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Localization of trehalase in flight muscle of the blowfly Phormia regina
ARCHIVES
OF
BIOCHEMISTRY
AND
Localization
BIOPHYSICS
of Trehalase Blowfly
W. DOUGLAS Laboratory
of Molecular Aging, Human Development,
146, 392-u)l
in Flight
Phormia
REED
(1971)
of the
regina
BERTRAM
AND
Muscle
SACKTOR
Gerontology Research Center, National Institute of Child Health National Institutes of Health, Baltimore, Maryland 112BQ Received
April
and
1, 1971
The ultrastructural localization of trehalase in the flight muscle of the blowfly, Phormia regina, has been investigated. Approximately 65yo of the total activity sediments with a low-speed pellet; 25% of the activity in the homogenate is soluble. The activity in the particulate fraction resides in the mitochondria. Isolated mitochondria have been fractionated by different procedures and the distribution of trehalase has been compared to the distributions of a-glycerophosphate dehydrogenase, hexokinase binding, malic dehydrogenase, and adenylate kinase, representing the inner and outer membranes, the matrix, and the space between the outer and inner membranes of the mitochondria, respectively. Trehalase is clearly separated from the enzyme markers of the matrix and the space between the two membranes. Its distribution closely resembles that of a-glycerophosphate dehydrogenase rather than that of hexokinase binding. Thus, these findings support the tentative conclusion that in blowfly flight muscle the mitochondrial trehalase is localized on the inner membrane. If the permeability of trehalose in mitochondria is the same as that of sucrose, then it is suggested that trehalase is situated on the outside of the mitochondrial inner membrane. The evidence to-date favors the view that the soluble trehalase activity in muscle homogenates represents a muscle enzyme distinct from the mitochondrial trehalase.
On initiation of flight, glycolytic flux in the blowfly, Phormia regina, increases lOOfold or more (1). During this rest-to-flight transition, flight muscle trehalase, which hydrolyzes trehalose (l-a-n-glucopyranosylar-n-glycopyranoside) to two glucose moieties, is activated (2). The mechanism of this control is being investigated. As part of this study, the subcellular localization of the enzyme in the muscle has been examined. Despite numerous reports of trehalase in various insect species and tissues, c.f., Wyatt (3), data on the precise subcellular localization of the enzyme are fragmentary and seemingly inconsistent. In general, two different trehalases, specific for trehalose, have been characterized and both may be present in the same insect (4, 5). One type is represented by the enzyme in intestine and blood; the other is found in muscle. The gut trehalase is soluble; the muscle enzyme is 392
largely associated with particles. The soluble enzyme has a lower pH optimum and a greater affinity for its substrate than has the particulate enzyme (3), but this view has recently been questioned (6). Some of the conflicting findings undoubtedly stem from the use in the different studies of the entire insect, or crude dissections of the insect; consequently, an indeterminant mixture of trehalases may have been examined. The hydrolysis of trehalose by muscle homogenates is well documented (7, 8). The enzyme has since been measured in muscle particulates, prepared primarily by differential centrifugation of homogenates in isotonic media. In the cecropia silkmoth and Blaberus roach, about half the total enzyme, with high specific activity, is found in the microsomal fraction (4, 5). Appreciable activity remains with a low-speed fraction that contains myofibrils, nuclei, and mitochon-
FLIGHT
MUSCLE
dria, but isolated mitochondria possess very little activity. In contrast, other investigations indicate that in the roach, Leucophaea, and in flies trehalase is located in mitochondria (6, 9, 10). In this paper, the ultrastructural localization of trehalase in flight muscle of Phormia regina has been examined in greater detail, using various techniques for separating particulates and coupling these fractionations with elect’ron microscopy and measurements of marker enzymes. MATERIALS
AND
METHODS
Blowflies, Pharmia regina, were maintained in laboratory culture as reported previously (II). Flight muscles were excised from flies, without regard to sex, 7-10 days after eclosion. Mitochondria were isolated by the general procedures described earlier (12, 13); however, the composition of the isolation medium and gravitationai forces varied with the experiment and these are stated in each case in the relevant table or figure. Mitochondria were treated with Digitonin or Lubrol by the method of Schnaitman and Greenawalt (14). Submitochondrial fractions were also prepared according to Sottocasa et al. (15) and Whereat et al. (IG). Samples of tissue and particulates were prepared for electron microscopy as described previously (17). Trehalase was measured spectrophotometrically (18) in a coupled assay containing 1.5 mM NADP, 1.1 mM ATP, 1.1 rnM MgC12, 20 mM phosphate buffer, pH 6.3, 8 mM trehalose, and 1.4 and 3.5 units of hexokinase and glucose-B-phosphate dehydrogenase, respectively. The mitochondrial a-glycerophosphate dehydrogenase was assayed polarographically with a Clark electrode in a m?d reaction containing 2 ml of 0.15 M KCl-10 Tris buffer, pH 7.4, 0.1 ml of phenazine methosulfate (5 mg/ml), 0.05 ml of 0.1 tif KCN, and 0.05 ml of 2.5 ag glycerophosphate (40% Z-aisomer). The mitochondrial malic dehydrogenase was measured as described by Schnaitman et al. (19), with particulate preparations being activated with Lubrol. Adenylate kinase and monoamine oxidase were assayed according to Schnaitman and Greenawalt (14). The binding of rat brain mitochondrial hexokinase to flight muscle mitoehondrial fractions was measured by the procedure of Kropp and Wilson (20). Protein was measured by the biuret reaction (21); crystalline bovine serum albumin was used as the standard . Trehalose and oxaloacetate were purchased from Calbiochem. a-Glycerophosphate was obtained from Eastern Chemical Corp. Nagarse was
393
TREHALASE
obtained through Enzyme Development Corp., New York. Other enzyme and nucleotides were purchased from Boehringer Mannheim Corp. RESULTS
When flight muscle is removed from the thorax of the blowfly, rinsed, homogenized gently in isotonic media, and fractionated by differen& centrifugation, trehalase activity is distributed in all fractions. As shown in Table I, however, over 60% of the total activity sediments with a lowspeed pellet. Less than 10 % is found with particles, which from an operational point of view is considered to be microsomal. The supernatant from t.he 100,000 g centrifugation always has approximately 25% of the tot,al activity; in 10 experiments t’he amount that is found ranges from 18 to 34%. This distribution in enzyme activity agrees with that’ reported by Hansen (10). Also noted in Table I is t’he estimate that Phormia flight, muscle trehalase has a specific activity of 0.2 pmoles of glucose formed X min-1 X mg-1 protein. This activity is sufficient to account for the rate of trehalose utilization by the blowfly, in viva, on init,iation of flight, (2). A survey electron micrograph of a fiber of the dorsal longitudinal flight. muscle of Phormia is illust,rated in Fig. 1. As described previously (22, 23), the myofibrils are cylindrical, about 2 p in diameter, set’ in sarcoplasm largely occupied by mitochondria. The mit.ochondria of the blowfly are ovoid TABLE
I
DISTRIBUTION OF TREH.IL.ISE ACTIVITY AFTER DIFFERENTML CENTRIFUGSTIOK OF FLIGHT MUSCLE HOMOGENITESO Fraction
Homogenate 50009 (5-min) pellet 100,OOOg (60.min) pellet 100,OOOg (60.min) supernatant
Trehalase activity Total units
9.3 6.0 O.ti 2.2
% 100 64 6 24
a Muscle of 80 thoraces were homogenized in an isolation medium containing 0.154 M KCl, 10 m&r Tris buffer, pH 7.4,1 mM EDTA, 2 mM EGTA, and 0.5% BSA. Units are expressed as pmoles of glucose formed X min-‘. Specific activity of trehalase in the homogenate was 0.2 units x mg-t protien.
394
REED AND SACKTOR
FIG. 1. A survey electron micrograph of Phormia flight muscle, in cross section, showing arrangement of mitochondria (Mit) about the myofibrils (mf). Other membranous structures evident are elements of the sarcoplasmic reticulum (SR) and the T-system (T). The T-system is comprised, in part, of elements of the plasma membrane which surround the invaginating tracheoles (tr). Glycogen rosettes (Gly) fill the sarcoplasmic spaces. The bar represents 1 cc.
and irregular in shape, up to 4 p in length, and are not aligned wit’h respect to myofibrillar striations. Each mitochondrion has a distinct outer limiting membrane and its inner membrane is arrayed in parallel plates, 30-35 cristae per micron (22). The large number of cristae is indicat’ive of the high respiratory activity of these mitochondria. It also indicates that the ratio of inner to outer membrane is extremely great. The sarcoplasmic
reticulum
of t#his asynchronous
muscle is remarkably reduced, representing only 0.2 % of the cell volume, which together with elements of the T-system and plasma membrane tubules, representing 0.5-l % of the fiber volume, form dyad associations adjoining the fibril surface (23). Glycogen
particles are located mostly in the intermyofibrillar sarcoplasm, with scattered deposits on the myofibrils (11). The low-speed pellet,, which possesses approximately 65% of the total trehalase activity and is obtained by the fractionation scheme given in Table I, consists primarily of myofibrils, mitochondria, and nuclei; the latter being relatively few in number. The small fraction of the total enzyme that precipitates at, 100,000 g for 1 hr cosediments with glycogen rosettes, reticular membranes, and tiny fragments of mitochondria and myofibrils. The remaining activity fails to sediment at, this higher force and is regarded to be nonparticulate. This soluble fraction will be considered later in this report,.
FLIGHT
MUSCLE
In order to resolve the question of the localization of t)he membrane-bound trehalase further, a direct comparison has been made between the enzymic activity in the dense particulate fract’ion, which contains a mixture of mitochondria and other large subcellular organelles, and that in a mitochondrial preparat,ion, which is essentially free of contaminants. As reported in Table II, there is no significant difference between the mixture of particulates sedimenting at 12,000 g and the isolated mitochondria in their relative trehalase act,ivities, . each having about 60% of the t.ot,al actlvlty in the homogenat’e. An electron micrograph of a typical mitochondrial pellet, prepared by t!he Nagarse procedure (13), is illustrated in Fig. 2. Myofibrils and nuclei are absent, and t’here is no evidence of extraneous membranes adhering to these isolated mitochondria (1). These findings suggest that TABLE II COMPARISON 0F THE TREHALASE ACTIVITY 1s THE DENSE PARTICULATE FRilCTION OF HOMOGIN ISOLATED MITOCHONENATES WITH TH.LT DRIBa Preparation
Homogenate “A” 12,000g (5.min) pellet 12,OOOg (5-min) supernatant Homogenate “B” Mitochondria 12,000g (5.min) supernatant
Trehalase Total units
activity %
0.75 0.49 0.15
100 66 20
3.8 2.1 1.2
100 56 32
a Each value is the average of four experiments. In the preparations, designated “A,” the flight muscles of five blowflies were homogenized in a “Virtis 23” homogenizer for 2 set in 1 ml of medbuffer, ium comprising 0.15 M KCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, and 0.5% bovine serum albumin. In the preparations, designated “B,” the muscles from 20 flies were ground by hand with three passes of a Potter-Elvejhem homogenizer in 5 ml of medium identical with that used in preparation “A,” except that 5 mg of Nagarse was added. The proteolytic enzyme was incubat,ed with the muscle suspension for 10 min at 0”. The digested muscle was filtered through cheesecloth and the mitochondria were sedimented at 12,000g for 5 min.
TREHALASE
393
t#he trehalase activity cosedimenting with low-speed centrifugation of homogenates is localized in mitochondria. It is also of interest to note from Fig. 2 that most of the mitochondria, as isolated, appear in the condensed configuration (24), although occasionally mitochondria in the orthodox form are seen. The view that trehalase is localized in mitochondria is supported by additional findings, shown in Table III, demonstrat,ing parallel dist,ributions of trehalase and the mitochondrial flavin-linked a-glycerophosphate dehydrogenase (12, 25) in the part’iculate fractions. When the pellets are repeatedly washed in the serum albumin-cont,aining medium and recenkifuged at. 10,000 g for 13 min, approximately 2% of the trehalase and 1% of the a-glycerophosphat’e dehydrogenase is found in the supernatant fraction after each cycle. This suggests that trehalase is not readily removed from mitochondria by this procedure, but. that a few mitochondrla are disrupted result’ing in smaller fragments that do not sediment at 10,000 g. On the other hand, when serum albumin is not included in the wash medium, up to 20 % of the trehalase may be removed whereas ordy 2 to 3% of t.he or-glycerophosphate dehydrogenase fails to sediment. The requirement for serum albumin in preserving mitochondrial integrity and in binding fatty acids produced from endogenous phospholipids by phospholipases in the mitochondria is well known (26-28). It, has yet t’o be determined, however, whet,her the solubilization of the mitochondrial trehalase by washing in the absence of albumin and this alt,eration in the mitochondria are interdependent or coincidently correlated. In order to determine the localization of trehalase within the mit’ochondrion, different techniques of subfractionat’ing mitochondriu were applied to the present, study. When flight muscle mit’ochondria are disrupted by swelling, shrinking, and sonication and t,he suspension is then fractionat,ed on a sucrosedensity gradient, according to methods described for mammalian mitochondria (13, IS), sarcosomal particles are distributed as illustjrat’ed in Fig. 3. A dense mitochondrial-
396
REED
FIG. 2. An electron pared by the Nagarse
micrograph procedure.
AND
of a typical
SACKTOR
pellet
of flight
muscle mitochondria,
pre-
FLIGHT
MUSCLE
TREHALASE
397
TABLE III IIISTRIBUTIONOFTREHALASEANDMITOCHONDRIIL CY-GL~~ER~~H~SPHATEDEHYDROGENASEIN THE PARTICULATE FRACTIONS n-GP Dehydrogenase
Trehalase Fraction
Total particulate 10,OOOg (15.min) Supernatant 10,OOOg (15-min) Pellet
Total units
%
Total units
6
1.73 0.06
100 4
20.0 0.2
100 1
1.54
89
19.2
96
a Flight muscles were homogenized in a medium containing 0.154 Y KCl, 20 mM potassium phosphate buffer, pH 7.2, 1 mM EGTA, and 0.5% BSA. The homogenate was centrifuged at 250,OOOg for 35 min. The precipitate was used as the total particulate fraction. The residue was resuspended in medium and recentrifuged at 10,OOOgfor 15 min. The unit of a-glycerophosphate (wGP) dehydrogenase activity is expressed as pg atoms oxygen X min+.
like pellet, designated Fraction 3, is found on the bottom of the tube. A lighter-colored brownish-red band, Fraction 2, is localized at the interface of the 1.2 and 1.0 M sucrose solutions. The opalescent mitochondrial medium (Fraction 1) remains on the top of the sucrose gradient.. No discernible band is formed at the medium-l.0 M sucrose interface, the locus where outer membrane fragments of mammalian mitochondria concentrate. Trehalase activity has been determined in the three fract’ions and its distribution compared to those of other enzymes whose int.ramitochondrial localizations are established (Table IV). cr-Glycerophosphate dehydrogenase has been used as the marker for the inner mitochondrial membrane (29, 30). Malic dehydrogenase is indicative of enzymes in the matrix. Adenylate kinase, prev~ously described in flight muscle mitocondria (31), has been reported in rat liver mitochondria to be in the space between the inner and out,er membranes (32). Two other activities, monoamine oxidase and rotenoneinsensitive KADH-cytochrome c reductase, useful markers for outer membranes of mammalian mitochondria, have activities too low t,o be measured with reliability in flight
#3- LJ
'! i
FIG. 3. JXagrammatic representation of the fractionation on a sucrose density gradient of disrupted flight muscle mitochondria. Further details are given in t.he text.
muscle mitochondria. As demonstrated in Table IV, trehalase and cu-glycerophosphate dehydrogenase are dist,ributed similarly in the t’hree fractions. The two enzymes are localized predominantly in Fraction 3. About 20% of their total activities are found in Fraction 2, whereas Fract.ion 1 has less than 10% of these enzymes. This indicates some fragmentation of the inner membrane to less dense particles t,hat band as Fraction 2 or remain in t’he mitochondrial medium. In contrast, Fraction 1 cont’ains 70% or more of the malic dehydrogenase and adenylate kinase activities, suggesting the rupture of many mitochondria and the release of enzymes from the matrix and space between the inner and outer membranes. Fraction 2 is essentially free of malic dehydrogenase. Fraction 3, however, apparently contains the inner membrane-matrix component and/ or intact mitochondria, in addition to the inner membrane. The finding that the’ recovery of malic dehydrogenase sums to more than lOO%, perhaps due t.o the failure of Lubrol to activate fully the dehydrogenase in all measurement,s (19), prevents quantitation of the mat’rix contamination in Fraction 3. The locus of the outer membrane in this sucrose gradient, can not be stat~ed with certaint,y. Subjecting mitochondria to freezing and thalving, followed by centrifugation, unequivocally separates trehalase from malic dehydrogenase. As shown in Table V, trehalase and a-glycerophosphate remain bound to the membranes whereas malic dehydrogenase is essentially completely released and
39s
REED
AND
SACKTOR
TABLE INTRAMITOCH~NDRI:~L
LOC~LIZ.\TI~N
OF
MITOCHONDRI~~
ON
.I
SUCROSE
Trehalase
AFTER DENSITY
(I GP dehydrogenase
Fraction
%>
Units
Disrupted mitochondria Fraction 1 Fraction 2 Fraction 3
IV
‘I’REHAL.ISE
7.81 0.41 1.47 5.04
100 5 19 65
Units
207 18 38 165
FRACTION.\TION
OF
DISRUPTED
GRADIENT’ Malic
dehydrogenase
Adenylate
kinase
%I
Units
%>
Units
%I
100 9 18 80
1970 1370 4 990
100 70
14.9 11.5 1.3 2.6
100 77 9 17
QMitochondria were isolated by the procedure described earlier (12) in a medium containing 0.07 M acid (HEPES) buffer, sucrose, 0.21 M mannitol, 2 mM N-2-hydroxyethylpiperazine-~~‘-2-ethane-sulfonic pH 7.4, and 0.05% BSA. The isolated mitochondria were disrupted by swelling, shrinking, and sonication according to Sottocasa et al. (15), as modified by Whereat et al. (16). The disrupted mitochondria were centrifuged at 140,OOOgfor 60 min, in a sucrose density gradient, shown in Fig. 3. Units of malic dehydrogenase are given as rmoles of oxaloacetate reduced X min- I. A unit of adenylate kinase represents a rmole of ADP converted X min-I. TABLE
V
INTRAMITOCRONDRI.~L TREIIALASE MITOCHONDRI.\
LOCALIZATION AFTER
OF
DISRUPTION RY
OF
FREEZINC
AND
THAWING~
WGP Trehalase Fraction
dehydrogenase
_-
Malic dehydrogenase ___.
Units I Y. Units I Y. Units I s1
_-
Intact mitochondria 3.9010061.2100 306100 Freeze-thaw, 144,OOOg 0.12 3 0.3
solubilized. This rules out the matrix as the locus for the mitochondrial trehalase. The data in the preceding experiments, although excluding the matrix and the space between the inner and outer membranes as the loci of the mitochondrial trehalase, do not clearly distinguish between the inner and outer membrane with respect to the site of the enzyme. This question has been examined by disrupting mitochondria with Digitonin, according t,o the procedure of Schnaitman and Greenawalt (14) for mammalian mitochondria, and by using as a marker for the outer membrane the recently reported prop-
erty of outer membranes to bind brain hexokinase (20). Hexokinase in rat brain mitochondria has been reported to be localized on the outer membrane (20, 33) and, when the enzyme is solubilized, it will rebind preferentially to the outer membrane of brain, liver, and other kinds of mitochondria (20, and J. E. Wilson, personal communication). As shown in Table VI, Digitonintreated flight muscle sarcosomes are disrupted, resulting in two particulate fractions; one, sedimenting at 12,000 y for 15 min, the other centrifuging down at 140,000 g for 60 min. cr-Glycerophosphate dehydrogenase, t’he inner membrane marker, is found mostly in the dense particulate fraction. When compared to int#act mitochondria, the specific activity of this enzyme in the 12,000 g pellet is increased, whereas its specific activity in the 140,000 g pellet is substantially lowered. The data in Table VI demonstrate that rat brain mitochondrial hexokinase does bind to insect flight muscle sarcosomes. Moreover, relative to intact mitochondria, the specific activity of the hexokinase binding capacity in t’he 140,000 g pellet is enhanced 2-fold whereas that in the dense particulate fraction is decreased almost in half. These findings suggest that the highspeed pellet is enriched with outer membranes and the low-speed pellet has concentrated the inner membranes. Quantitation of the relative enrichments is precluded, however, due to the poor recovery of the
FLIGHT
MUSCLE TABLE
INTRAMITOCHONDRL~L
LOC.ILIZATION
Fraction Intact mitochondria Digit,onin-t,reated, 12,OOOg (15 min) supernatant 140,OOOg (60-min) pellet of 12,000g supernatant 140,OOOg (60.min) supernatant of 12,OOOgsupernatant Digitonin-t,reated, 12,OOOg (15min) pellet
399
TBEHALASE VI
OF TREH~LME AFTER DIGITONIN” Trehalase
OF MITOCHON~RI.\
L)ISRUPTION
WITH
sp act
Units
::
sp act
Hexokinase binding Units % sp act
100 17
0.17 0.07
340 22
100 6
1.5 0.2
61.5 -
100 -
0.28 -
1.5
4
0.11
9
3
0.7
7.6
12
0.52
5.6
15
0.16
12
4
0.3
-
15.3
40
0.17
234
70
2.5
16.8
Units
%
38.1 6.5
c-GP dehydrogenase
-
27
0.16
(1Mitochondria were isolat,ed as described in Table IV. The final suspension of the mitochondria was made very concentrated, 100 mg of protein/ml. The mitochondrial suspension was treated with 1.2y0 Digitonin by the method of Schnaitman and Greenawalt (14). The binding of solubilized rat brain mitochondrial hexokinase to flight muscle mitochondrial part&dates was carried out according to Kropp and Wilson (20), except that the hexokinase bound by the particulates was estimated by assay of the reisolated particulates rather than by measurement of the disappearance of hexokinase activity from the supernatant fraction of the incubation reaction. A unit of hexokinase represents apmole of glucose-6phosphate formed X mi0. Specific activities (sp act) are given as units X mg-’ protein.
hexokinase-binding capacity in the submitochondrial fract’ions, perhaps because of the inability to measure binding in the highspeed supernatant fractions and to differences in the assay procedure in the present study from t,hat described by Kropp and Wilson (20). Despite these uncertainties, it is evident from Table VI that the distribution of t’rehalase in the various submitochondrial fractions closely resembles that of cY-glycerophosphate dehydrogenase rather than that of hexokinase binding. There is 10 times more trehalase in the 12,000 g pellet than in the 140,000 g particulate. In addition, the specific activity of trehalase in the high-speed pellet is decreased. This agrees with the decrease in specific activity of oc-glycerophosphate dehydrogenase in t’his fraction and is in contradistinct’ion with the increase in the specific activity of hexokinase binding. Thus, these findings favor the tentative conclusion that in blowfly flight muscle the mitochondrial trehalase is localized in t,he inner membrane of the sarcosome. It is noted in Table VI that after Digitonin treatment a substantial amount of trehalase, 6.5 units, is found in t,he 12,000 g supernatant fraction, but most of this, 5.6 units, has been solubilized or fails to sediment at 140,000 g
for 60 min. Other agents that disrupt mit’ochondria, such as Triton X100, Lubrol, deoxycholate, and sodium dodecyl sulfate, also effect considerable solubilization of the mitochondrial trehalase. As reported in Table I, about 25 % of the total trehalase of the muscle homogenate remains in the supernatant after centrifugation at 100,000 g for 60 min. Alternative explanations have been offered as to the derivation of this soluble activity and the question continues to be the subject of some controversy (1, 3-6, 10). To illustrate : (a) the soluble enzyme may represent a distinct muscle trehalase, differing from the membrane-bound enzyme; (b) all the muscle trehalase is particulate, but that 25 % of the activity is washed off during homogenization and centrifugation procedures; and (c) the soluble enzyme stems from the hemolymph which adheres to the muscle or its entrapped in the extracellular spaces. In the course of the present study, observations have been made that seem relevant t,o this issue. Values for the K, of the soluble and particulate trehalase range from 0.6 t)o 1.3 rnxr and 0.9 to 1.9 mu, respectively. These values are not, significantly different. Therefore, this result is inconsistent with t’he view of the
400
REED AND SACKTOR
presence in the muscle preparation of a true membrane-bound muscle trehalase with a high K, and a soluble blood or intestinal enzyme with a low K,. Rising of the excised muscle in medium does not noticeably reduce the amount of soluble enzyme. In addition, the 100,000 g supernatant, although containing about 25 % of the total trehalase activity, has essentially none of the mitochondrial a-glycerophosphate dehydrogenase activity. Moreover, trehalase is found abundantly in the soluble fraction even though the muscle is homogenized in serum albumin-containing media, which, as noted earlier, largely prevents the solubilization of the mitochondrial trehalase. Repeated centrifuging at low speeds and resuspending the pellet in fresh media containing albumin does remove about 2% of the trehalase each time, but it has been found that 60% of this trehalase will sediment at 100,000 g. About 1% of the total mitochondrial a-glycerophosphate dehydrogenase behaves in the same fashion. These findings suggest that, although preparative procedures may cause limited rupture of the mitochondria, such disruption hardly accounts for the presence in t#he original 100,000 g supernatant (Table I) of onefourt,h of the total trehalase activity. While acknowledging that more rigorous evidence has yet to be developed before the problem is settled, the present data tend to support the hypothesis for two distinct muscle trehalases in t,he blowfly. DISCUSSION
Previous studies have demonstrated that flight muscle trehalase is activated on initiation of contraction (2). As reported in Table I, the specific activity of the muscle trehalase is about 0.2 units X mg’ protein. It has been calculated that this activity is sufficient to account for the rate of trehalose utilization at the beginning of flight (1, 2). This observation, plus the finding that in the blowfly, in contrast to other kinds of insects (4, 5, 9, 34), trehalase activity is not enhanced by various physical and chemical treatments, i.e., freezing and thawing (Table V) and detergents (Table VI), that tend to disrupt membrane structure, suggest that the observed activity probably represents
the decontrolled or active rate of the enzyme. Thus, the demonstration that blowfly flight muscle trehalase is localized in mitochondria focuses the problem of the control of trehalase activity on the rapid transitions taking place in the sarcosomes in situ, when the insect goes from rest to flight. The present study also describes the ultrastructural localization of trehalase with greater precision than known heretofore. The distribution of trehalase in the major mitochondrial compartments has now been examined. The data in Tables IV and V clearly distinguish bet,ween the distributions of malic dehydrogenase and adenylate kinase from that of trehalase and provide good vidence to rule out the matrix and the space bet’ween the outer and inner membranes as the site of trehalase. Clean separation of the inner and out’er membranes has proved to be difficult because of the paucity of outer relative to inner membrane (Figs. 2 and 3) and to the extremely low activities in flight muscle mitochondria of monoamine oxidase and rotenone-insensitive NADHcytochrome c reductase, the established outer membrane markers in mammalian mitochondria. Nevertheless, by comparing the distribution of trehalase with those of a-glycerophosphate dehydrogenase and hexokinase binding in fractions derived from Digitonin-disrupted sarcosomes (Table VI), the hypothesis that trehalase is localized on the inner membrane gains support. It is implicit in this tentative conclusion that trehalose is readily accessible to the inner membrane, and this implies that the disaccharide is permeable through the mitochondrial outer membrane. A precedent is to be found with the disaccharide, sucrose, which freely enters the space between the inner and outer membranes (35). However, sucrose does not significantly enter the matrix. If trehalose behaves similarly in this respect, then it is suggested that trehalase is situated on the outside of the mitochondrial inner membrane. REFERENCES 1. SACKTOR, B., Advan. Insect Physiol. (1970). 2. SACKTOR, B. AND WORMSER-SHAVIT, Biol. Chem. 241, 624 (1966).
7, 267
E., J.
FLIGHT
MUSCLE
3. WYATT, G. R., Advan. Znsect Physiol. 4, 237 (1967). 4. GUSSIN A. E. S., AND WYATT, G. R., Arch. Biochem. Biophys. 112, 626 (1965). 5. GILBY, A.R., WYATT,S.S., AND WYATT,G. R., Ada B&him. Polonica 14, 83 (1967). F. CLEMEXTS, A. N., PIGE, J., BORCK, K., AND VAN DOBEN, A. J. J., J. Insect Physiot. 16, 1389 (1970). 7. SACKT~R, B., J. Biophys. Biochem. Cytol. 1, 29 (1955). 8. CLEGO. J. S., .%NDEVANS, D. R., J. Exp. Biol. 38, ii1 (1961). 9. ZE~E, E. C., AND MC&IAN, W. H., J. Cell. Camp. Physiol. 53, 21 (1959). 10. HANREN, K., Biochem. 2. 344, 15 (1966). 11. CHILDRESS, C. C., SACKTOR, B., GROSSMAN, I. W., AND BUEDING, E., J. Cell Biol. 46, 83 (1970). 12. Sacx~o~, B., AND COCHRAN, D. G., Arch. Biochem. Biophys. 74, 266 (1958). 13. HANSFORD, R. G., AND CHAPPELL, J. B., Biochem. Biophys. Res. Commun. 27, 686 (1967). 14. SCHN.4ITM.4N, C., .4ND GREENAWALT, J. W., J. Cell Biol. 38, 158 (1968). 15. SOTTOC.ISA, G. L. KUYLENSTIERNA, B., ERNSTER, L., AND BERSTRAND, A., J. Cell Biol. 32, 415 (1967) 16. WHEREAT, A. F., ORISHIUO, M. W., NELSON, J., AND PHILLIPS, S J., J. Biol. Chem. 244, 6498 (1969) 17. BERGER, S., .4ND SACIiTOR, B., J. Cell Biol. 47, 637 (1970). 18. SACKTOR, B., Proc. Vat. Acad. Sci. U.S.A. 6O,lCKI7 (1968).
TREHALASE
401
C. A., ERWIN, V. G., AND 19. SCHNAITMIN, GREENIWY~LT, J. W., J. Cell Biol. 32, 719 (1967). 20. KROPP, E. S., AND WILSON, J. E., Biochem. Biophys. Res. Commun. 33, 74, (1970). 21. HANSFORD, R. G. AND SACICTOR, B., J. Biol. Chem. 246, 991 (1970). 22. SMITH, D. S., J. Cell Biol. 19, 115 (1963). 23. SMITH D. S., AND SXKTOI~, B., Tissue & Cell 2, 355 (1970) 24. HACKENBROCIC, C. It., J. Cell Biol. 30, 269 (1966). 25. CHANCE, B., AND SACKTOR, B., Arch. Biochem. Biophys. 76, 509 (1958). 26. SACXTOR, B., J. Gen. Physiol. 37, 343 (1954). 27. S~CI~TOR, B., O'NEILL, J. J., AND COCHRSN, D. G., J. Biol. Chem. 233, 1233 (1958). 28. WOJTCZAK, L. AND WOJT~ZAK, A. B.; Biochim. Biophys. Acta 39, 277 (1960). 29. KLINGENBERG, M., Eur. J. Biochem. 13, 247 (1970) 30. I~ONNELLAN, J. F., BARKER, M. D., WOOD, J., AND BEECHEY, R. B., Biochem. J. 120, 467 (1970) 31. SSCKTOR, B., J. Gen. Physiol. 36, 371 (1953). 32. S~HNAITMIIN, C. A., AND PEDERSEN, P. L., Biochem. Biophys. Res. Commun. 30, 428 (19G8). 33. CRAVEX, P. A., GOLDBLATT, P. J., AND BASFORD, R. E., Biochemistry 8, 3525 (1969). 14, 179 34. STEVENSON, E., J. Insect Physiol. (1968). 35. HANSFORD, R. G., AND CHAPPELL, J. B., Biochem. Biophys. Res. Commun. 30, 643 (1908).
OF
BIOCHEMISTRY
AND
Localization
BIOPHYSICS
of Trehalase Blowfly
W. DOUGLAS Laboratory
of Molecular Aging, Human Development,
146, 392-u)l
in Flight
Phormia
REED
(1971)
of the
regina
BERTRAM
AND
Muscle
SACKTOR
Gerontology Research Center, National Institute of Child Health National Institutes of Health, Baltimore, Maryland 112BQ Received
April
and
1, 1971
The ultrastructural localization of trehalase in the flight muscle of the blowfly, Phormia regina, has been investigated. Approximately 65yo of the total activity sediments with a low-speed pellet; 25% of the activity in the homogenate is soluble. The activity in the particulate fraction resides in the mitochondria. Isolated mitochondria have been fractionated by different procedures and the distribution of trehalase has been compared to the distributions of a-glycerophosphate dehydrogenase, hexokinase binding, malic dehydrogenase, and adenylate kinase, representing the inner and outer membranes, the matrix, and the space between the outer and inner membranes of the mitochondria, respectively. Trehalase is clearly separated from the enzyme markers of the matrix and the space between the two membranes. Its distribution closely resembles that of a-glycerophosphate dehydrogenase rather than that of hexokinase binding. Thus, these findings support the tentative conclusion that in blowfly flight muscle the mitochondrial trehalase is localized on the inner membrane. If the permeability of trehalose in mitochondria is the same as that of sucrose, then it is suggested that trehalase is situated on the outside of the mitochondrial inner membrane. The evidence to-date favors the view that the soluble trehalase activity in muscle homogenates represents a muscle enzyme distinct from the mitochondrial trehalase.
On initiation of flight, glycolytic flux in the blowfly, Phormia regina, increases lOOfold or more (1). During this rest-to-flight transition, flight muscle trehalase, which hydrolyzes trehalose (l-a-n-glucopyranosylar-n-glycopyranoside) to two glucose moieties, is activated (2). The mechanism of this control is being investigated. As part of this study, the subcellular localization of the enzyme in the muscle has been examined. Despite numerous reports of trehalase in various insect species and tissues, c.f., Wyatt (3), data on the precise subcellular localization of the enzyme are fragmentary and seemingly inconsistent. In general, two different trehalases, specific for trehalose, have been characterized and both may be present in the same insect (4, 5). One type is represented by the enzyme in intestine and blood; the other is found in muscle. The gut trehalase is soluble; the muscle enzyme is 392
largely associated with particles. The soluble enzyme has a lower pH optimum and a greater affinity for its substrate than has the particulate enzyme (3), but this view has recently been questioned (6). Some of the conflicting findings undoubtedly stem from the use in the different studies of the entire insect, or crude dissections of the insect; consequently, an indeterminant mixture of trehalases may have been examined. The hydrolysis of trehalose by muscle homogenates is well documented (7, 8). The enzyme has since been measured in muscle particulates, prepared primarily by differential centrifugation of homogenates in isotonic media. In the cecropia silkmoth and Blaberus roach, about half the total enzyme, with high specific activity, is found in the microsomal fraction (4, 5). Appreciable activity remains with a low-speed fraction that contains myofibrils, nuclei, and mitochon-
FLIGHT
MUSCLE
dria, but isolated mitochondria possess very little activity. In contrast, other investigations indicate that in the roach, Leucophaea, and in flies trehalase is located in mitochondria (6, 9, 10). In this paper, the ultrastructural localization of trehalase in flight muscle of Phormia regina has been examined in greater detail, using various techniques for separating particulates and coupling these fractionations with elect’ron microscopy and measurements of marker enzymes. MATERIALS
AND
METHODS
Blowflies, Pharmia regina, were maintained in laboratory culture as reported previously (II). Flight muscles were excised from flies, without regard to sex, 7-10 days after eclosion. Mitochondria were isolated by the general procedures described earlier (12, 13); however, the composition of the isolation medium and gravitationai forces varied with the experiment and these are stated in each case in the relevant table or figure. Mitochondria were treated with Digitonin or Lubrol by the method of Schnaitman and Greenawalt (14). Submitochondrial fractions were also prepared according to Sottocasa et al. (15) and Whereat et al. (IG). Samples of tissue and particulates were prepared for electron microscopy as described previously (17). Trehalase was measured spectrophotometrically (18) in a coupled assay containing 1.5 mM NADP, 1.1 mM ATP, 1.1 rnM MgC12, 20 mM phosphate buffer, pH 6.3, 8 mM trehalose, and 1.4 and 3.5 units of hexokinase and glucose-B-phosphate dehydrogenase, respectively. The mitochondrial a-glycerophosphate dehydrogenase was assayed polarographically with a Clark electrode in a m?d reaction containing 2 ml of 0.15 M KCl-10 Tris buffer, pH 7.4, 0.1 ml of phenazine methosulfate (5 mg/ml), 0.05 ml of 0.1 tif KCN, and 0.05 ml of 2.5 ag glycerophosphate (40% Z-aisomer). The mitochondrial malic dehydrogenase was measured as described by Schnaitman et al. (19), with particulate preparations being activated with Lubrol. Adenylate kinase and monoamine oxidase were assayed according to Schnaitman and Greenawalt (14). The binding of rat brain mitochondrial hexokinase to flight muscle mitoehondrial fractions was measured by the procedure of Kropp and Wilson (20). Protein was measured by the biuret reaction (21); crystalline bovine serum albumin was used as the standard . Trehalose and oxaloacetate were purchased from Calbiochem. a-Glycerophosphate was obtained from Eastern Chemical Corp. Nagarse was
393
TREHALASE
obtained through Enzyme Development Corp., New York. Other enzyme and nucleotides were purchased from Boehringer Mannheim Corp. RESULTS
When flight muscle is removed from the thorax of the blowfly, rinsed, homogenized gently in isotonic media, and fractionated by differen& centrifugation, trehalase activity is distributed in all fractions. As shown in Table I, however, over 60% of the total activity sediments with a lowspeed pellet. Less than 10 % is found with particles, which from an operational point of view is considered to be microsomal. The supernatant from t.he 100,000 g centrifugation always has approximately 25% of the tot,al activity; in 10 experiments t’he amount that is found ranges from 18 to 34%. This distribution in enzyme activity agrees with that’ reported by Hansen (10). Also noted in Table I is t’he estimate that Phormia flight, muscle trehalase has a specific activity of 0.2 pmoles of glucose formed X min-1 X mg-1 protein. This activity is sufficient to account for the rate of trehalose utilization by the blowfly, in viva, on init,iation of flight, (2). A survey electron micrograph of a fiber of the dorsal longitudinal flight. muscle of Phormia is illust,rated in Fig. 1. As described previously (22, 23), the myofibrils are cylindrical, about 2 p in diameter, set’ in sarcoplasm largely occupied by mitochondria. The mit.ochondria of the blowfly are ovoid TABLE
I
DISTRIBUTION OF TREH.IL.ISE ACTIVITY AFTER DIFFERENTML CENTRIFUGSTIOK OF FLIGHT MUSCLE HOMOGENITESO Fraction
Homogenate 50009 (5-min) pellet 100,OOOg (60.min) pellet 100,OOOg (60.min) supernatant
Trehalase activity Total units
9.3 6.0 O.ti 2.2
% 100 64 6 24
a Muscle of 80 thoraces were homogenized in an isolation medium containing 0.154 M KCl, 10 m&r Tris buffer, pH 7.4,1 mM EDTA, 2 mM EGTA, and 0.5% BSA. Units are expressed as pmoles of glucose formed X min-‘. Specific activity of trehalase in the homogenate was 0.2 units x mg-t protien.
394
REED AND SACKTOR
FIG. 1. A survey electron micrograph of Phormia flight muscle, in cross section, showing arrangement of mitochondria (Mit) about the myofibrils (mf). Other membranous structures evident are elements of the sarcoplasmic reticulum (SR) and the T-system (T). The T-system is comprised, in part, of elements of the plasma membrane which surround the invaginating tracheoles (tr). Glycogen rosettes (Gly) fill the sarcoplasmic spaces. The bar represents 1 cc.
and irregular in shape, up to 4 p in length, and are not aligned wit’h respect to myofibrillar striations. Each mitochondrion has a distinct outer limiting membrane and its inner membrane is arrayed in parallel plates, 30-35 cristae per micron (22). The large number of cristae is indicat’ive of the high respiratory activity of these mitochondria. It also indicates that the ratio of inner to outer membrane is extremely great. The sarcoplasmic
reticulum
of t#his asynchronous
muscle is remarkably reduced, representing only 0.2 % of the cell volume, which together with elements of the T-system and plasma membrane tubules, representing 0.5-l % of the fiber volume, form dyad associations adjoining the fibril surface (23). Glycogen
particles are located mostly in the intermyofibrillar sarcoplasm, with scattered deposits on the myofibrils (11). The low-speed pellet,, which possesses approximately 65% of the total trehalase activity and is obtained by the fractionation scheme given in Table I, consists primarily of myofibrils, mitochondria, and nuclei; the latter being relatively few in number. The small fraction of the total enzyme that precipitates at, 100,000 g for 1 hr cosediments with glycogen rosettes, reticular membranes, and tiny fragments of mitochondria and myofibrils. The remaining activity fails to sediment at, this higher force and is regarded to be nonparticulate. This soluble fraction will be considered later in this report,.
FLIGHT
MUSCLE
In order to resolve the question of the localization of t)he membrane-bound trehalase further, a direct comparison has been made between the enzymic activity in the dense particulate fract’ion, which contains a mixture of mitochondria and other large subcellular organelles, and that in a mitochondrial preparat,ion, which is essentially free of contaminants. As reported in Table II, there is no significant difference between the mixture of particulates sedimenting at 12,000 g and the isolated mitochondria in their relative trehalase act,ivities, . each having about 60% of the t.ot,al actlvlty in the homogenat’e. An electron micrograph of a typical mitochondrial pellet, prepared by t!he Nagarse procedure (13), is illustrated in Fig. 2. Myofibrils and nuclei are absent, and t’here is no evidence of extraneous membranes adhering to these isolated mitochondria (1). These findings suggest that TABLE II COMPARISON 0F THE TREHALASE ACTIVITY 1s THE DENSE PARTICULATE FRilCTION OF HOMOGIN ISOLATED MITOCHONENATES WITH TH.LT DRIBa Preparation
Homogenate “A” 12,000g (5.min) pellet 12,OOOg (5-min) supernatant Homogenate “B” Mitochondria 12,000g (5.min) supernatant
Trehalase Total units
activity %
0.75 0.49 0.15
100 66 20
3.8 2.1 1.2
100 56 32
a Each value is the average of four experiments. In the preparations, designated “A,” the flight muscles of five blowflies were homogenized in a “Virtis 23” homogenizer for 2 set in 1 ml of medbuffer, ium comprising 0.15 M KCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, and 0.5% bovine serum albumin. In the preparations, designated “B,” the muscles from 20 flies were ground by hand with three passes of a Potter-Elvejhem homogenizer in 5 ml of medium identical with that used in preparation “A,” except that 5 mg of Nagarse was added. The proteolytic enzyme was incubat,ed with the muscle suspension for 10 min at 0”. The digested muscle was filtered through cheesecloth and the mitochondria were sedimented at 12,000g for 5 min.
TREHALASE
393
t#he trehalase activity cosedimenting with low-speed centrifugation of homogenates is localized in mitochondria. It is also of interest to note from Fig. 2 that most of the mitochondria, as isolated, appear in the condensed configuration (24), although occasionally mitochondria in the orthodox form are seen. The view that trehalase is localized in mitochondria is supported by additional findings, shown in Table III, demonstrat,ing parallel dist,ributions of trehalase and the mitochondrial flavin-linked a-glycerophosphate dehydrogenase (12, 25) in the part’iculate fractions. When the pellets are repeatedly washed in the serum albumin-cont,aining medium and recenkifuged at. 10,000 g for 13 min, approximately 2% of the trehalase and 1% of the a-glycerophosphat’e dehydrogenase is found in the supernatant fraction after each cycle. This suggests that trehalase is not readily removed from mitochondria by this procedure, but. that a few mitochondrla are disrupted result’ing in smaller fragments that do not sediment at 10,000 g. On the other hand, when serum albumin is not included in the wash medium, up to 20 % of the trehalase may be removed whereas ordy 2 to 3% of t.he or-glycerophosphate dehydrogenase fails to sediment. The requirement for serum albumin in preserving mitochondrial integrity and in binding fatty acids produced from endogenous phospholipids by phospholipases in the mitochondria is well known (26-28). It, has yet t’o be determined, however, whet,her the solubilization of the mitochondrial trehalase by washing in the absence of albumin and this alt,eration in the mitochondria are interdependent or coincidently correlated. In order to determine the localization of trehalase within the mit’ochondrion, different techniques of subfractionat’ing mitochondriu were applied to the present, study. When flight muscle mit’ochondria are disrupted by swelling, shrinking, and sonication and t,he suspension is then fractionat,ed on a sucrosedensity gradient, according to methods described for mammalian mitochondria (13, IS), sarcosomal particles are distributed as illustjrat’ed in Fig. 3. A dense mitochondrial-
396
REED
FIG. 2. An electron pared by the Nagarse
micrograph procedure.
AND
of a typical
SACKTOR
pellet
of flight
muscle mitochondria,
pre-
FLIGHT
MUSCLE
TREHALASE
397
TABLE III IIISTRIBUTIONOFTREHALASEANDMITOCHONDRIIL CY-GL~~ER~~H~SPHATEDEHYDROGENASEIN THE PARTICULATE FRACTIONS n-GP Dehydrogenase
Trehalase Fraction
Total particulate 10,OOOg (15.min) Supernatant 10,OOOg (15-min) Pellet
Total units
%
Total units
6
1.73 0.06
100 4
20.0 0.2
100 1
1.54
89
19.2
96
a Flight muscles were homogenized in a medium containing 0.154 Y KCl, 20 mM potassium phosphate buffer, pH 7.2, 1 mM EGTA, and 0.5% BSA. The homogenate was centrifuged at 250,OOOg for 35 min. The precipitate was used as the total particulate fraction. The residue was resuspended in medium and recentrifuged at 10,OOOgfor 15 min. The unit of a-glycerophosphate (wGP) dehydrogenase activity is expressed as pg atoms oxygen X min+.
like pellet, designated Fraction 3, is found on the bottom of the tube. A lighter-colored brownish-red band, Fraction 2, is localized at the interface of the 1.2 and 1.0 M sucrose solutions. The opalescent mitochondrial medium (Fraction 1) remains on the top of the sucrose gradient.. No discernible band is formed at the medium-l.0 M sucrose interface, the locus where outer membrane fragments of mammalian mitochondria concentrate. Trehalase activity has been determined in the three fract’ions and its distribution compared to those of other enzymes whose int.ramitochondrial localizations are established (Table IV). cr-Glycerophosphate dehydrogenase has been used as the marker for the inner mitochondrial membrane (29, 30). Malic dehydrogenase is indicative of enzymes in the matrix. Adenylate kinase, prev~ously described in flight muscle mitocondria (31), has been reported in rat liver mitochondria to be in the space between the inner and out,er membranes (32). Two other activities, monoamine oxidase and rotenoneinsensitive KADH-cytochrome c reductase, useful markers for outer membranes of mammalian mitochondria, have activities too low t,o be measured with reliability in flight
#3- LJ
'! i
FIG. 3. JXagrammatic representation of the fractionation on a sucrose density gradient of disrupted flight muscle mitochondria. Further details are given in t.he text.
muscle mitochondria. As demonstrated in Table IV, trehalase and cu-glycerophosphate dehydrogenase are dist,ributed similarly in the t’hree fractions. The two enzymes are localized predominantly in Fraction 3. About 20% of their total activities are found in Fraction 2, whereas Fract.ion 1 has less than 10% of these enzymes. This indicates some fragmentation of the inner membrane to less dense particles t,hat band as Fraction 2 or remain in t’he mitochondrial medium. In contrast, Fraction 1 cont’ains 70% or more of the malic dehydrogenase and adenylate kinase activities, suggesting the rupture of many mitochondria and the release of enzymes from the matrix and space between the inner and outer membranes. Fraction 2 is essentially free of malic dehydrogenase. Fraction 3, however, apparently contains the inner membrane-matrix component and/ or intact mitochondria, in addition to the inner membrane. The finding that the’ recovery of malic dehydrogenase sums to more than lOO%, perhaps due t.o the failure of Lubrol to activate fully the dehydrogenase in all measurement,s (19), prevents quantitation of the mat’rix contamination in Fraction 3. The locus of the outer membrane in this sucrose gradient, can not be stat~ed with certaint,y. Subjecting mitochondria to freezing and thalving, followed by centrifugation, unequivocally separates trehalase from malic dehydrogenase. As shown in Table V, trehalase and a-glycerophosphate remain bound to the membranes whereas malic dehydrogenase is essentially completely released and
39s
REED
AND
SACKTOR
TABLE INTRAMITOCH~NDRI:~L
LOC~LIZ.\TI~N
OF
MITOCHONDRI~~
ON
.I
SUCROSE
Trehalase
AFTER DENSITY
(I GP dehydrogenase
Fraction
%>
Units
Disrupted mitochondria Fraction 1 Fraction 2 Fraction 3
IV
‘I’REHAL.ISE
7.81 0.41 1.47 5.04
100 5 19 65
Units
207 18 38 165
FRACTION.\TION
OF
DISRUPTED
GRADIENT’ Malic
dehydrogenase
Adenylate
kinase
%I
Units
%>
Units
%I
100 9 18 80
1970 1370 4 990
100 70
14.9 11.5 1.3 2.6
100 77 9 17
QMitochondria were isolated by the procedure described earlier (12) in a medium containing 0.07 M acid (HEPES) buffer, sucrose, 0.21 M mannitol, 2 mM N-2-hydroxyethylpiperazine-~~‘-2-ethane-sulfonic pH 7.4, and 0.05% BSA. The isolated mitochondria were disrupted by swelling, shrinking, and sonication according to Sottocasa et al. (15), as modified by Whereat et al. (16). The disrupted mitochondria were centrifuged at 140,OOOgfor 60 min, in a sucrose density gradient, shown in Fig. 3. Units of malic dehydrogenase are given as rmoles of oxaloacetate reduced X min- I. A unit of adenylate kinase represents a rmole of ADP converted X min-I. TABLE
V
INTRAMITOCRONDRI.~L TREIIALASE MITOCHONDRI.\
LOCALIZATION AFTER
OF
DISRUPTION RY
OF
FREEZINC
AND
THAWING~
WGP Trehalase Fraction
dehydrogenase
_-
Malic dehydrogenase ___.
Units I Y. Units I Y. Units I s1
_-
Intact mitochondria 3.9010061.2100 306100 Freeze-thaw, 144,OOOg 0.12 3 0.3
solubilized. This rules out the matrix as the locus for the mitochondrial trehalase. The data in the preceding experiments, although excluding the matrix and the space between the inner and outer membranes as the loci of the mitochondrial trehalase, do not clearly distinguish between the inner and outer membrane with respect to the site of the enzyme. This question has been examined by disrupting mitochondria with Digitonin, according t,o the procedure of Schnaitman and Greenawalt (14) for mammalian mitochondria, and by using as a marker for the outer membrane the recently reported prop-
erty of outer membranes to bind brain hexokinase (20). Hexokinase in rat brain mitochondria has been reported to be localized on the outer membrane (20, 33) and, when the enzyme is solubilized, it will rebind preferentially to the outer membrane of brain, liver, and other kinds of mitochondria (20, and J. E. Wilson, personal communication). As shown in Table VI, Digitonintreated flight muscle sarcosomes are disrupted, resulting in two particulate fractions; one, sedimenting at 12,000 y for 15 min, the other centrifuging down at 140,000 g for 60 min. cr-Glycerophosphate dehydrogenase, t’he inner membrane marker, is found mostly in the dense particulate fraction. When compared to int#act mitochondria, the specific activity of this enzyme in the 12,000 g pellet is increased, whereas its specific activity in the 140,000 g pellet is substantially lowered. The data in Table VI demonstrate that rat brain mitochondrial hexokinase does bind to insect flight muscle sarcosomes. Moreover, relative to intact mitochondria, the specific activity of the hexokinase binding capacity in t’he 140,000 g pellet is enhanced 2-fold whereas that in the dense particulate fraction is decreased almost in half. These findings suggest that the highspeed pellet is enriched with outer membranes and the low-speed pellet has concentrated the inner membranes. Quantitation of the relative enrichments is precluded, however, due to the poor recovery of the
FLIGHT
MUSCLE TABLE
INTRAMITOCHONDRL~L
LOC.ILIZATION
Fraction Intact mitochondria Digit,onin-t,reated, 12,OOOg (15 min) supernatant 140,OOOg (60-min) pellet of 12,000g supernatant 140,OOOg (60.min) supernatant of 12,OOOgsupernatant Digitonin-t,reated, 12,OOOg (15min) pellet
399
TBEHALASE VI
OF TREH~LME AFTER DIGITONIN” Trehalase
OF MITOCHON~RI.\
L)ISRUPTION
WITH
sp act
Units
::
sp act
Hexokinase binding Units % sp act
100 17
0.17 0.07
340 22
100 6
1.5 0.2
61.5 -
100 -
0.28 -
1.5
4
0.11
9
3
0.7
7.6
12
0.52
5.6
15
0.16
12
4
0.3
-
15.3
40
0.17
234
70
2.5
16.8
Units
%
38.1 6.5
c-GP dehydrogenase
-
27
0.16
(1Mitochondria were isolat,ed as described in Table IV. The final suspension of the mitochondria was made very concentrated, 100 mg of protein/ml. The mitochondrial suspension was treated with 1.2y0 Digitonin by the method of Schnaitman and Greenawalt (14). The binding of solubilized rat brain mitochondrial hexokinase to flight muscle mitochondrial part&dates was carried out according to Kropp and Wilson (20), except that the hexokinase bound by the particulates was estimated by assay of the reisolated particulates rather than by measurement of the disappearance of hexokinase activity from the supernatant fraction of the incubation reaction. A unit of hexokinase represents apmole of glucose-6phosphate formed X mi0. Specific activities (sp act) are given as units X mg-’ protein.
hexokinase-binding capacity in the submitochondrial fract’ions, perhaps because of the inability to measure binding in the highspeed supernatant fractions and to differences in the assay procedure in the present study from t,hat described by Kropp and Wilson (20). Despite these uncertainties, it is evident from Table VI that the distribution of t’rehalase in the various submitochondrial fractions closely resembles that of cY-glycerophosphate dehydrogenase rather than that of hexokinase binding. There is 10 times more trehalase in the 12,000 g pellet than in the 140,000 g particulate. In addition, the specific activity of trehalase in the high-speed pellet is decreased. This agrees with the decrease in specific activity of oc-glycerophosphate dehydrogenase in t’his fraction and is in contradistinct’ion with the increase in the specific activity of hexokinase binding. Thus, these findings favor the tentative conclusion that in blowfly flight muscle the mitochondrial trehalase is localized in t,he inner membrane of the sarcosome. It is noted in Table VI that after Digitonin treatment a substantial amount of trehalase, 6.5 units, is found in t,he 12,000 g supernatant fraction, but most of this, 5.6 units, has been solubilized or fails to sediment at 140,000 g
for 60 min. Other agents that disrupt mit’ochondria, such as Triton X100, Lubrol, deoxycholate, and sodium dodecyl sulfate, also effect considerable solubilization of the mitochondrial trehalase. As reported in Table I, about 25 % of the total trehalase of the muscle homogenate remains in the supernatant after centrifugation at 100,000 g for 60 min. Alternative explanations have been offered as to the derivation of this soluble activity and the question continues to be the subject of some controversy (1, 3-6, 10). To illustrate : (a) the soluble enzyme may represent a distinct muscle trehalase, differing from the membrane-bound enzyme; (b) all the muscle trehalase is particulate, but that 25 % of the activity is washed off during homogenization and centrifugation procedures; and (c) the soluble enzyme stems from the hemolymph which adheres to the muscle or its entrapped in the extracellular spaces. In the course of the present study, observations have been made that seem relevant t,o this issue. Values for the K, of the soluble and particulate trehalase range from 0.6 t)o 1.3 rnxr and 0.9 to 1.9 mu, respectively. These values are not, significantly different. Therefore, this result is inconsistent with t’he view of the
400
REED AND SACKTOR
presence in the muscle preparation of a true membrane-bound muscle trehalase with a high K, and a soluble blood or intestinal enzyme with a low K,. Rising of the excised muscle in medium does not noticeably reduce the amount of soluble enzyme. In addition, the 100,000 g supernatant, although containing about 25 % of the total trehalase activity, has essentially none of the mitochondrial a-glycerophosphate dehydrogenase activity. Moreover, trehalase is found abundantly in the soluble fraction even though the muscle is homogenized in serum albumin-containing media, which, as noted earlier, largely prevents the solubilization of the mitochondrial trehalase. Repeated centrifuging at low speeds and resuspending the pellet in fresh media containing albumin does remove about 2% of the trehalase each time, but it has been found that 60% of this trehalase will sediment at 100,000 g. About 1% of the total mitochondrial a-glycerophosphate dehydrogenase behaves in the same fashion. These findings suggest that, although preparative procedures may cause limited rupture of the mitochondria, such disruption hardly accounts for the presence in t#he original 100,000 g supernatant (Table I) of onefourt,h of the total trehalase activity. While acknowledging that more rigorous evidence has yet to be developed before the problem is settled, the present data tend to support the hypothesis for two distinct muscle trehalases in t,he blowfly. DISCUSSION
Previous studies have demonstrated that flight muscle trehalase is activated on initiation of contraction (2). As reported in Table I, the specific activity of the muscle trehalase is about 0.2 units X mg’ protein. It has been calculated that this activity is sufficient to account for the rate of trehalose utilization at the beginning of flight (1, 2). This observation, plus the finding that in the blowfly, in contrast to other kinds of insects (4, 5, 9, 34), trehalase activity is not enhanced by various physical and chemical treatments, i.e., freezing and thawing (Table V) and detergents (Table VI), that tend to disrupt membrane structure, suggest that the observed activity probably represents
the decontrolled or active rate of the enzyme. Thus, the demonstration that blowfly flight muscle trehalase is localized in mitochondria focuses the problem of the control of trehalase activity on the rapid transitions taking place in the sarcosomes in situ, when the insect goes from rest to flight. The present study also describes the ultrastructural localization of trehalase with greater precision than known heretofore. The distribution of trehalase in the major mitochondrial compartments has now been examined. The data in Tables IV and V clearly distinguish bet,ween the distributions of malic dehydrogenase and adenylate kinase from that of trehalase and provide good vidence to rule out the matrix and the space bet’ween the outer and inner membranes as the site of trehalase. Clean separation of the inner and out’er membranes has proved to be difficult because of the paucity of outer relative to inner membrane (Figs. 2 and 3) and to the extremely low activities in flight muscle mitochondria of monoamine oxidase and rotenone-insensitive NADHcytochrome c reductase, the established outer membrane markers in mammalian mitochondria. Nevertheless, by comparing the distribution of trehalase with those of a-glycerophosphate dehydrogenase and hexokinase binding in fractions derived from Digitonin-disrupted sarcosomes (Table VI), the hypothesis that trehalase is localized on the inner membrane gains support. It is implicit in this tentative conclusion that trehalose is readily accessible to the inner membrane, and this implies that the disaccharide is permeable through the mitochondrial outer membrane. A precedent is to be found with the disaccharide, sucrose, which freely enters the space between the inner and outer membranes (35). However, sucrose does not significantly enter the matrix. If trehalose behaves similarly in this respect, then it is suggested that trehalase is situated on the outside of the mitochondrial inner membrane. REFERENCES 1. SACKTOR, B., Advan. Insect Physiol. (1970). 2. SACKTOR, B. AND WORMSER-SHAVIT, Biol. Chem. 241, 624 (1966).
7, 267
E., J.
FLIGHT
MUSCLE
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