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The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves

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The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves MILESTONES IN NEPHROLOGY J Am Soc Nephrol 9: 217()-2l77. 1998 The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves JENS CHR. SKOU Institute ofPhysiology, Universit’ ofAarhus (Denmark) with COrnflleflts by JENS CHR. SKOU andJOSEPH F. HOFFMAN Reprinted from Biochirn. Biophys. Acta. 23: 394-401, 1957 Stimulation of a nerve leads to an influx of sodium ions into the fibre and hence to an increase in the intra-axonab sodium AUTHOR COMMENTARY concentratio&. Normal conditions are restored by an outward Jens Cht Skou transport of the sodium ions, and this process requires energy because the efflux takes place against an electrochemical gra- Institute ofBiophysics, University dient. The mechanism of this transport is not known. In experiments with giant axons from Sepia officinalic and ofAarhus, Denmark from lligoforbesi, HoIu.iN 1Nl) KE\NES2 found that dinitrophe- nob, azide and cyanide inhibit the active transport of sodium ions out of the nerve; this inhibition is revetsible. In the con- centrations used all three substances also inhibit the oxydative phosphorybation which takes place in mitochondria; dinitro- 1953 I wrote to professor David Nachmansohn at phenol and azide do so through an uncoupling of the phos- I Columbia University in New York and asked if I could photylation4, and cyanide through an inhibition of the oxy- come in August and September and prepare some acetyl- dation5. CALDWELI observed correspondingly that addition of cholinesterase from electric eel. I wanted to test the effect of these substances, in the concentrations used by HODGKIN AND surface pressure on the activity of an enzyme incorporated KE\NES, led to a reduction of the content of energy-rich phos- into a monomolecular layer of nerve membrane lipids on a phate esters in the axoplasm ofgiant axons. This seems to mdi- cate that energy-rich phosphate esters are somehow involved in water surface. I was a medical doctor who was interested in surgery, had taken a break in my clinical training, and got a the active transport of sodium ions out of the nerve fibres. In this connexion it is of interest that LIBET7 and ABOOD position at the Institute of Physiology at Aarhus University ANt) GnRn8 weie able to demonstrate an adenosine triphos- in order to write a thesis on the mechanism of action of local phatase (ATPase) in the sheath of giant axons. A further study anesthetics. I used a monomolecular layer of nerve mem- on the ATPase in nerves and its possible robe in the active out- brane lipids on a Krebs-Ringer water surface as a model for ward transport of sodium ions seems warranted. the nerve membrane. Local anesthetics placed in the water According to LIBET, the ATPase in the sheath ofgiant axons is phase penetrated the monolayer, and there was a correlation calcium-activated, while the experiments by MooD AND GERARD between the anesthetic potency and the increase in pressure suggest that it is activated by magnesium and located in submi- in the monolayer. This suggested to me that local anesthet- croscopic particles. In peripheral nerves from the rat the latter ics, by an effect on the lipid part of the nerve membrane, authors found both a calcium- and a magnesium-activated blocked for a conformational change in the proteins in the ATPase. The calcium-activated enzyme was predominantly bocat- membrane, leading to the increase in permeability for sodi- ed in the mitochondria, while the magnesium-activated, as in urn, and thereby blocking the nerve impulse. To test this, I giant axons, was mainly located in the submicroscopic particles. needed a lipid monolayer with a protein incorporated, and to Giant axons were not available to us. In preliminary exper- show that penetration of local anesthetics into the lipid part irnents we found that a homogenate of leg nerves from the of the monolayer influenced the conformation of the protein. shore crab (Carcinus inaenas) contained both a calcium- and The protein responsible for the permeability increase for a magnesium-activated ATPase, and that their localization sodium was unknown; instead, I decided to form a mono- was sirnilai to that of the ATPase found by AB0OD AND GERARD layer of lipids with an enzyme incorporated, and use a in rat-nerve homogenates. For our study we have chosen the change in enzymatic activity as an indication of a confor- magnesium-activated enzyme, because it resembles the mag- mational change of the protein. For the experiments, I need- nesium-activated ATPase from the sheath of giant axons in ed a membrane-bound enzyme with a high activity. Acetyl- that it is located in submicroscopic particles. cholinesterase was a candidate. The present investigation is concerned with the effect on In his reply, Professor Nachmansohn said that he intend- the enzyme activity exerted by the cations normally present in ed to spend August at the Marine Biological Station in the tissue-sodium , potassium , magnesium and calcium. Milestones in Nephrology 2171 EXPERIMENTAL Woods Hole, and suggested that I join him there. In The ATPase %%?55 prepared bs homogenization and subsequent differ- ential centrifugation5 of leg nerves froni the shore crab ( Garcinus September we could then return to New York and prepare mam as). the enzyme. I did not know what I would do in Woods Hole The isolated nerves were washed and honiogenized in 10 volumes of but accepted. This was a lucky choice. It was a great experi- 0.25 M ice-cold sucrose httffered with histidine, 30 niM/l, pH 7.6. The admixture of alkali metal ions was avoided by using the histidine base, ence to spend a month in surroundings where scientists and the pH was adjusted by addition of 1 N HC1. The homogenate was came from all over the world to work during the summer, centrifuged in a Servall angle centrifuge at OC. Fragments and stroma because there was access to squids and thereby to giant were removed by centrifugation at 2,000 X g for I 5 minutes, IllitoChon- dna and s()Ilse submicroscopic particles by centrifiigation at 10,000 X g axons. I did not take part in the experimental work, but for I 5 minutes. The supernatant was ceiitrifuged at 20,000 X g (maxi- looked, listened, and learned. In between I read scientific mum available) for 3 hours. During this centnfugation the temperature rose from 0’ to 8-10#{176}C. papers, and in one written by Nachmansohn it was men- The sediment after this last centrifugation was suspended in a vol- tioned that in 1948 Libet had shown that there is an ATP unie of 0.25 /i,f ice-cold buffered sucrose corresponclmg to one half of hydrolyzing enzyme in the sheath part of the giant axon (1). the volume of the original homogenate. This suspensioll was centrifuged at 10,000 x g for 10 minutes in order to remove an remaining mito- I knew that ATP was the energy source in cells, and won- cliondria. The final supernatant was used as enzyme solution in the dered why there was an AlP hydrobyzing enzyme in the experIments: it contained from one half to two thirds of the activity orig- nerve membrane. Furthermore, being a membrane-bound inably present iii the homogenate. The enzvtne was relatively unstable; when it was stored in a refrigera- enzyme it could be a candidate for my monolayer work. tor at 4-5#{176}C, its activity fell to oe half in 3-4 days. I decided to take a book at the enzyme when I returned The disodium or diharium salts of ATP and the barium salt of ADP to Aarhus, a decision that turned out to have far-reaching (Sigma products) were converted into free acids, the Na salt by passage through an Ansherlite 120 (H forni) column and the barium salts by consequences. precipitation with equimolar amounts of sulphuric acid and subsequent In September I prepared acetylchobinesterase at Cobum- passage through au Ansl)erlite 1 20 colunn to renave residual hariuni iOns. The free acids were neutralized to pH 7 with a 1 A! solution of 2- bia University, and then returned to Aarhus where I contin- arino-2-I11ethvl-l .3-propanediol. In the concentrations used, this sub- ued my monolayer experiments. During the autumn of 1954 stance does not affect the enzyme activity and is without influence on the I started to look for the nerve membrane enzyme. I had phosphate determination. ATP and ADP were (letermined spectrophotonietricallv” and h mea- no access to giant axons, but decided to use crab nerves surement of 7’P; Na and K were determined ssith a flame spectropho- instead, and arranged with a fisherman to deliver some tometer, Beckman DU, flame attachment No. 920(). The pH was mea- crabs. The membrane fragments of sciatic crab nerves sured witls a glass electrode, Radiometer PHM 3. Inorganic phosphate was determined by the method of FIsKE ANt) StIsBRow1#{176} with aniidol as showed a Mg2tactivated ATPase activity, which was slight- the reducing agent. ly increased by Na, while K had no effect. Then I ran into The activity of the enzyme was determined in a volume of I .0 ml con- problems. I was unable to reproduce my results, as the mea- taming 0. 1 I1l of the above-i’nentioned enzynse solution. The reaction mixture was buffered with histidine, 30 mM/b, pH 7.2. Unless otherwise sured enzyme activity varied. I continued, but was interrupt- stated, the mixture contained 3 mM ATP/l. The cations were added as ed by the Christmas holidays, the spring semester teaching, solution of their chloride. All experiments were performed at 36#{176}C. and it was After a lO-mintite temperature equilibration the experiment was started and summer holidays, not until the autumn of by the addition of enzyme, and the rnixttire was inctibated for 15 or 30 1955 that I got the answer, namely that the enzyme for activ- minutes, according to the reaction velocity.
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