PARTX. THE LIPID BILAYERMEMBRANE AS
AN ASSAY FOR ION-CONDUCTANCE-INDUCING MATERIALS ISOLATED FROM BIOLOGICAL MEMBRANES
THE INTERACTION OF H E M O C Y A N I N AND L Y M P H O C Y T E S WITH LIPID BILAYER M E M B R A N E S Robert Blumenthal Laboratory of Theoretical Biology National Cancer Institute National Institutes of Health Bethesda, Maryland 2001 4
INTRODUCTION Two different examples of the use of the lipid bilayer as an assay for ion-conductance-inducing materials are shown. In the case of hemocyanin, a giant respiratory protein dissolved in the hemolymph of Mollusca and Arthropoda, Pant and Conrad‘ had shown that the aggregated form produces electrical conductance increases of as much as six orders of magnitude when added to the aqueous solution after forming the lipid bilayer membrane. Alvarez, Diaz, and Latorre’ carried out discrete current fluctuation analysis of the hemocyanin-induced conductance and showed that the ion transport is mediated by a channel mechanism. The dissociated form of hemocyanin also induces conductance changes in lipid bilayer membranes, which is more likely to be mediated by a “defect” mechani~m.~ In the case of lymphocytes, it h’ad been postulated that the killing of target cells is caused by an initial permeability change to cations4 and subsequent colloid osmotic lysis. The bilayer experiments indicate th,at the permeability change can be induced without any contribution of target cell cytoplasm or membrane proteins.’
HEMOCYANIN Molluscan hemocyanins are cylindrical molecules with a diameter of about 340 A and a height ranging from 140 A to 680 A, built up from 3 to 12 rows of subunits.‘ Hemocyanin readily dissociates and aggregates upon changing the pH, Ca2+concentration, and hemocyanin concentration.’ Aggregated hemocyanin, which has a sedimentation coefficient of about 100 S, dissociates into half-molecules at pH above 7 or below 5, and next into smaller units, which are tenth molecules. At still higher pH values. the tenths dissociate into units, which are found to be twentieth molecules. Three-dimensional image reconstruction* of hemocyanin indicates that the subunits are arranged in a helical order giving rise to a structure with an internal polar core or “channel.” The differences between the dose response curves of associated (A) and dissociated (D) hemocyanins are shown in FIGURE1. The associated form is a dispersion of commercial (Schwarz-Mann) key-hole limpet hemocyanin in deionized w.ater. The dispersion contains a mixture of twentieth, 476
Blumenthal : Hemocyanin and Lymphocytes on Bilayers
477
FIGURE1 . Conductance-concentration curve for associated ( A ) and dissociated (D) hernocyanin in 100 mM KCl, pH = 7. The vertical bars ( l e f t ) represent conductance increases within four minutes after increasing the associated hemocyanin concentration; the dots (right) represent dissociated hem* cyanin-induced conductance.
tenth, half, and whole aggregates.' The higher-order concentration-dependence of the conductance indicates that a number of units aggregate to form a channel. The straight portion of the conductance-concentration curve yields a slope of six on a logarithmic scale which is an average value for the insertion of one, two, ten and twenty units into the bilayer to form a channel. The higher order concentration-dependence has been observed in the case of the other well-known channel-formers, nystatin' and alamethicin.'O Dissociated hemocyanin, prepared according to the method of Weigle" consists of twentieth units, which have irreversibly lost their capacity to reaggregate. The dose-response curves in FIGURE1 show three marked differences between associated and dissociated hemocyanin. For the same hernocyanin concentration at 80 pg/ml there is about two orders of magnitude difference in conductance increase with associated hemocyanin. This effect is even more dramatic when we take into account that the dissociated hemocyanin is about tenfold more concentrated than the associated hemocyanin on a molar basis, because the molecular weight of associated hemocyanin is about tenfold that of dissociated hemocyanin. The other difference is that upon addition of the dissociated hemocyanin to the solution, a steady-state conductance level is reached immediately, whereas with associated hemocyanin concentrations greater than 40 pg/ml the conductance keeps increasing at a slower rate after a large stepwise increase after addition of the hemocyanin in the external solution. The later conductance increase is actually indicated in FIGURE1 by the vertical bars, which represent a conductance change within four minutes. The third difference is that the straight portion of the conductance-concentration curve yields a slope of six on a logarithmic scale in the case of associated hernocyanin, whereas the dissociated hemocyanin yields a slope of one. Upon addition of associated hemocyanin to the bathing solution, the current increases in discrete steps ("formation bumps") at a given voltage, with conductances of about 0.1 nmho in 100 mM KCI (FIGURE 2 ) . Discrete conductance steps induced by associated hemocyanin were originally observed by Alvarez, Diaz, and Latorre,' who showed that the voltage-dependence of the conductance-step fluctuation correlates very well with the voltage-dependence of the conductance of a many-channel membrane. Discrete conductance steps were not observed with dissociated hemocyanin.
Annals New York Academy of Sciences
47 8 I
4r
[ 50mV
_I_.
, ,
P
,,.-
FIGURE2. Stepwise increase in current (middle truce), driven by a constant voltage (upper truce), after adding 20 pg/ml associated hemocyanin in 100 mM KCl, pH = 7. The lower trace marks one-second intervals. The rectification in the current-voltage characteristic for associated hemocyanin, which had been observed by Pant and Conrad’ and by Alvarez et u1.t is shown in FIGURE 3. Dissociated hemocyanin, however, exhibits a linear 3. current-voltage characteristic as shown in FIGURE The nature of the conductance induced into the lipid bilayer membrane by associated hemocyanin (discrete conductance fluctuations, higher-order dose-response curve and rectification) and its three-dimensional structure’ indicate that ion transport is mediated by a channel mechanism. The single subunit of dissociated hemocyanin, on the other hand, which has no possibility of forming a polar core, together with the nature of its conductance induced into the lipid bilayer (absence of discrete conductance steps, first-order doseresponse curve and linear current-voltage curve) points to an alternative mechanism mediating the ion transport. As shown in FIGURE 4 insertion of a single subunit into the lipid matrix could give rise to a “crack” and conductance could be induced by a “defect” mechanism. In that case the lipids do not completely surround the inserted protein and resulting leaks could cause ion permeation. The protein could possess some hydrophobic moiety that attaches to the bilayer. The possibility of a “defect” mechanism for ion transport
FIGURE3. Current-voltage curves for dissociated ( D ) and associated (A) hernocyanin-doped membranes. The curve marked AT represents the current and voltage taken from a triangular voltage sweep at a rate of 15.2 mV/sec and the curve marked As is obtained from the steady-state current, driven by voltage pulses lasting 20 seconds. The experiments were in 100 mM KCl, pH = 7; associated hemocyanin was at 40 pg/ ml, dissociated hemocyanin at 100 pg/ml. The current of the dissociated hemocyanin-doped membrane was normalized to that of associated hemocyanin-doped membrane.
Blumenthal:
Hemocyanin and Lymphocytes on Bilayers
419
FIGURE 4. Model for the “defect” mechanism of ion transport through a lipid bilayer membrane. The protein consists of a single subunit and contains a small hydrophobic area (shaded). The incomplete insertion of the protein into the lipid matrix gives rise to a leak through which ions could flow.
of the “closed” channel of excitability-inducing material (EIM) has been suggested by the experiments of Latorre et aL1’ on the temperature-dependence of the conductance of the “open” and “closed” channels of EIM. Latorre et a1.I’ found that the conductance of the open channel increased as expected from the temperature-dependence of the mobility of ions in water, whereas the conductance of the closed channel decreased with increasing temperature. The experiments indicate that the closed channel conductance is mediated by a “defect” mechanism and that at higher temperature the increased lipid fluidity “seals” the crack around the protein. Del Castillo et d.Iahad reported previously on the conductance induced into thin lipid films by enzymes or antibodies and the enhancement of the conductance caused by substrate-enzyme or antigen-antibody interaction. I believe that those conductances are caused by a defective mechanism and that the interaction with substrate or antigen enhances the size of the defect.
LYMPHOCYTES In order to study more closely the first stages of lymphocyte-mediated cytolysis of target cells, Henkart and Blumenthals prepared black lipid membranes consisting of oxidized cholesterol and DNP-phosphatidylethanolamine, synthesized from bacterial phosphatidylethanolamine by the method of Kinsky.” The lipid bilayers were formed across a 330-,Ahole in a thin bottom Teflon cup that is immersed in a small lucite chamber. The antigenic membrane was coated by injecting 5-,AAvolume of 1-2 mglml purified anti-TNP antibody directly above the membrane followed by 20-minute incubation. Lymphocytes were then injected in a 5-pl volume (of a suspension of lo’ cells/ml) down onto the bilayer membrane, became visible in the microscope immediately, and finished settling within five minutes as shown in FIGURE5. The lipid bilayer could support dozens of lymphocytes for periods of more than an hour before membrane-breakage occurred. The conductance of the lipid bilayer membrane was monitored at all stages of antibody and lymphocyte incubation. As shown in FIGURE 6, the “antigenic” oxidized cholesterol-DNP phosphatidylethanolamine bilayer had a conductance between 10-0 and lo-*mho/cma. Exposure to the antibody had no effect on this conductance. (The antigen-antibodyinduced conductance observed by Del Castillo et al.” is very dependent upon
480
Annals New York Academy of Sciences
FIGURE 5. Lymphocytes resting on the antibody-coated lipid bilayer. This photograph was taken through the phase microscope during the course of an experiment. A torus containing a thick layer of lipid, including bubbles, surrounds the bilayer, which appears dark under phase optics. Approximately 50 lymphocytes can be seen on the bilayer.
the lipid forming the bilayer. With the lipids we used we never observed a significant antibody-antigen-induced conductance change). A marked conductance change was observed within 2-20 minutes after lymphocytes settled on the membrane. Two types of patterns of conductance that change with time, when the bilayer membrane was clamped at a fixed voltage, are shown 6. The conductance increase was voltage-dependent: the increases in FIGURE only occurred when the membrane potential was positive on the lymphocyte side of the membrane. Since the electrical potential difference across cellular membranes is generally positive on the outside with respect to the inside of the cell, the polarity conditions for lymphocyte-mediated killing are the same in the lipid bilayer as in the target cell. Observation of discrete conductance steps and the absence of a lymphocyte-mediated conductance change in thick membranes indicated that the response is mediated by a channel mechanism. The system has all its controls built in, as shown in TABLE1. Lymphocytes resting on the bilayer in the absence of antibody did not induce significant conductance changes, which is analogous to the cytotoxicity of target cells, which is also antibody-dependent. The other controls shown in TABLE1 (F(ab)3 antibodies, which do not contain the Fc fragment recognized by the lymphocyte
Blumenthal : Hemocyanin and Lymphocytes on Bilayers
C
Noanti TNP
r35 t
481
IMlCl
lalcl
10-7
and lymphocytes lacking the Fc receptor) also correlated well with the targetcell-killing experiments. T h e experiments show that lymphocyte- and antibodydependence of membrane conductance increase is identical to that of target c ~ l lysis l and the results support the hypothesis that the membrane-permeability increase is the initial event in lymphocyte-mediated cytolysis. The experiments d o not distinguish yet whether the channel-forming material is released from TABLE 1
ANTIBODYAND LYMPHOCYTE-DEPENDENCE OF MEMBRANE CONDUCTANCE INCREASE
Antibody
Lymphocytes
Percentage of W r Release by Tnp-RBC (No. of Exp.*)
Rabbit anti-Tnp
Human PBL Human PBL Human PBL None Fc receptor negative
100 0-2 (4) 4-5 (2) -1-8 (4) 3-15 (4)
None Anti-Tnp-F(ab), Anti-Tnp Anti-Tnp
Membrane Conductance Increase (No. of Exp.) 25OX-20,OOOX (12) 0-1OX (6) 0 (1) 0-5X (2f) 0 (1)
* Relative to complete system (line 1). f In each experiment the membrane is incubated with antibody alone for 20 minutes; conductance increases greater than 5x were never seen. In two experiments the membranes were incubated with antibody alone for one hour.
482
Annals New York Academy of Sciences
the lymphocyte and subsequently inserted into the bilayer or whether the conductance change is induced while the channel-forming material is still attached to the lymphocyte surface. The experiments on the interaction of lymphocytes with the antigen-antibody-coated lipid bilayer membrane clearly show that the permeability change and consequently cytolysis can be induced without any involvement of target-cell cytoplasm or membrane proteins. The system thus provides us with a new approach to study the molecular nature of lymphocyte-mediated cytolysis and the molecular basis of the specificity of the response. References 1972. J. Membr. Biol. 8: 357-362. 1. PANT,H. C. & P. CONRAD. O., E. DIAZ & R. LATORRE.1975. Biochim. Biophys. Acta 3 8 9 4442. ALVAREZ, 448. R. 1975. Submitted for publication. 3. BLUMENTHAL, J. C. & K. T. BRUNNER. 1974. Adv. Immunol. 18: 67-132. 4. CEROTTINI, 5. HENKART, P. & R. BLUMENTHAL. 1975. Proc. Nat. Acad. Sci. U.S.A. 72: 27892793. 6. FERNANDEZ-MORAN, H., E. F. J. VAN BRUGGEN & M. OHTSUKI. 1966. J. Mol. Biol. 16: 191-207. 7. KONINGS,W. N., R. J. SIEZEN& M. GRABER.1969. Biochim. Biophys. Acta 194: 376-385. 8. MELLEMA,J. E. & A. KLUG. 1972. Nature (London) 239: 146-150. 9. FINKELSTEIN,A. & A. Chss 1968. J. Gen. Physiol. 52: 145S-172S. 10. MUELLER,P. & D. 0. RUDIN. 1968. Nature (London) 217: 713-719. 11. WEIGLE,W. 0. 1964. Immunochemistry 1: 295-302. 12. LATORRE, R., 0. ALVAREZ& P. VERDUGO.1974. Biochim. Biophys. Acta 3 7 6 361-365. 13. DELCASTILLO,J. A. RODRIQUEZ, C. A. ROMERO& V. SANCHEZ. 1966. Science 153: 185-188. 14. UEMURA,K. & S. C. KINSKY.1972. Biochemistry 11: 4085-4094.
AN ASSAY FOR ION-CONDUCTANCE-INDUCING MATERIALS ISOLATED FROM BIOLOGICAL MEMBRANES
THE INTERACTION OF H E M O C Y A N I N AND L Y M P H O C Y T E S WITH LIPID BILAYER M E M B R A N E S Robert Blumenthal Laboratory of Theoretical Biology National Cancer Institute National Institutes of Health Bethesda, Maryland 2001 4
INTRODUCTION Two different examples of the use of the lipid bilayer as an assay for ion-conductance-inducing materials are shown. In the case of hemocyanin, a giant respiratory protein dissolved in the hemolymph of Mollusca and Arthropoda, Pant and Conrad‘ had shown that the aggregated form produces electrical conductance increases of as much as six orders of magnitude when added to the aqueous solution after forming the lipid bilayer membrane. Alvarez, Diaz, and Latorre’ carried out discrete current fluctuation analysis of the hemocyanin-induced conductance and showed that the ion transport is mediated by a channel mechanism. The dissociated form of hemocyanin also induces conductance changes in lipid bilayer membranes, which is more likely to be mediated by a “defect” mechani~m.~ In the case of lymphocytes, it h’ad been postulated that the killing of target cells is caused by an initial permeability change to cations4 and subsequent colloid osmotic lysis. The bilayer experiments indicate th,at the permeability change can be induced without any contribution of target cell cytoplasm or membrane proteins.’
HEMOCYANIN Molluscan hemocyanins are cylindrical molecules with a diameter of about 340 A and a height ranging from 140 A to 680 A, built up from 3 to 12 rows of subunits.‘ Hemocyanin readily dissociates and aggregates upon changing the pH, Ca2+concentration, and hemocyanin concentration.’ Aggregated hemocyanin, which has a sedimentation coefficient of about 100 S, dissociates into half-molecules at pH above 7 or below 5, and next into smaller units, which are tenth molecules. At still higher pH values. the tenths dissociate into units, which are found to be twentieth molecules. Three-dimensional image reconstruction* of hemocyanin indicates that the subunits are arranged in a helical order giving rise to a structure with an internal polar core or “channel.” The differences between the dose response curves of associated (A) and dissociated (D) hemocyanins are shown in FIGURE1. The associated form is a dispersion of commercial (Schwarz-Mann) key-hole limpet hemocyanin in deionized w.ater. The dispersion contains a mixture of twentieth, 476
Blumenthal : Hemocyanin and Lymphocytes on Bilayers
477
FIGURE1 . Conductance-concentration curve for associated ( A ) and dissociated (D) hernocyanin in 100 mM KCl, pH = 7. The vertical bars ( l e f t ) represent conductance increases within four minutes after increasing the associated hemocyanin concentration; the dots (right) represent dissociated hem* cyanin-induced conductance.
tenth, half, and whole aggregates.' The higher-order concentration-dependence of the conductance indicates that a number of units aggregate to form a channel. The straight portion of the conductance-concentration curve yields a slope of six on a logarithmic scale which is an average value for the insertion of one, two, ten and twenty units into the bilayer to form a channel. The higher order concentration-dependence has been observed in the case of the other well-known channel-formers, nystatin' and alamethicin.'O Dissociated hemocyanin, prepared according to the method of Weigle" consists of twentieth units, which have irreversibly lost their capacity to reaggregate. The dose-response curves in FIGURE1 show three marked differences between associated and dissociated hemocyanin. For the same hernocyanin concentration at 80 pg/ml there is about two orders of magnitude difference in conductance increase with associated hemocyanin. This effect is even more dramatic when we take into account that the dissociated hemocyanin is about tenfold more concentrated than the associated hemocyanin on a molar basis, because the molecular weight of associated hemocyanin is about tenfold that of dissociated hemocyanin. The other difference is that upon addition of the dissociated hemocyanin to the solution, a steady-state conductance level is reached immediately, whereas with associated hemocyanin concentrations greater than 40 pg/ml the conductance keeps increasing at a slower rate after a large stepwise increase after addition of the hemocyanin in the external solution. The later conductance increase is actually indicated in FIGURE1 by the vertical bars, which represent a conductance change within four minutes. The third difference is that the straight portion of the conductance-concentration curve yields a slope of six on a logarithmic scale in the case of associated hernocyanin, whereas the dissociated hemocyanin yields a slope of one. Upon addition of associated hemocyanin to the bathing solution, the current increases in discrete steps ("formation bumps") at a given voltage, with conductances of about 0.1 nmho in 100 mM KCI (FIGURE 2 ) . Discrete conductance steps induced by associated hemocyanin were originally observed by Alvarez, Diaz, and Latorre,' who showed that the voltage-dependence of the conductance-step fluctuation correlates very well with the voltage-dependence of the conductance of a many-channel membrane. Discrete conductance steps were not observed with dissociated hemocyanin.
Annals New York Academy of Sciences
47 8 I
4r
[ 50mV
_I_.
, ,
P
,,.-
FIGURE2. Stepwise increase in current (middle truce), driven by a constant voltage (upper truce), after adding 20 pg/ml associated hemocyanin in 100 mM KCl, pH = 7. The lower trace marks one-second intervals. The rectification in the current-voltage characteristic for associated hemocyanin, which had been observed by Pant and Conrad’ and by Alvarez et u1.t is shown in FIGURE 3. Dissociated hemocyanin, however, exhibits a linear 3. current-voltage characteristic as shown in FIGURE The nature of the conductance induced into the lipid bilayer membrane by associated hemocyanin (discrete conductance fluctuations, higher-order dose-response curve and rectification) and its three-dimensional structure’ indicate that ion transport is mediated by a channel mechanism. The single subunit of dissociated hemocyanin, on the other hand, which has no possibility of forming a polar core, together with the nature of its conductance induced into the lipid bilayer (absence of discrete conductance steps, first-order doseresponse curve and linear current-voltage curve) points to an alternative mechanism mediating the ion transport. As shown in FIGURE 4 insertion of a single subunit into the lipid matrix could give rise to a “crack” and conductance could be induced by a “defect” mechanism. In that case the lipids do not completely surround the inserted protein and resulting leaks could cause ion permeation. The protein could possess some hydrophobic moiety that attaches to the bilayer. The possibility of a “defect” mechanism for ion transport
FIGURE3. Current-voltage curves for dissociated ( D ) and associated (A) hernocyanin-doped membranes. The curve marked AT represents the current and voltage taken from a triangular voltage sweep at a rate of 15.2 mV/sec and the curve marked As is obtained from the steady-state current, driven by voltage pulses lasting 20 seconds. The experiments were in 100 mM KCl, pH = 7; associated hemocyanin was at 40 pg/ ml, dissociated hemocyanin at 100 pg/ml. The current of the dissociated hemocyanin-doped membrane was normalized to that of associated hemocyanin-doped membrane.
Blumenthal:
Hemocyanin and Lymphocytes on Bilayers
419
FIGURE 4. Model for the “defect” mechanism of ion transport through a lipid bilayer membrane. The protein consists of a single subunit and contains a small hydrophobic area (shaded). The incomplete insertion of the protein into the lipid matrix gives rise to a leak through which ions could flow.
of the “closed” channel of excitability-inducing material (EIM) has been suggested by the experiments of Latorre et aL1’ on the temperature-dependence of the conductance of the “open” and “closed” channels of EIM. Latorre et a1.I’ found that the conductance of the open channel increased as expected from the temperature-dependence of the mobility of ions in water, whereas the conductance of the closed channel decreased with increasing temperature. The experiments indicate that the closed channel conductance is mediated by a “defect” mechanism and that at higher temperature the increased lipid fluidity “seals” the crack around the protein. Del Castillo et d.Iahad reported previously on the conductance induced into thin lipid films by enzymes or antibodies and the enhancement of the conductance caused by substrate-enzyme or antigen-antibody interaction. I believe that those conductances are caused by a defective mechanism and that the interaction with substrate or antigen enhances the size of the defect.
LYMPHOCYTES In order to study more closely the first stages of lymphocyte-mediated cytolysis of target cells, Henkart and Blumenthals prepared black lipid membranes consisting of oxidized cholesterol and DNP-phosphatidylethanolamine, synthesized from bacterial phosphatidylethanolamine by the method of Kinsky.” The lipid bilayers were formed across a 330-,Ahole in a thin bottom Teflon cup that is immersed in a small lucite chamber. The antigenic membrane was coated by injecting 5-,AAvolume of 1-2 mglml purified anti-TNP antibody directly above the membrane followed by 20-minute incubation. Lymphocytes were then injected in a 5-pl volume (of a suspension of lo’ cells/ml) down onto the bilayer membrane, became visible in the microscope immediately, and finished settling within five minutes as shown in FIGURE5. The lipid bilayer could support dozens of lymphocytes for periods of more than an hour before membrane-breakage occurred. The conductance of the lipid bilayer membrane was monitored at all stages of antibody and lymphocyte incubation. As shown in FIGURE 6, the “antigenic” oxidized cholesterol-DNP phosphatidylethanolamine bilayer had a conductance between 10-0 and lo-*mho/cma. Exposure to the antibody had no effect on this conductance. (The antigen-antibodyinduced conductance observed by Del Castillo et al.” is very dependent upon
480
Annals New York Academy of Sciences
FIGURE 5. Lymphocytes resting on the antibody-coated lipid bilayer. This photograph was taken through the phase microscope during the course of an experiment. A torus containing a thick layer of lipid, including bubbles, surrounds the bilayer, which appears dark under phase optics. Approximately 50 lymphocytes can be seen on the bilayer.
the lipid forming the bilayer. With the lipids we used we never observed a significant antibody-antigen-induced conductance change). A marked conductance change was observed within 2-20 minutes after lymphocytes settled on the membrane. Two types of patterns of conductance that change with time, when the bilayer membrane was clamped at a fixed voltage, are shown 6. The conductance increase was voltage-dependent: the increases in FIGURE only occurred when the membrane potential was positive on the lymphocyte side of the membrane. Since the electrical potential difference across cellular membranes is generally positive on the outside with respect to the inside of the cell, the polarity conditions for lymphocyte-mediated killing are the same in the lipid bilayer as in the target cell. Observation of discrete conductance steps and the absence of a lymphocyte-mediated conductance change in thick membranes indicated that the response is mediated by a channel mechanism. The system has all its controls built in, as shown in TABLE1. Lymphocytes resting on the bilayer in the absence of antibody did not induce significant conductance changes, which is analogous to the cytotoxicity of target cells, which is also antibody-dependent. The other controls shown in TABLE1 (F(ab)3 antibodies, which do not contain the Fc fragment recognized by the lymphocyte
Blumenthal : Hemocyanin and Lymphocytes on Bilayers
C
Noanti TNP
r35 t
481
IMlCl
lalcl
10-7
and lymphocytes lacking the Fc receptor) also correlated well with the targetcell-killing experiments. T h e experiments show that lymphocyte- and antibodydependence of membrane conductance increase is identical to that of target c ~ l lysis l and the results support the hypothesis that the membrane-permeability increase is the initial event in lymphocyte-mediated cytolysis. The experiments d o not distinguish yet whether the channel-forming material is released from TABLE 1
ANTIBODYAND LYMPHOCYTE-DEPENDENCE OF MEMBRANE CONDUCTANCE INCREASE
Antibody
Lymphocytes
Percentage of W r Release by Tnp-RBC (No. of Exp.*)
Rabbit anti-Tnp
Human PBL Human PBL Human PBL None Fc receptor negative
100 0-2 (4) 4-5 (2) -1-8 (4) 3-15 (4)
None Anti-Tnp-F(ab), Anti-Tnp Anti-Tnp
Membrane Conductance Increase (No. of Exp.) 25OX-20,OOOX (12) 0-1OX (6) 0 (1) 0-5X (2f) 0 (1)
* Relative to complete system (line 1). f In each experiment the membrane is incubated with antibody alone for 20 minutes; conductance increases greater than 5x were never seen. In two experiments the membranes were incubated with antibody alone for one hour.
482
Annals New York Academy of Sciences
the lymphocyte and subsequently inserted into the bilayer or whether the conductance change is induced while the channel-forming material is still attached to the lymphocyte surface. The experiments on the interaction of lymphocytes with the antigen-antibody-coated lipid bilayer membrane clearly show that the permeability change and consequently cytolysis can be induced without any involvement of target-cell cytoplasm or membrane proteins. The system thus provides us with a new approach to study the molecular nature of lymphocyte-mediated cytolysis and the molecular basis of the specificity of the response. References 1972. J. Membr. Biol. 8: 357-362. 1. PANT,H. C. & P. CONRAD. O., E. DIAZ & R. LATORRE.1975. Biochim. Biophys. Acta 3 8 9 4442. ALVAREZ, 448. R. 1975. Submitted for publication. 3. BLUMENTHAL, J. C. & K. T. BRUNNER. 1974. Adv. Immunol. 18: 67-132. 4. CEROTTINI, 5. HENKART, P. & R. BLUMENTHAL. 1975. Proc. Nat. Acad. Sci. U.S.A. 72: 27892793. 6. FERNANDEZ-MORAN, H., E. F. J. VAN BRUGGEN & M. OHTSUKI. 1966. J. Mol. Biol. 16: 191-207. 7. KONINGS,W. N., R. J. SIEZEN& M. GRABER.1969. Biochim. Biophys. Acta 194: 376-385. 8. MELLEMA,J. E. & A. KLUG. 1972. Nature (London) 239: 146-150. 9. FINKELSTEIN,A. & A. Chss 1968. J. Gen. Physiol. 52: 145S-172S. 10. MUELLER,P. & D. 0. RUDIN. 1968. Nature (London) 217: 713-719. 11. WEIGLE,W. 0. 1964. Immunochemistry 1: 295-302. 12. LATORRE, R., 0. ALVAREZ& P. VERDUGO.1974. Biochim. Biophys. Acta 3 7 6 361-365. 13. DELCASTILLO,J. A. RODRIQUEZ, C. A. ROMERO& V. SANCHEZ. 1966. Science 153: 185-188. 14. UEMURA,K. & S. C. KINSKY.1972. Biochemistry 11: 4085-4094.
