Tobacco Documents Online

a TIiT•. COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. Memorandum To: Dr. S.C. Sommers From: D.H. Ford NK@1168002 Re: Site visit with Dr. A. McLaughlin, Brookhaven National Laboratory, Upton, N.Y. bctober 13th, 1982 Grant No. 1493; Title: 'Interaction of Divalent Cations with Model and Biological Membranes.' Overview: As stated in Dr. McLaughlin's proposal, his objective; are to examine the molecular mechanisms by which divalent cations such as calcium (Ca) interact with bilayer membranes formed from phospholipids and glycolipids (model membranes). Thus, he will be determining the divalent cation binding sites and their ability to indurce lateral phase separation in membranes formed from taix- tureA of neutral and charged lipids. This should eventually en- hance our understanding of the significance of the interaction of such cations with the bilayer component of cellular and sub- cellular membranes. A second related objective concerns his in- terest in examining t he interaction of Ca with the chromaffin granule membranes so as to induce release of the granule compo- nentg (ATP and epinephrine). He believes that Ca binds to the surface of the chromaffin granule membrane to induce a K flux which is coupled to the flux of a proton to cause an increase in osmotic pressure and the subsequent disruption of the membrane causing exocytosis of the contents of the granule (ATP and epinephrine). Biological significance: Important in understanding release of secretory products into blood or extracellular spaces; important in understanding role of Ca and other ions in determining surface potential of such membranes as neurons and muscles in relation to N4 and C1 channels and conductance. A basic question in s uch studies is: When divalent cations change the electrical potential of a membrane receptor site, do the cations bind to the charge on a mer•brane or near it or to a specific receptor or do they merely screen the charge or receptor on the membrane? Techonolgy of NMR: Dr. McLaughlin provided an elegant lucid review of Nuclear Magnetic Resonance, which is a spectroscopic technique based on a static magnetic field and a radio frequency field causing a rotat ional effect on the nucleus of an-atom of carbon, phosphorus or hydrogen to provide a measurable perturba- tion of the field as a function of that particular radio frequency. This can provide information about the physical characteristics of a molecule (more precisely, specific atoms - C, P,'H- within a molecule).

HK111680a3 Advantages: Gives more information than U.V. spectrometry relative to the action of protons of H, P. and C. The character- istic movements of the protons of these atoms are very sensitive to environmental changes (i.e., pH, presence of divalent cations such as Ca or Co. and motional constraints within the nucleus of an atom. Occasionally not all atoms will react the same way to the radio frequency, as happens with ATP. o( and j' are pea!s from ADP, additiornal7j , kp peak wi ll form when ATP is scsnned, The a( peak is sensitive to p.4 ahadges ` - J /o y.w.. ArP Disadvanta es: Low sensitivity; thus need s large a~mounts of sample (20-30 mgs ml of a protein or 5-10mg/mi with membranes. Also, sometimes provides too much information (L.e., from all the protons wittdn a protein). The Model: Initial studies have used an a'rtifical bilayer membrane system, althcugh he is also using chromaffin granules within cells andhas a purified preparation from cells. In a related study he has been using the granules within intact platelets. The model system can be used to check the -validity of the Guey-Chapman-Stern theory, which deals with the surface potentials of the membranes of biological sy stems. This theory depends on the concept that the positive and negative charges which exist on opposite sides of a bilayer membrane form a con- tinuous field of either + or - charges over the entire surface. Some of the data assembled by McLaughlin and others suggest the charges may be collected together in clusters, le aving large areas of the membrane surface uncharged. Thus, this concep t needs to be confirmed before studies can proceed utilizing the theory. Using a paramagie tic series of cations (have large,effects on NMR signals) instead of diamagnetic,cations (have small effects on the signal), Dr. McLauglin should be able to d etermine what bccurs when a cation binds loosely or tightly to a membrane. Should be able to determine types of ligands bound, type of com- plex bound to, and lifetime of complex. While he is interested gpecifically in effects of Ca, he is using Co*as a test cation for h number of technical reasons. This has nece ssitated that he demonstrate that Co act in a manner similar to Ca in his system. This it appears to do, acting the same as Ca at Na and K channels, with the phosphodiester and carbonyl groups, though it-binds more firmly than Ca to biological molecules and is not transported (so is toxic). * CE++ is diamagnetic and produces veak signa ls wheras Co++ is paramagnetic (unpaired electron) and gives go od signals.

..E~_..:.-.:.-... -3- NK11168004 What 2ffect does a paramagnetie cation such as Co have on the NMR signal, such as obtained from the P,in ATP. Shifts sig- nal to left and broadens the signal base. See figure: Enlarged view ~ NMR signal from AMP with Co ~ .b NMR signal from free A*f Signal Shift to left when Co added I I The addition of Co to the solutimn containing AM P changed the magnetic moment of the proton of the P nucleus. (The greater the motion of the nuclear proton, the narrower the NNE signal. Thus, can obtain information on binding, stochiometry and struct- ural chemistry. Results: Tn the phosphatidyl series Dr. McLaughlin has used both a P and a C NMIt signal in conjunction with the presence of Co. Has been able to determine the number of COOH gr oups bound to Co and their lifetiihe in the molecule. (Studies are still only prelim3nary~). 3as also used a number of large divalent cations (toxins such as curare and ethamethonium, hexamethonium and de- camethQniunt) to determine surface potential in r elation to ~la channels and conductance. Preliminary data su¢gest that the Na channels are not behaving as if they have single point charges. So iar; most of the time applied to the study ap pears to have been to confirm that Co may be used in the system in place of Ca without producing nhissleading data. In the study with the chtomaffin granules, has tried purified granules and encountered some technical difficulties. A lot of ATP was observed in the granules (expected) and it was observed that a change in pH changed the peak of P from A TP. Addition of Co to the system did not appear to change the pH, as might have been expected if the flux of K and the proton fr om P were to act together to increase the internal osmolarity of the granule. There- fore, pH may not be involved with changes in osm otic pressure (as he theorizes) to lead to extrusion of the Rranul es. The failure to observe this may have been caused by the purification of the granules,due to a lass of calrnodulin, thereby pr eventing a response to Ca (or Ca acting liki Ca). Now planning to w ork on intact cells wherein the calmodulin will not have been remove d during purifi- cation of the granules. Also has st;arted workin R with platelets as a modell system, since they are easier to handle. Has found that 0.5 M sodium and asperin inhibit the release of the granules. When he adds thrombin to the solution, the signal pea k for ATP is lost from tlie cytoplasm of the platelet, while the ATP in the granules is still present and unchanged. Further, there was no change in pH c,-iused by the addition of Co. This too sugge sts that a change in

-4- 0 NK®1168005 pH within the granules is not associated with extrusion"of the con- tained granules. Would appear he may have to change his theory. Future: Government cutbacks in funding for research are having an effect at Brookhaven. This had led Dr. McLaughlin to consider shifting his affiliation to Stonybrook SUNY (has a partial affiliation at present). This would require moving his laboratory and cause, lie estimates, about a 3 month delay in the work. If he moves, he plans to take all his equipment with him. his would lead to a need to refit the magnet of the NMR apparatus at a cost of,about $15,000 plus a cost for cooling the magnet of $5000. Despite this shift, it would appear likely that he would complete his project within the requested 3 years. Comment: A bright young man with a clear understanding of the strenRths and weakensses of NMR and how it may be applied to increase our knowledge of the function of biological membranes. His current atudy with platelets would appear to open up con- siderations for other sutdies related to clotting mechanisms. Recommend continued support. D.H. Ford DHF/rg