- Email: [email protected]
The Na+,K+-activated adenosine triphosphatase in degenerating peripheral nerve
ARCHIVES
OF
BIOCHEMISTRY
AND
The Na+,K+-Activated
BIOPHYSICS
117,
98-105
Adenosine
Triphosphatase
Peripheral
Monash
in Degenerating
Nerve
H. S. BACHELARD’ Departments of Biochemistry and Anatomy,
(1966)
and G. D. SILVA
University,
P. 0. Box 92, Clayton, Victoria,
Australia
Received December 28, 1965 At various times (2-32 days) after unilateral injury to cat sciatic nerve, the rate of hydrolysis of ATP was determined. The Na+,K+-ATPase activity, which decreased from 2 to 8 days after injury, increased to above control levels in the degenerating nerve from 16 to 32 days after injury. The decreased Na+,K+-ATPase activity of the injured nerve was inhibited by ouabain to an extent slightly less than the inhibition of the normal activity. Subfractions of sucrose homogenates of segments of the 4-day injured nerve were prepared by differential centrifugation, and the enzymic activities were compared with those in preparations from equivalent segments of normal nerve from the same animals. In both injured and normal nerves, the Na+,K+-ATPase activity was localized mainly in the subfraction which sedimented between 10,000 and 100,OOOg(P3). Decreased enzymic activity was observed in all subfractions of the injured nerve. The diminished activity in subfraction P3 and the nonparticulate fraction of the 4-day injured nerve was partially restored by exogenous phospholipids.
When a peripheral nerve is crushed or cut, those portions of the nerve near the injury lose, within 24 days, the ability to conduct electrical impulses (1). Possibly involved in this rapid loss of conductivity is the Na+,K+-activated, Mg++-dependent ATP hydrolyzing system first described by Skou (2, 3) in crab leg nerve particulate fractions and considered (2-8) to be concerned with ion-transport mechanisms essential for the maintenance of the physiological functions of the tissues in which it occurs. The requirement for active cation transport in excitable tissues has generated great interest in this ATPase, which has been shown to occur in the membrane subfractions of peripheral nerve (2) and cerebral cortex (5). In their studies on the levels of various enzymes in degenerating nerves, Rossiter and his co-workers (9) reported that there
was no change in the rate of hydrolysis of ATP in the presence of Ca++ from 16 to 32 days after injury; the activity during the early stages of degeneration was not recorded. However, these studies were performed before it was realized that Ca++ is inhibitory (2) to the Na+, K+, Mg++ATPase system. The levels of other enzymes have been shown to be changed during the early stages of Wallerian degeneration. Within 3 days of section of rat sciatic nerve, Adams and Tuqan (10) observed increased proteinase activity which was considered to be due to liberation of endogenous proteolytic enzymes with subsequent liberation of proteinbound lipid so that the membrane lipoproteins became dissociated. The activities of acetyl cholinesterase (11) and alkaline phosphatase (9) have been shown to decrease during the stages of degeneration associated with gross morphological changes in the nerve and the loss of
1 Present address: Department of Biochemistry, Institute of Psychiatry, Maudsley Hospital, London, S.E.5. 98
ATPASE
IN DEGENERATING
sphingomyelin and other phospholipids (12). The increased levels of acid phosphatase, Snucleotidase, and p-glucuronidase during later stages have been attributed to the proliferation of macrophages (9) and Schwann cells (13) around the site of injury. This communication reports the resuIts of studies on the activity of the Na+,K+activated, ouabain-sensitive ATPnse in cat sciatic nerve at various times (2-32 days) after unilateral injury to the nerve. For a more detailed study of the enzymic activity in subfractions, prepared by differential centrifugation from sucrose homogenates of segments of the injured nerve, the 4-day period was used. The 4-day period was chosen in view of the rapid loss of conductivity during the early stages of degeneration, in order to study the activity during this period, when t,he lipoprotein of membranes is considered to undergo dissociation (lo), and before gross changes in morphology or proliferation of supporting cells (13) occurred to any extent. The effects of added phospholipids on the ATPase activity in degenerating nerve were studied in view of the recent observations (14-16) that phospholipids seem to be required for full activity of the enzyme system. METHODS Xerve i,njwy. Adult cats were immunized with Vaxitas FE vaccine (1.5 ml) 5 days preoperatively. Under light intraperitoneal Nembutal anaesthesia, a postero-lateral incision was made in the back of one thigh, the fascia 1at.a was incised, and t,he sciatic nerve was exposed. Unilateral damage was effected, midway between the greater sciatic notch and the popliteal fossa, either by transection or by crushing. The nerve W;LScrushed with a fine pair of artery forceps, 1 mm in width, and the pressure was maintained for 3 minutes, with resultant flattening and blanching at the site of crush. A loose black silk suture was placed over the site of crush to facilitate localization at the subsequent operation. Hemostasis was obtained and the fascia and skin were sutured in layers. Penicillin (400,000 units) was administered postoperatively daily for 2 days. During the operation, care was taken to ensure that the noninjured portions of the nerve were not handled, and the nerve was kept moist with exudate. il t t,he required time after nerve injury, the cats were again anaest,hetized with Nembutal. The sciatic nerves on both sides were carefully exposed
NERVl~:
!1!1
and the site of injury on the treated IIPI’VO rvas Iocated. Adhering connect.ivc tissue was removed and the proximal and distal parts were excised and immediately frozen by immersion in liquid N,. Each of the 2 frozen parts of the nerve was chopped into halves to yield 4 segments: A, distal end; H, dist,al t.o illjury; C, prr)xirn:tl t.r) il>jury; I), proxima1 end. At the same time a similar length (ci cm) 01’t,hc opposite sciatic nerve was removed, frozen, and subsequently chopped intr, 4 segments equivalent in length to the segments from i.hr injured nerve. Homogenates and subJractions. The frozen segments were weighed on an ASE Torsion balailct! and ground in a Teflon-pestle homogenizer (A. l-l Thomas Co., clearance 0.134.18 mmj at 2000 rpm in ice-cold 0.25 M sucrose, lo a tinal concent r:ttiorl of 100 mg nerve per milliliter. The homogenate was centrifuged at 1000~ (;tvg) for 10 minutes in a Servall RC 2 (M 34 head 1. The pellet was washed once by resuspension in half of the original volume of sucrose and reccntrifllgetl to yield fraction “P,.” Th e combined s~~pernnttt~~~ fraction and washings were centrifrtged at 10.000~~ for 15 minutes in the Servall for fra(xtion ‘(I’.‘,” iI1 which was also collected the floaf ing “nlyelill layer.” This pellet was also washed as above. Thr: “P,” fraction was prepared by cent,rilliging the: combined supernatant, fraction and washings at 100,OOOg for 1 hr in 2 ml tubes in the No. 40 head OI a Spinco model Ii Illtracelltrifuge. The sclperll:ttant fraction was designated “S.” Each pellet was resuspended in 0.25 M sllcrose to a final vol~~rnt~01 1 ml/l00 rng original I issilc. All fr:lc.t.icms wtlI*’ stored at -20” until use. ATPase assay. Tris-ATP, 15 nlM, pH 7.4. was prepared from Nar ATP by the method drsrribctl previously (5). For the ATPase assay, the final incllbation mixtllrc ront.:tined: tisslle fractic,n; 3 mM Tris-ATP, pH 7.4; 3 111.~MgC12; and f>iiIIPI 100 rnl\f Tris-HCl, pH 7.4, 100 IXM NaCI, ~0 WM KCI, or 230 111~Tris-HCl, pH 7.4. Pu’onctla,vrnit* ATP control t ltbes were prepared for hot h ilrc*llb:ltion mixtures. All constitueltts except, the tisslle fract,ion and ATP were mixed in a volllrne of 0.X ml in tubes at 0”. Immediately after the addit iorl of tissue fraction the tubes were equilibrated :tt :i’i” for 5 minutes and the reaction was started by the addition of 15 111~ Tris-ATP (0.2 ml). The v~)l~ur~es of t,issue fractions normally used were: hoiriogenate (H), 10 ~1; 7’1, 50 ~1; P2’,,50 ~1; PS: 20 ~1; 5: 100 gl. ‘Test)s with H alld P3 confirmed that hydrolysis of t.he ATP was linear wit,h t,irnc, rip to 15 minutes; IO-minute incllbation times u-~r(: routinely used and the reactions were stclpped by the addition of lo:;, (w/v) trichloroa~t ic ;rcnitl (1 ml) at 0”. Inorganic phosphatr was derrrrllined after iso-but anol-benzellc extract ion of t hr 1,110s.
100
BACHELARD
phomolybdate as described by Deul and McIlwain (17). Ouabain inhibition was tested at a final concenat a final tration of 0.1 IIIM (5); Ca++ inhibition concentration of G mM CaClz (2). Naf and K+ were determined with an EEL “type A” flame photometer. Protein. The Lowry method (18) for the determination of protein was used with crystalline bovine serum albumin as standard. Identical concentrations of t,he sucrose in which the various subcellular fractions had been suspended were included in the blank and standard tubes. Occasionally with subfract,ions high in lipid content, the final solutions after addition of all reagents were faintly turbid. These were clarified by centrifugation of the final reaction mixture at 25009 for 10 minutes in a Servall “SSI” centrifuge. Checks using added standard confirmed that no color was lost during this procedure. Treatment with phospholipids. The phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, and sphingomyelin) were dissolved, without further purification, as a mixture in chloroform-methanol (4:l v/v) to a final concentration of 10 mg of each per milliliter. A sufficient quantity of the mixture for a group of incubations was pipetted into a test tube and the solvents were removed by warming in a stream of Nz. The phospholipid smear was thoroughly dispersed in the incubation medium containing all of the constituents for the ATPase assay except Tris-ATP and the tissue fraction. Samples of 0.8 ml were placed in the relevant incubation tubes at 0”. The assay was then carried out m described above. The volume of the original phospholipid mixture used was calculated so that the final amount in each incubation tube was 200 pg (50 fig of each). In the enzymic assays the phospholipid control tubes gave elevated color yields. The increase in optical density was 0.05 (compared with an OD of 0.04-0.08 from the ATP control tubes). Reagents. Reagents not otherwise specified were of analytic reagent quality. Tris 121, NhATP, and ouabain were obtained from the Sigma Chemical Co., St. Louis, Missouri; phosphatidyl choline and phosphatidyl ethanolamine from California Corporation for Biochemical Research, Los Angeles, California; phosphatidyl serine and sphingomyelin from L. Light and Co., Colnbrook, Bucks. Vaxitas FE vaccine (Tasman Vaccine Laboratories Ltd., New Zealand) was purchased from Veterinary Supplies Ltd., South Melbourne, Australia. RESULTS
Na+, K+-ATPase activity of normal sciatic nerve. The activity of the ATP-hydrolyzing
AND
SILVA TABLE NAP, K+-ATPASE
I
ACTIVITY SCIATIC NERVE
OF NORMAL
Homogenates were prepared and assayed for ATPase activity wit’h 3 mu Mg++ in the presence or absence of 30 mM KC1 and 100 mM NaCl as described in Methods. Results are expressed as mean value f SD (No.). ATPase activity Addition
-
Na+, K+ Na+, K+ onabain (0.1
0.81 f 1.08 f 0.56 f
0.15 (12) 0.18 (12) 0.09 (8)
9.7 12.9 3.9
rnM)
Na+, KC, Ca++ (6 mM)
0.66 z!z 0.14 (7)
8.0
system in normal sciatic nerve of the cat was tested with Mgff and in the presence or absence of Na+ and K+ (Table I). The cardiac glycoside, ouabain, which generally is known (5) to inhibit the increased ATPase activity due to Na+ and K+ in the presence of Mg++, decreased the activity in these preparations to below the level observed in the absence of Na+ and K+. The fact that the activity in the presence of ouabain, Mg++, Na+, and K+ was lower than the activity in the presence of Mg++ alone may be due to the presence of endogenous lSa+ and K+ in the nerve homogenates, which would be expected to have an activating effect, i.e., the observed level, in the presence of Mg++ alone, may be elevated due to endogenous Na+ and K+. Direct estimations on 8 homogenates yielded the following results: Na+, 15 f 3 mM; K+, 9 f 2 mM. The 0.25 M sucrose used in the preparation of the homogenates contained less than 0.095 mM Na+ or K+. Jarnefelt (19) has suggested that brain microsomal ATPase preparations contain bound endogenous Na+ and K+ which can be removed by treatment with deoxycholate. The activity in the presence of Mg++, Na+ and K+ recorded in Table I (1.08 pmoles ATP hydrolyzed per milligram fresh nerve per hour) is higher than the activity previously reported for cat sciatic nerve by Bonting and co-workers (20) who found a
ATPASE IX DEGESEItATING
Ol 0
# 5
I 20
1 15
IO
Days
after
nerve
101
NERVE
25
30
I 35
injury
FIG. 1. Na+,K+-ATPase activities in normal and damaged sciatic nerves. The enzymic activities were estimated with 3 mM MgCl,, 30 mM KCI, and 100 mM NaCl. Untreat)ed nerve (m); damaged nerve: segment B, distal to injury (0); damagednerve, segment C, proximal to injury (0).
slight inhibition (10%) by ouabain and an activation by Ca++. In the present study, Ca++ was inhibitory, in accordance with the report by Skou (2) on the Ca++ inhibition on Na+, Ii+-ATPase in peripheral nerve. Na +, K+-A Tl’ase activity of damaged sciatic nerve. The activity of the ATPase in the presence of added Mg++, Sa+, and B+ in the damaged sciatic nerve at various times (2-32 days) after crushing, was compared with the activity in the normal untreated nerves from the same cats. The levels of enzymic activity in both distal and proximal parts of the crushed nerve (Fig. l), expressed as micromoles ATP hydrolyzed per milligram protein per hour, were well below the control activities at the earliest time studied (2 days), and remained at approximately the same level until S days. By 16 days after injury the activities had reached levels higher than those of the control nerves. During the period of nerve degeneration studied, a pronounced increase in the weight of the tissue occurred-the weight increased from a normal value of 60-70 mg per centimet,er to SO-110 rng per centimeter in the damaged nerves in the vicinity of the injury.
since enzymic activity (xxAccordingly, pressed on a basis of the protein content of the nerve may not make allowance for t,hc weight changes of the nerve, the results have also been expressed on the basis of the a&ivity per milligram fresh nerve and the activity per centimeter nerve (Fig. 2); a similar relationship between normal and damaged nerve can be observed. This indicates t#hat the ATPase activity was genuinely lower than normal in the injured nerve during thu first 8 days; the results do not merely reflects changes in the weight or protein c*ontent’ of the nerve (13). Ouabain inhibited the Xa+, I<+-.~1TPasc activity in the injured nerve to a slightly lesser extent than in the normal nerve (Tnble 11). Na+ , K+-A TPase in subfractions qf I)o~~ma1 and injured nef-ves. The distributions of the Na+, K+-ATPase activity in the subfractions prepared by differential centrifugation of homogenates of the untreated and damaged sciatic nerves 4 days after nerve-section are shown in Table III. There was little variation in the activities observed in t.hcb homogenates (H) for different segments from the normal nerve. Similarly, only slight,
102
BACHELARD
AND SILVA TABLE
II
OUABAIN INHIBITION OF NA+, K+-ATPASE NORMAL AND CRUSHED SCIATIC NERVE
IN
Homogenates of the nerve segments were assayed for ATPase activity in the presence of added Mg++, Na+, and K+ as described in Methods. The crushed and control nerves were taken from cats 8 days after crushing. Each value is the average of the preparations from 2 cats. ATPase activity (~moles Nerve segment
Control Damaged “B” Damaged “C”
01
5
10
15
20
25
30
35
Days after nerve injury
Fra. 2. Na+,K+-ATPase activities in normal and damaged sciatic nerves. The results shown in Fig. 1 are expressed on a basis of nerve weight (mg) or length (cm). Untreated nerve (m); damaged nerve, segment B, distal to injury (e); damaged nerve, segment C, proximal to injury (0).
variations were observed in the P3fraction of the segments from the normal nerve. This fraction, which sedimented between 10,000 and lOO,OOOg,was the most enriched in enxymic activity; the mean value of 61 Imoles ATP hydrolyzed per milligram protein per hour was comparable with the activity observed in membrane-enriched subfractions from cerebral cortex microsome preparations (5, 15) (50-75 pmoles per milligram protein per hour). In the nerve 4 days after injury, the ATPase system was less active in the homogenates from all segments of the damaged nerve than in the normal nerve. The lowest activity occurred in the segment (B) immediately distal to the cut. The activity in the segment (C) immediately proximal to the cut was also noticeably diminished. The smallest effect was apparent in segment Din the area further from the site of injury on the proxima1 side.
(Distal) (Proximal)
P//mg protein/hour)
NO ouabain
0.1 mM ouabain
11.2 3.6 4.8
5.7 (49) 2.1 (42) 3.2 (34)
(7, inhibition)
The changes in enzymic activity in the subfractions from the segments of the degenerating nerve followed closely those in the whole homogenates. The activity in Pa, the richest source of the enzyme system in these preparations, fell to below 40% of the control level in segment B, immediately distal to the injury and almost no activity could be detected in the soluble fraction (S) from that segment. As was found with the homogenates, the least change in activity was observed for subfraction Pa from the proximal segment furthest from the site of injury (D). Effect of phospholipids on the ATPase in sectioned nerve. The ability of a phospho-
lipid mixture to restore the dimished enzymic activity of the P3 and the soluble fraction was tested, and partial restoration was effected in both fractions (Table IV). The fractions chosen for this aspect of the study were those in which the greatest decreasein enzymic activity had been observed The activity of the Pa fraction of segment B (immediately distal to the injury) was slightly increased, but a more successful restoration occurred in fraction Ps of segment C (immediately proximal to the injury) in which the original activity was less depleted. A similar pattern emerged for the soluble fractions: segment B, which suffered the greatest depletion of original activity was not affected by the phospholipids whereas the activity of segment C was considerably restored.
ATPASE
IN UECEKERATING TABLE
NA+,
K+-ATPASE
ACTIVITY
ru’EI
1 o:j
III
IN SUBFRACTIONS
OF NORMAL
AND
I~AMAGEU NERVE
Subfractions of the segments of normal and damaged nerves from cats 4 days after unilateral nerve sections were prepared and assayed for ATPase activity as described in Methods. The enzymic activity proteitl was estimated with added Mg ++, Naf and K+, and is expressed as rmoles ATP hgdrolyzed/mg hour. Each value is the mean of preparations from 2 cats. Segment
.A” Normal Sectioned cc,‘;,Normal) B Normal Sectioned Cc,?;Normal) C Normal Sectioned (7; Normal) 1) Normal Sectioned (I;; Normal)
Hb
10.1 7 G (75) 13.0 3.4
P,
16.0 3.4
P2
11.3 7.0
pa
:,
65.0 3-l. 1 (52) (il.0 23 7
Ii.:3
@ti)
WI
(61)
(39)
13.9 5.4 (39) 11.0 8.4 (76)
11.3 5.5 (49 i
16.5 12.0 (74 )
61 .o “i.!) (-l-G) 57.0 50.5 !88 )
1.7 (Sfii
x.i 0.2 121
x 5 1.4 (16) 10.4 ‘)-.- ,> (21 I
h A: distal end; B: distal IO sect,ion; C: proximal to section; 11: proximal ettd. b H: Homogenate; Pi: subfraction which sedimented at 1000 g for 10 minutes; Py sttbfraction which sedimented between 1000 g, 10 minutes and 10,000 g, 15 minutes; Pa: subfract,ion which sedimented between 10,000 g, 15 minutes and 100,000 g, GO minutes; S: soluble fraction as sttpernatattt fractiott after 100,000 g, GO minutes. 11TSCUSSION
The marked loss of NaT,K+-activated, Ng++-dependent ATPase activity during the early stages of peripheral nerve degeneration, when conductivity is lost, is of interest since enzyme systems of this type have been suggested (2-S) to be associated with ion transport mechanisms necessary to maintain the physiological function of the tissue. It is possible that t’he activity remains diminished for longer periods but is masked by an increased activity due to the proliferation of macrophages (13) in the vicinity of the injury. The distribution of the ATPase activity in the segments of the degenerating nerve resembles the pattern observed for acetyl cholinesterase (11) in that the most marked diminution of activity occurred in that part of the nerve immediately distal to the injury and the least effect was found in the proximal segment furthest from the site of injury. The results presented here indicate that the decrease in ATPase activity was not due to changes in weight or protein content of the injured nerve (13), since the activity per centimeter nerve and the activity per unit
protein were both greatly dimished. The changes in activity of all of the particulate fractions suggest that general changes in the organisation of membrane systems iri the tissue have occurred, as might be expected if dissociation of the membrane lipoproteinq (10) has occurred. The partial restoration of the depleted enzymic activity by relatively large amounts of a phospholipid mixture (0.2 mg were used per 2-5 mg original nerve) provides additional evidence in support of recent reports that ATPases of this type require phospholipids, which are considered to be associated with the native enzyme systems. Phospholipase C, which acts on sphingomyelin (21) as well as on lecit.hins, has been shown to inhibit the Naf, K+-ATPases of erythrocytes (14) and cerebral cortex (15). Tanaka and Abood (16) reported a considerable reduction in ATPase activity in membrane preparations from rat cerebral cortex after removal of the endogenous phospholipids by treatment with deoxycholate and ammonium sulfate fractionation. Restoration of enzymic activity was obtained by the addition of large amounts of exogenous phospholipids,
104
BACHELARD TABLE
EFFECT
NA+,
IV
OF PHOSPHOLIPIDS K+-ATPASE ACTIVITY OF SECTIONED
ON THE DECREASED OF SUBFRACTIONS NERVE
The phospholipids were added as described in Methods to a final concentration of 200 gg (50 I.rgof each phospholipid) per assay. The amount of tissue protein used was Pa, 1520 rg (representing 2 mg of nerve); S, 200-250 pg. (representing 5 mg of nerve). ATPase assays were carried out with 3 mM Mg&, 30 mM KC1, and 100 mM NaCl; each value is the mean of two nerve preparations. ATPase
Segment&&
p&ypX’ormal
B*
Ps
C
Pa
B
S
c
s
+ + + +
54.5 55.8 63.7 63.0 7.9 8.2 6.7 6.5
Activity (Irmoles protein/hour) Sectioned
23.3 32.2 29.8 47.0 0.0” 0.0” 0.9 3.3
P/mg
NOi&lj
(43) (58) (47) (74)
(13) (51)
a Not detectable. * B: distal to section; C: proximal to section. Pa: subfraction which sediment,ed between 10,000 g, 15 minutes; and 100,000 g, 60 minutes ; S : soluble fraction as supernate after 100,000 g, 60 minutes.
phosphatidyl serine, or phoslecithins, phatidyl ethanolamine. In this study, the highest restoration was effected by 10 mg lecithin per 25 mg rat brain. These workers concluded that phospholipids are essential for optimal activity of the ATPase. The restoration of enzymic activity by phospholipids observed in the present study was more effective in the less severely damaged segments of the injured nerve (Table IV). In the areas of the nerve which had suffered the greatest decrease in activity, loss of endogenous phospholipid may have contributed to the loss of activity, although measurable losses of phospholipids in degenerating nerves have not usually been possible until later stages of degeneration have been reached (12). However, as Adams and Tuqan (10) suggested, fine changes in the configuration of lipoprotein structures, or dissociation of the lipoproteins of the membranes, might occur well before changes in phospholipid levels can be detected by
AND SILVA
chemical analysis. Samples of the segments from the injured and normal nerves used in this study are currently being examined in the electron microscope with a view to correlating changes in membrane structure and integrity with the observed changes in enzymic activity and also to gauge the extent of proliferation of macrophages and fibroblasts over the relevant time period. The similarity between the changes in ATPase and acetyl cholinesterase (11) activities during early periods of degeneration suggests that it would be of interest to attempt a study of the activity of the ATPase system during nerve regeneration. A local axonal synthesis of acetyl cholinesterase has been reported (22) ; the ATPase could also be of potential interest in studying protein synthesis in peripheral nerve. However, it is doubtful if the biochemical techniques which were the basis of the present report would give sufficient resolution; unlike acetyl cholinesterase, ATPases of this type occur widely (20) and discrimination between activity in the nerve itself and activity in surrounding tissues, proliferating macrophages and fibroblasts would be difficult. However, the lower ATPase activity during early degeneration does indicate the value to be obtained from a study of the activity during regeneration; the required resolution may be obtained in association with the use of histochemical electron microscopic techniques such as those described by Barrnett (23), when the necessary quantitation of such methods has been developed. ACKNOWLEDGMENTS We are indebted to Dr. A. Wilson of the Anatomy Department, Monash University, for his helpful advice; and to Mr. H. Ueckert and Mr. P. Moritz for their skilled technical assistance. REFERENCES L., Ph~sioE. Revs. 36, 441 (1956). J. C., Biochim. Biophy,. Acta 23, 394 (1957). 3. SKOU, J. C., Biochim. Biophys. Acta 42, 6 (1960). 4. SKOU, J. C., “Symposium onMembrane Transport and Metabolism, Prague, 1961.” p. 228. 5. SCHWARTZ, A., BACHELARD, H. S., AND McILWAIN, H., Biochem. J. 84, 626 (1962). 1. GUTH,
2. SKOU,
ATPASE 6. 7. 8. 9. 10. 11. 12. 13.
14. 15.
IN DEGENERATING
GLYNN, I. M., J. Phyaiol. 160, 18P (1962). WHITTAM, H ., Rio&em. J. 82, 205 (1962). WHITTAM, It., Biochem. J. 84, 110 (1962). HOLUNGER, 1). M., ROSSWER, R. J., AND UPMALIS, H., Riochem. J. 62, 652 (1952). ADAMS, C. W. M., AND TUQAN, N. A., J. Neurochew 6, 334 (1961). &LENA, J., AND LUBRINSKA, L., Physiol. Bohem. 11, 261 (1962). LoG.~N,J. E., MANNELL, W. A., AND ROSSITER, K. J., Riochena. J. 61,482 (1952). JOHNSON, A. C., MCNABB, A. R., AND ROSSI'PER, R. J., Arch. :Veurol. Psychiat. Chicago 64, 105 (1950). SCHATZMANN, H. J., Nature 196, 677 (1962). SWANSON, P. I>., BRADFORD, H. I?., AND MCILWAIN, H., Biochem. J. 92, 235 (1964).
NERVE
I 0.i
16. TANAKA, R., AND ABOOD, L. G., Arch. Ridiem Biophys. 108, 47 (1964). 17. DEUL, D. H., AND MCILWAIN, H., J. .VCU,Y)them. 8, 246 (1961). 18. L~\vRY, 0. H., ROSEBROUGH, N. J.. FARI<, A. L., AND RANDALL, R. J., J. fiiol. C’hem. 193, 265 (1951). 19. JAILXEFELT, J., Biochem. Bioph!Js. Ken. Commun. 17, 330 (1964). 20. BONWNG. S. L., SIMON, Ii. i)., ANI) HA\+-KIXS, N. &I., Arch. Biochem. Biophys. 95, 415 (1961). 21. HANAHAN, 1). J., ASD VERCAMEK, I:.. ./. :I,,/. Chem. Sot. 76, 1804 (1954). 22. KOENIG, E., J. Neurochem. 12, 343 (1965). 23. BAI~IISETT', R. J., J. Ro~J. Microscop. Sm. 83, 143 (1964).
OF
BIOCHEMISTRY
AND
The Na+,K+-Activated
BIOPHYSICS
117,
98-105
Adenosine
Triphosphatase
Peripheral
Monash
in Degenerating
Nerve
H. S. BACHELARD’ Departments of Biochemistry and Anatomy,
(1966)
and G. D. SILVA
University,
P. 0. Box 92, Clayton, Victoria,
Australia
Received December 28, 1965 At various times (2-32 days) after unilateral injury to cat sciatic nerve, the rate of hydrolysis of ATP was determined. The Na+,K+-ATPase activity, which decreased from 2 to 8 days after injury, increased to above control levels in the degenerating nerve from 16 to 32 days after injury. The decreased Na+,K+-ATPase activity of the injured nerve was inhibited by ouabain to an extent slightly less than the inhibition of the normal activity. Subfractions of sucrose homogenates of segments of the 4-day injured nerve were prepared by differential centrifugation, and the enzymic activities were compared with those in preparations from equivalent segments of normal nerve from the same animals. In both injured and normal nerves, the Na+,K+-ATPase activity was localized mainly in the subfraction which sedimented between 10,000 and 100,OOOg(P3). Decreased enzymic activity was observed in all subfractions of the injured nerve. The diminished activity in subfraction P3 and the nonparticulate fraction of the 4-day injured nerve was partially restored by exogenous phospholipids.
When a peripheral nerve is crushed or cut, those portions of the nerve near the injury lose, within 24 days, the ability to conduct electrical impulses (1). Possibly involved in this rapid loss of conductivity is the Na+,K+-activated, Mg++-dependent ATP hydrolyzing system first described by Skou (2, 3) in crab leg nerve particulate fractions and considered (2-8) to be concerned with ion-transport mechanisms essential for the maintenance of the physiological functions of the tissues in which it occurs. The requirement for active cation transport in excitable tissues has generated great interest in this ATPase, which has been shown to occur in the membrane subfractions of peripheral nerve (2) and cerebral cortex (5). In their studies on the levels of various enzymes in degenerating nerves, Rossiter and his co-workers (9) reported that there
was no change in the rate of hydrolysis of ATP in the presence of Ca++ from 16 to 32 days after injury; the activity during the early stages of degeneration was not recorded. However, these studies were performed before it was realized that Ca++ is inhibitory (2) to the Na+, K+, Mg++ATPase system. The levels of other enzymes have been shown to be changed during the early stages of Wallerian degeneration. Within 3 days of section of rat sciatic nerve, Adams and Tuqan (10) observed increased proteinase activity which was considered to be due to liberation of endogenous proteolytic enzymes with subsequent liberation of proteinbound lipid so that the membrane lipoproteins became dissociated. The activities of acetyl cholinesterase (11) and alkaline phosphatase (9) have been shown to decrease during the stages of degeneration associated with gross morphological changes in the nerve and the loss of
1 Present address: Department of Biochemistry, Institute of Psychiatry, Maudsley Hospital, London, S.E.5. 98
ATPASE
IN DEGENERATING
sphingomyelin and other phospholipids (12). The increased levels of acid phosphatase, Snucleotidase, and p-glucuronidase during later stages have been attributed to the proliferation of macrophages (9) and Schwann cells (13) around the site of injury. This communication reports the resuIts of studies on the activity of the Na+,K+activated, ouabain-sensitive ATPnse in cat sciatic nerve at various times (2-32 days) after unilateral injury to the nerve. For a more detailed study of the enzymic activity in subfractions, prepared by differential centrifugation from sucrose homogenates of segments of the injured nerve, the 4-day period was used. The 4-day period was chosen in view of the rapid loss of conductivity during the early stages of degeneration, in order to study the activity during this period, when t,he lipoprotein of membranes is considered to undergo dissociation (lo), and before gross changes in morphology or proliferation of supporting cells (13) occurred to any extent. The effects of added phospholipids on the ATPase activity in degenerating nerve were studied in view of the recent observations (14-16) that phospholipids seem to be required for full activity of the enzyme system. METHODS Xerve i,njwy. Adult cats were immunized with Vaxitas FE vaccine (1.5 ml) 5 days preoperatively. Under light intraperitoneal Nembutal anaesthesia, a postero-lateral incision was made in the back of one thigh, the fascia 1at.a was incised, and t,he sciatic nerve was exposed. Unilateral damage was effected, midway between the greater sciatic notch and the popliteal fossa, either by transection or by crushing. The nerve W;LScrushed with a fine pair of artery forceps, 1 mm in width, and the pressure was maintained for 3 minutes, with resultant flattening and blanching at the site of crush. A loose black silk suture was placed over the site of crush to facilitate localization at the subsequent operation. Hemostasis was obtained and the fascia and skin were sutured in layers. Penicillin (400,000 units) was administered postoperatively daily for 2 days. During the operation, care was taken to ensure that the noninjured portions of the nerve were not handled, and the nerve was kept moist with exudate. il t t,he required time after nerve injury, the cats were again anaest,hetized with Nembutal. The sciatic nerves on both sides were carefully exposed
NERVl~:
!1!1
and the site of injury on the treated IIPI’VO rvas Iocated. Adhering connect.ivc tissue was removed and the proximal and distal parts were excised and immediately frozen by immersion in liquid N,. Each of the 2 frozen parts of the nerve was chopped into halves to yield 4 segments: A, distal end; H, dist,al t.o illjury; C, prr)xirn:tl t.r) il>jury; I), proxima1 end. At the same time a similar length (ci cm) 01’t,hc opposite sciatic nerve was removed, frozen, and subsequently chopped intr, 4 segments equivalent in length to the segments from i.hr injured nerve. Homogenates and subJractions. The frozen segments were weighed on an ASE Torsion balailct! and ground in a Teflon-pestle homogenizer (A. l-l Thomas Co., clearance 0.134.18 mmj at 2000 rpm in ice-cold 0.25 M sucrose, lo a tinal concent r:ttiorl of 100 mg nerve per milliliter. The homogenate was centrifuged at 1000~ (;tvg) for 10 minutes in a Servall RC 2 (M 34 head 1. The pellet was washed once by resuspension in half of the original volume of sucrose and reccntrifllgetl to yield fraction “P,.” Th e combined s~~pernnttt~~~ fraction and washings were centrifrtged at 10.000~~ for 15 minutes in the Servall for fra(xtion ‘(I’.‘,” iI1 which was also collected the floaf ing “nlyelill layer.” This pellet was also washed as above. Thr: “P,” fraction was prepared by cent,rilliging the: combined supernatant, fraction and washings at 100,OOOg for 1 hr in 2 ml tubes in the No. 40 head OI a Spinco model Ii Illtracelltrifuge. The sclperll:ttant fraction was designated “S.” Each pellet was resuspended in 0.25 M sllcrose to a final vol~~rnt~01 1 ml/l00 rng original I issilc. All fr:lc.t.icms wtlI*’ stored at -20” until use. ATPase assay. Tris-ATP, 15 nlM, pH 7.4. was prepared from Nar ATP by the method drsrribctl previously (5). For the ATPase assay, the final incllbation mixtllrc ront.:tined: tisslle fractic,n; 3 mM Tris-ATP, pH 7.4; 3 111.~MgC12; and f>iiIIPI 100 rnl\f Tris-HCl, pH 7.4, 100 IXM NaCI, ~0 WM KCI, or 230 111~Tris-HCl, pH 7.4. Pu’onctla,vrnit* ATP control t ltbes were prepared for hot h ilrc*llb:ltion mixtures. All constitueltts except, the tisslle fract,ion and ATP were mixed in a volllrne of 0.X ml in tubes at 0”. Immediately after the addit iorl of tissue fraction the tubes were equilibrated :tt :i’i” for 5 minutes and the reaction was started by the addition of 15 111~ Tris-ATP (0.2 ml). The v~)l~ur~es of t,issue fractions normally used were: hoiriogenate (H), 10 ~1; 7’1, 50 ~1; P2’,,50 ~1; PS: 20 ~1; 5: 100 gl. ‘Test)s with H alld P3 confirmed that hydrolysis of t.he ATP was linear wit,h t,irnc, rip to 15 minutes; IO-minute incllbation times u-~r(: routinely used and the reactions were stclpped by the addition of lo:;, (w/v) trichloroa~t ic ;rcnitl (1 ml) at 0”. Inorganic phosphatr was derrrrllined after iso-but anol-benzellc extract ion of t hr 1,110s.
100
BACHELARD
phomolybdate as described by Deul and McIlwain (17). Ouabain inhibition was tested at a final concenat a final tration of 0.1 IIIM (5); Ca++ inhibition concentration of G mM CaClz (2). Naf and K+ were determined with an EEL “type A” flame photometer. Protein. The Lowry method (18) for the determination of protein was used with crystalline bovine serum albumin as standard. Identical concentrations of t,he sucrose in which the various subcellular fractions had been suspended were included in the blank and standard tubes. Occasionally with subfract,ions high in lipid content, the final solutions after addition of all reagents were faintly turbid. These were clarified by centrifugation of the final reaction mixture at 25009 for 10 minutes in a Servall “SSI” centrifuge. Checks using added standard confirmed that no color was lost during this procedure. Treatment with phospholipids. The phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, and sphingomyelin) were dissolved, without further purification, as a mixture in chloroform-methanol (4:l v/v) to a final concentration of 10 mg of each per milliliter. A sufficient quantity of the mixture for a group of incubations was pipetted into a test tube and the solvents were removed by warming in a stream of Nz. The phospholipid smear was thoroughly dispersed in the incubation medium containing all of the constituents for the ATPase assay except Tris-ATP and the tissue fraction. Samples of 0.8 ml were placed in the relevant incubation tubes at 0”. The assay was then carried out m described above. The volume of the original phospholipid mixture used was calculated so that the final amount in each incubation tube was 200 pg (50 fig of each). In the enzymic assays the phospholipid control tubes gave elevated color yields. The increase in optical density was 0.05 (compared with an OD of 0.04-0.08 from the ATP control tubes). Reagents. Reagents not otherwise specified were of analytic reagent quality. Tris 121, NhATP, and ouabain were obtained from the Sigma Chemical Co., St. Louis, Missouri; phosphatidyl choline and phosphatidyl ethanolamine from California Corporation for Biochemical Research, Los Angeles, California; phosphatidyl serine and sphingomyelin from L. Light and Co., Colnbrook, Bucks. Vaxitas FE vaccine (Tasman Vaccine Laboratories Ltd., New Zealand) was purchased from Veterinary Supplies Ltd., South Melbourne, Australia. RESULTS
Na+, K+-ATPase activity of normal sciatic nerve. The activity of the ATP-hydrolyzing
AND
SILVA TABLE NAP, K+-ATPASE
I
ACTIVITY SCIATIC NERVE
OF NORMAL
Homogenates were prepared and assayed for ATPase activity wit’h 3 mu Mg++ in the presence or absence of 30 mM KC1 and 100 mM NaCl as described in Methods. Results are expressed as mean value f SD (No.). ATPase activity Addition
-
Na+, K+ Na+, K+ onabain (0.1
0.81 f 1.08 f 0.56 f
0.15 (12) 0.18 (12) 0.09 (8)
9.7 12.9 3.9
rnM)
Na+, KC, Ca++ (6 mM)
0.66 z!z 0.14 (7)
8.0
system in normal sciatic nerve of the cat was tested with Mgff and in the presence or absence of Na+ and K+ (Table I). The cardiac glycoside, ouabain, which generally is known (5) to inhibit the increased ATPase activity due to Na+ and K+ in the presence of Mg++, decreased the activity in these preparations to below the level observed in the absence of Na+ and K+. The fact that the activity in the presence of ouabain, Mg++, Na+, and K+ was lower than the activity in the presence of Mg++ alone may be due to the presence of endogenous lSa+ and K+ in the nerve homogenates, which would be expected to have an activating effect, i.e., the observed level, in the presence of Mg++ alone, may be elevated due to endogenous Na+ and K+. Direct estimations on 8 homogenates yielded the following results: Na+, 15 f 3 mM; K+, 9 f 2 mM. The 0.25 M sucrose used in the preparation of the homogenates contained less than 0.095 mM Na+ or K+. Jarnefelt (19) has suggested that brain microsomal ATPase preparations contain bound endogenous Na+ and K+ which can be removed by treatment with deoxycholate. The activity in the presence of Mg++, Na+ and K+ recorded in Table I (1.08 pmoles ATP hydrolyzed per milligram fresh nerve per hour) is higher than the activity previously reported for cat sciatic nerve by Bonting and co-workers (20) who found a
ATPASE IX DEGESEItATING
Ol 0
# 5
I 20
1 15
IO
Days
after
nerve
101
NERVE
25
30
I 35
injury
FIG. 1. Na+,K+-ATPase activities in normal and damaged sciatic nerves. The enzymic activities were estimated with 3 mM MgCl,, 30 mM KCI, and 100 mM NaCl. Untreat)ed nerve (m); damaged nerve: segment B, distal to injury (0); damagednerve, segment C, proximal to injury (0).
slight inhibition (10%) by ouabain and an activation by Ca++. In the present study, Ca++ was inhibitory, in accordance with the report by Skou (2) on the Ca++ inhibition on Na+, Ii+-ATPase in peripheral nerve. Na +, K+-A Tl’ase activity of damaged sciatic nerve. The activity of the ATPase in the presence of added Mg++, Sa+, and B+ in the damaged sciatic nerve at various times (2-32 days) after crushing, was compared with the activity in the normal untreated nerves from the same cats. The levels of enzymic activity in both distal and proximal parts of the crushed nerve (Fig. l), expressed as micromoles ATP hydrolyzed per milligram protein per hour, were well below the control activities at the earliest time studied (2 days), and remained at approximately the same level until S days. By 16 days after injury the activities had reached levels higher than those of the control nerves. During the period of nerve degeneration studied, a pronounced increase in the weight of the tissue occurred-the weight increased from a normal value of 60-70 mg per centimet,er to SO-110 rng per centimeter in the damaged nerves in the vicinity of the injury.
since enzymic activity (xxAccordingly, pressed on a basis of the protein content of the nerve may not make allowance for t,hc weight changes of the nerve, the results have also been expressed on the basis of the a&ivity per milligram fresh nerve and the activity per centimeter nerve (Fig. 2); a similar relationship between normal and damaged nerve can be observed. This indicates t#hat the ATPase activity was genuinely lower than normal in the injured nerve during thu first 8 days; the results do not merely reflects changes in the weight or protein c*ontent’ of the nerve (13). Ouabain inhibited the Xa+, I<+-.~1TPasc activity in the injured nerve to a slightly lesser extent than in the normal nerve (Tnble 11). Na+ , K+-A TPase in subfractions qf I)o~~ma1 and injured nef-ves. The distributions of the Na+, K+-ATPase activity in the subfractions prepared by differential centrifugation of homogenates of the untreated and damaged sciatic nerves 4 days after nerve-section are shown in Table III. There was little variation in the activities observed in t.hcb homogenates (H) for different segments from the normal nerve. Similarly, only slight,
102
BACHELARD
AND SILVA TABLE
II
OUABAIN INHIBITION OF NA+, K+-ATPASE NORMAL AND CRUSHED SCIATIC NERVE
IN
Homogenates of the nerve segments were assayed for ATPase activity in the presence of added Mg++, Na+, and K+ as described in Methods. The crushed and control nerves were taken from cats 8 days after crushing. Each value is the average of the preparations from 2 cats. ATPase activity (~moles Nerve segment
Control Damaged “B” Damaged “C”
01
5
10
15
20
25
30
35
Days after nerve injury
Fra. 2. Na+,K+-ATPase activities in normal and damaged sciatic nerves. The results shown in Fig. 1 are expressed on a basis of nerve weight (mg) or length (cm). Untreated nerve (m); damaged nerve, segment B, distal to injury (e); damaged nerve, segment C, proximal to injury (0).
variations were observed in the P3fraction of the segments from the normal nerve. This fraction, which sedimented between 10,000 and lOO,OOOg,was the most enriched in enxymic activity; the mean value of 61 Imoles ATP hydrolyzed per milligram protein per hour was comparable with the activity observed in membrane-enriched subfractions from cerebral cortex microsome preparations (5, 15) (50-75 pmoles per milligram protein per hour). In the nerve 4 days after injury, the ATPase system was less active in the homogenates from all segments of the damaged nerve than in the normal nerve. The lowest activity occurred in the segment (B) immediately distal to the cut. The activity in the segment (C) immediately proximal to the cut was also noticeably diminished. The smallest effect was apparent in segment Din the area further from the site of injury on the proxima1 side.
(Distal) (Proximal)
P//mg protein/hour)
NO ouabain
0.1 mM ouabain
11.2 3.6 4.8
5.7 (49) 2.1 (42) 3.2 (34)
(7, inhibition)
The changes in enzymic activity in the subfractions from the segments of the degenerating nerve followed closely those in the whole homogenates. The activity in Pa, the richest source of the enzyme system in these preparations, fell to below 40% of the control level in segment B, immediately distal to the injury and almost no activity could be detected in the soluble fraction (S) from that segment. As was found with the homogenates, the least change in activity was observed for subfraction Pa from the proximal segment furthest from the site of injury (D). Effect of phospholipids on the ATPase in sectioned nerve. The ability of a phospho-
lipid mixture to restore the dimished enzymic activity of the P3 and the soluble fraction was tested, and partial restoration was effected in both fractions (Table IV). The fractions chosen for this aspect of the study were those in which the greatest decreasein enzymic activity had been observed The activity of the Pa fraction of segment B (immediately distal to the injury) was slightly increased, but a more successful restoration occurred in fraction Ps of segment C (immediately proximal to the injury) in which the original activity was less depleted. A similar pattern emerged for the soluble fractions: segment B, which suffered the greatest depletion of original activity was not affected by the phospholipids whereas the activity of segment C was considerably restored.
ATPASE
IN UECEKERATING TABLE
NA+,
K+-ATPASE
ACTIVITY
ru’EI
1 o:j
III
IN SUBFRACTIONS
OF NORMAL
AND
I~AMAGEU NERVE
Subfractions of the segments of normal and damaged nerves from cats 4 days after unilateral nerve sections were prepared and assayed for ATPase activity as described in Methods. The enzymic activity proteitl was estimated with added Mg ++, Naf and K+, and is expressed as rmoles ATP hgdrolyzed/mg hour. Each value is the mean of preparations from 2 cats. Segment
.A” Normal Sectioned cc,‘;,Normal) B Normal Sectioned Cc,?;Normal) C Normal Sectioned (7; Normal) 1) Normal Sectioned (I;; Normal)
Hb
10.1 7 G (75) 13.0 3.4
P,
16.0 3.4
P2
11.3 7.0
pa
:,
65.0 3-l. 1 (52) (il.0 23 7
Ii.:3
@ti)
WI
(61)
(39)
13.9 5.4 (39) 11.0 8.4 (76)
11.3 5.5 (49 i
16.5 12.0 (74 )
61 .o “i.!) (-l-G) 57.0 50.5 !88 )
1.7 (Sfii
x.i 0.2 121
x 5 1.4 (16) 10.4 ‘)-.- ,> (21 I
h A: distal end; B: distal IO sect,ion; C: proximal to section; 11: proximal ettd. b H: Homogenate; Pi: subfraction which sedimented at 1000 g for 10 minutes; Py sttbfraction which sedimented between 1000 g, 10 minutes and 10,000 g, 15 minutes; Pa: subfract,ion which sedimented between 10,000 g, 15 minutes and 100,000 g, GO minutes; S: soluble fraction as sttpernatattt fractiott after 100,000 g, GO minutes. 11TSCUSSION
The marked loss of NaT,K+-activated, Ng++-dependent ATPase activity during the early stages of peripheral nerve degeneration, when conductivity is lost, is of interest since enzyme systems of this type have been suggested (2-S) to be associated with ion transport mechanisms necessary to maintain the physiological function of the tissue. It is possible that t’he activity remains diminished for longer periods but is masked by an increased activity due to the proliferation of macrophages (13) in the vicinity of the injury. The distribution of the ATPase activity in the segments of the degenerating nerve resembles the pattern observed for acetyl cholinesterase (11) in that the most marked diminution of activity occurred in that part of the nerve immediately distal to the injury and the least effect was found in the proximal segment furthest from the site of injury. The results presented here indicate that the decrease in ATPase activity was not due to changes in weight or protein content of the injured nerve (13), since the activity per centimeter nerve and the activity per unit
protein were both greatly dimished. The changes in activity of all of the particulate fractions suggest that general changes in the organisation of membrane systems iri the tissue have occurred, as might be expected if dissociation of the membrane lipoproteinq (10) has occurred. The partial restoration of the depleted enzymic activity by relatively large amounts of a phospholipid mixture (0.2 mg were used per 2-5 mg original nerve) provides additional evidence in support of recent reports that ATPases of this type require phospholipids, which are considered to be associated with the native enzyme systems. Phospholipase C, which acts on sphingomyelin (21) as well as on lecit.hins, has been shown to inhibit the Naf, K+-ATPases of erythrocytes (14) and cerebral cortex (15). Tanaka and Abood (16) reported a considerable reduction in ATPase activity in membrane preparations from rat cerebral cortex after removal of the endogenous phospholipids by treatment with deoxycholate and ammonium sulfate fractionation. Restoration of enzymic activity was obtained by the addition of large amounts of exogenous phospholipids,
104
BACHELARD TABLE
EFFECT
NA+,
IV
OF PHOSPHOLIPIDS K+-ATPASE ACTIVITY OF SECTIONED
ON THE DECREASED OF SUBFRACTIONS NERVE
The phospholipids were added as described in Methods to a final concentration of 200 gg (50 I.rgof each phospholipid) per assay. The amount of tissue protein used was Pa, 1520 rg (representing 2 mg of nerve); S, 200-250 pg. (representing 5 mg of nerve). ATPase assays were carried out with 3 mM Mg&, 30 mM KC1, and 100 mM NaCl; each value is the mean of two nerve preparations. ATPase
Segment&&
p&ypX’ormal
B*
Ps
C
Pa
B
S
c
s
+ + + +
54.5 55.8 63.7 63.0 7.9 8.2 6.7 6.5
Activity (Irmoles protein/hour) Sectioned
23.3 32.2 29.8 47.0 0.0” 0.0” 0.9 3.3
P/mg
NOi&lj
(43) (58) (47) (74)
(13) (51)
a Not detectable. * B: distal to section; C: proximal to section. Pa: subfraction which sediment,ed between 10,000 g, 15 minutes; and 100,000 g, 60 minutes ; S : soluble fraction as supernate after 100,000 g, 60 minutes.
phosphatidyl serine, or phoslecithins, phatidyl ethanolamine. In this study, the highest restoration was effected by 10 mg lecithin per 25 mg rat brain. These workers concluded that phospholipids are essential for optimal activity of the ATPase. The restoration of enzymic activity by phospholipids observed in the present study was more effective in the less severely damaged segments of the injured nerve (Table IV). In the areas of the nerve which had suffered the greatest decrease in activity, loss of endogenous phospholipid may have contributed to the loss of activity, although measurable losses of phospholipids in degenerating nerves have not usually been possible until later stages of degeneration have been reached (12). However, as Adams and Tuqan (10) suggested, fine changes in the configuration of lipoprotein structures, or dissociation of the lipoproteins of the membranes, might occur well before changes in phospholipid levels can be detected by
AND SILVA
chemical analysis. Samples of the segments from the injured and normal nerves used in this study are currently being examined in the electron microscope with a view to correlating changes in membrane structure and integrity with the observed changes in enzymic activity and also to gauge the extent of proliferation of macrophages and fibroblasts over the relevant time period. The similarity between the changes in ATPase and acetyl cholinesterase (11) activities during early periods of degeneration suggests that it would be of interest to attempt a study of the activity of the ATPase system during nerve regeneration. A local axonal synthesis of acetyl cholinesterase has been reported (22) ; the ATPase could also be of potential interest in studying protein synthesis in peripheral nerve. However, it is doubtful if the biochemical techniques which were the basis of the present report would give sufficient resolution; unlike acetyl cholinesterase, ATPases of this type occur widely (20) and discrimination between activity in the nerve itself and activity in surrounding tissues, proliferating macrophages and fibroblasts would be difficult. However, the lower ATPase activity during early degeneration does indicate the value to be obtained from a study of the activity during regeneration; the required resolution may be obtained in association with the use of histochemical electron microscopic techniques such as those described by Barrnett (23), when the necessary quantitation of such methods has been developed. ACKNOWLEDGMENTS We are indebted to Dr. A. Wilson of the Anatomy Department, Monash University, for his helpful advice; and to Mr. H. Ueckert and Mr. P. Moritz for their skilled technical assistance. REFERENCES L., Ph~sioE. Revs. 36, 441 (1956). J. C., Biochim. Biophy,. Acta 23, 394 (1957). 3. SKOU, J. C., Biochim. Biophys. Acta 42, 6 (1960). 4. SKOU, J. C., “Symposium onMembrane Transport and Metabolism, Prague, 1961.” p. 228. 5. SCHWARTZ, A., BACHELARD, H. S., AND McILWAIN, H., Biochem. J. 84, 626 (1962). 1. GUTH,
2. SKOU,
ATPASE 6. 7. 8. 9. 10. 11. 12. 13.
14. 15.
IN DEGENERATING
GLYNN, I. M., J. Phyaiol. 160, 18P (1962). WHITTAM, H ., Rio&em. J. 82, 205 (1962). WHITTAM, It., Biochem. J. 84, 110 (1962). HOLUNGER, 1). M., ROSSWER, R. J., AND UPMALIS, H., Riochem. J. 62, 652 (1952). ADAMS, C. W. M., AND TUQAN, N. A., J. Neurochew 6, 334 (1961). &LENA, J., AND LUBRINSKA, L., Physiol. Bohem. 11, 261 (1962). LoG.~N,J. E., MANNELL, W. A., AND ROSSITER, K. J., Riochena. J. 61,482 (1952). JOHNSON, A. C., MCNABB, A. R., AND ROSSI'PER, R. J., Arch. :Veurol. Psychiat. Chicago 64, 105 (1950). SCHATZMANN, H. J., Nature 196, 677 (1962). SWANSON, P. I>., BRADFORD, H. I?., AND MCILWAIN, H., Biochem. J. 92, 235 (1964).
NERVE
I 0.i
16. TANAKA, R., AND ABOOD, L. G., Arch. Ridiem Biophys. 108, 47 (1964). 17. DEUL, D. H., AND MCILWAIN, H., J. .VCU,Y)them. 8, 246 (1961). 18. L~\vRY, 0. H., ROSEBROUGH, N. J.. FARI<, A. L., AND RANDALL, R. J., J. fiiol. C’hem. 193, 265 (1951). 19. JAILXEFELT, J., Biochem. Bioph!Js. Ken. Commun. 17, 330 (1964). 20. BONWNG. S. L., SIMON, Ii. i)., ANI) HA\+-KIXS, N. &I., Arch. Biochem. Biophys. 95, 415 (1961). 21. HANAHAN, 1). J., ASD VERCAMEK, I:.. ./. :I,,/. Chem. Sot. 76, 1804 (1954). 22. KOENIG, E., J. Neurochem. 12, 343 (1965). 23. BAI~IISETT', R. J., J. Ro~J. Microscop. Sm. 83, 143 (1964).