris

Conformational changes of purified (Na+ + K+)-ATPase detected by a sulfhydryl fluorescence probe.

The fluorescent sulfhydryl reagent S-mercuric-N-dansyl cysteine (Dn-Cys-Hg+) has been used to label a purified preparation of the (Na+ + K+)-ATPase obtained from the electric organ of Electrophorous electricus. The labelled (Na+ +K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), although reversibly inhibited, was capable of undergoing conformational changes associated with the active enzyme that could be monitored fluorometrically. The presence of ligands (Na+ + Mg2+ + ATP or Mg2+ + Pi) which are known to convert the enzyme from the E-1 state to the E-2-P state brought about large (97--100%) increases in fluorescence of the dimethylaminonaphthalene sulfonyl (Dn) label. An E-2 state could be achieved by the addition of Mg2+ which caused only a 32.3% increase in fluorescence over the E-1 state. Neither AMP nor TTP with or without Mg2+ or Na+ or Pi added without Mg2+ had any effect on the Dn fluorescence. If the enzyme was denatured, no fluorescence changes were observed. Small changes in the polarization of fluorescence of the Dn moiety were observed under all the conditions used. These small polarization changes and the large increases in the fluorescence intensity suggest that the enzyme can change conformational states in the presence of appropriate ligands and these conformational changes may take place in a relatively limited region of the proteins structure.

Ouabain binding sites and the (Na + ,K + )-ATPase of brain microsomal membranes.

Abstract Beef brain microsomes bound approximately 180–220 pmoles of [ 3 H]ouabain per mg of protein in the presence of either MgCl 2 and inorganic phosphate or ATP, MgCl 2 and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na + ,K + )-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO 4 , sodium dodecyl sulfate, p- chloromercuribenzoate , urea. ultrasonication, heating, pH and phospholinase C. The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO 4 as evidenced by increased binding of [ 3 H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.

Interaction of a new fluorescent reagent with sulfhydryl groups of the (Na++K+)-stimulated ATPase

The (Na+ + K+)-ATPase enzyme of rat brain microsomes can be reversibly inhibited by a new fluorescent sulfhydryl (SH) probe, dimethylaminoaphthalenecysteine-Hg+ (Dn-cys-Hg+). This reagent is more reactive than N-ethylamaleimide (MalNEt) toward membrane sulfhydryl groups. A procedure using the two SH reagents sequentially seems to permit a more selective labelling of the SH groups involved in the (Na+ + K+)-ATPase than is possible by using MalNEt alone. Brain microsomes treated by this method incorporate the fluorescent label within or near the active site of the enzyme. When the probe was bound a blue shift of its fluorescence emission maximum (from 540 to 495 nm) and a 20-fold increase in relative fluorescence occurred. This indicates that the Dn moiety is within a very non-polar region of the membrane.

Interactions between fluorescent labeled phosphatidyl serine and cations

Phosphatidyl serine (PS) was reacted with dimethylaminoanphthalenesulfonyl (DNS) chloride to produce a fluorescent phospholipid derivative (DNS--PS). By comparison of the emission maximum of the fluorochrome in solvents of varying dielectric constants and in aqueous suspension it was concluded that the DNS moiety of sonicated DNS--PS micells was within the glycerol region of the lipid. Addition of CaC12 to aqueous suspensions of DNS--PS micells caused a shift in the emission maximum from 524 nm to 490 nm, a two-fold increase in the relative fluorescence and an increase in the polarization of fluorescence and finally aggregation. MgC12 produced similar but smaller changes in the fluorescence parameters. In the presence of calcium the thermal transition of the fatty acid tail region could be monitored as an increase in the fluorescence intensity of the DNS group. Without calcium an almost linear decrease in relative fluorescence was observed. These data suggested that calcium caused the fluorochrome to be shifted from the glycerol region of the lipid structure into the fatty acid tail region. These observations indicate how the conformation of a biological membrane might be altered by the interaction between acidic phospholipids and calcium.

Altering erythrocyte membrane composition with phospholipid exchange protein

Abstract 1. 1. In establishing a protocol for the use of a phospholipid (PL) † exchange protein (PLEP) preparation to incorporate fluorescent lipids into membrane fragments, the potential for the PLEP preparation to alter membrane composition was investigated. 2. 2. Cholesterol was removed from erythrocyte ghosts and PL content was increased after incubation of the membranes with phosphatidylcholine (PC) vesicles and a nonspecific bovine liver PLEP preparation. 3. 3. The elevated PL/cholesterol ratio of the ghosts decreased fluorescence polarization of diphenylhexatriene (DPH), indicating an increased average membrane fluidity. 4. 4. The increased PL/cholesterol ratio and reduced DPH fluorescence polarization were prevented by including cholesterol in the PC vesicles during the initial incubation of ghosts with PLEP preparation and lipid. 5. 5. In addition, the PC content of ghost membranes was elevated after incubation with PLEP preparation and either PC or mixed PC-cholesterol vesicles.

Incorporation of cis-parinaric acid, a fluorescent fatty acid, into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg2+ or Ca2+ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [3H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.

Interactions of adrenergic compounds with brain membrane constituents

Abstract The specific activity of the Na + , K + -ATPase of rat brain synaptosomes was modulated by a series of adrenergic compounds in a manner related to each compounds partition between aqueous and organic solvents. The more organic-soluble compounds, the β-2 adrenergic blockers propranolol, pronethalol and butoxamine, inhibited the enzyme between 13 and 30 per cent. The more aqueous-soluble compounds, the agonists, epinephrine, norepinephrine and isoproterenol, and the β-1 blockers, practolol and acebutolol, stimulated the enzymatic activity by 30 per cent. These effects may be due to non-specific membrane interactions rather than to specific receptor effects. Optical measurements with pure protein and phospholipid indicated that the aqueous-soluble compounds bound to protein while the organic-soluble compounds interacted with acidic phospholipid phosphatidyl serine. The possible consequences of the compounds binding with acidic phospholipids and the resulting effect on membrane properties are discussed.

Origin of the γ polypeptide of the Na+/K+-ATPase

The Na+/K+-ATPase purified from lamb kidney contains a gamma polypeptide fraction which is a collection of fragments derived from the alpha and beta polypeptides of the enzyme. This fraction has the solubility characteristics of a proteolipid and was isolated either by high performance liquid chromatography (size exclusion chromatography) in 1% sodium dodecyl sulfate or by sequential organic extraction of purified lamb kidney Na+/K+-ATPase. Formation of gamma polypeptide(s) from detergent solubilized holoenzyme was accelerated by sulfhydryl containing reagents and was unaffected by addition of inhibitors of proteolytic enzymes. Treatment of the holoenzyme with the photoaffinity reagent N-(2-nitro-4-azidophenyl)[3H]ouabain ([3H]NAP-ouabain) labeled the alpha polypeptide and the gamma polypeptide fraction but not the beta polypeptide. Amino acid sequence analysis of one gamma polypeptide preparation revealed homology of one component of this fraction with the N-terminus of the beta subunit of the Na+/K+-ATPase. Amino acid analysis of two preparations of proteolipid showed similar amino acid compositions with a peptide derived from the alpha subunit. The insolubility and complexity of the gamma polypeptide(s)/proteolipid fraction appears to preclude a conclusive sequence analysis of all components of this fraction.

Lipid Regions of Na,K-ATPase Examined with Fluorescent Lipid Probes

Publisher Summary This chapter examines the interactions between lipids and Na, K-ATPase without disruption of the native membrane lipid. Fluorescent derivatives of cholesterol (dehydroergosterol, DHE) and phospholipids ([ 3 H]dansylphosphatidylethanolamine, DNS-PE; and [ 3 H] dansylphosphatidylserine, DNS-PS) were synthesized and incorporated into Electrophorous electricus Na, K-ATPase using a phospholipid exchange protein (PLEP) preparation. From data gathered on fluorescent lipid probes, the chapter (1) describes interactions between membrane lipid and Na, K-ATPase, and (2) compares Na, K-ATPase with E. electricus membranes enriched in acetyl cholinesterase (AChE) and with erythrocyte ghosts to determine if these interactions are specific for the Na, K-ATPase or if they can be generalized to other membrane proteins. Lipid probes were transferred to Na, K-ATPase by incubating the lipid vesicles and Na, K-ATPase with PLEP. Na, K-ATPase was pelleted by centrifugation. Nonspecific “adherence” of lipid substrate to Na,K-ATPase membranes was calculated from the loss of [ 14 C] triolein in the supernatant. Greater than 80% of the Na, K-ATPase activity was recovered. The chapter also graphically represents the polarization of the lipid probes in a membrane, which is because of membrane protein or for probing concentration. Fluorescence polarization was determined in an SLM 4800 nanosecond spectrofluorometer.

Preparation of a ouabain-binding membrane fraction from brain

Abstract A procedure employing 2 M NaClO 4 is presented that allows the mild disruption of brain microsomal membranes and yields three distinct fractions. One fraction has a relatively high ouabain-binding capacity but low (Na + , K + )-ATPase activity. This fraction was shown by polyacrylamide gel electrophoresis to be very similar in protein composition to that of the initial microsomes but the phospholipid content was lower. It is suggested that the enzyme in this fraction lacks either necessary phospholipids or a membrane matrix needed for complete activity. The presence of enzyme as indicated by the binding of this specific inhibitor may be useful in further purification of the (Na + , K + )-ATPase enzyme system.