Subcellular fractionation of fleshfly flight muscle in attempts to isolate synaptosomes and to establish the location of glutamate enzymes

Insect Biochem., 1974, Vol. 4, PP. 243 to 265. Pergamon Press. Printed in Great Britain

SUBCELLULAR MUSCLE TO

FRACTIONATION

IN ATTEMPTS

ESTABLISH

THE

TO

OF FLESHFLY

ISOLATE

LOCATION

FLIGHT

SYNAPTOSOMES

OF GLUTAMATE

243

AND

ENZYMES

J O H N F. D O N N E L L A N , D O N A L D W. J E N N E R , AND A L I S O N R A M S E Y Woodstock Laboratory, Shell Research Ltd., Sittingbourne, Kent, England

(Received 2 June x973; revised IO September 1973) ABSTRACT i. A description is given of the methods used in attempts to isolate neuromuscular junctions (synaptosomes) from the flight muscle of the fleshfly Sarcophaga barbata. T h e failure to obtain a subcellular fraction enriched in synaptosomes is attributed to the fragmentation of the neuromuscular junctions during the initial homogenization of the muscle. 2. T h e subcellular location in the flight muscle cell of a number of enzymes involved in glutamate metabolism has been established. Controlled disruption of flight muscle mitochondria has been used to determine the mitochondrial location of three glutamate-metabolizing enzymes.

THE iontophoretic application of L-glutamic acid to insect nerve-muscle preparations gives responses consistent with the hypothesis that this amino-acid is the excitatory neuromuscular transmitter (Usherwood and Machili, 1966 and r 968 ; Baranek and Miller, i968 ) . Furthermore motor stimulation of such preparations has been reported to cause the release of the proposed neurotransmitter (Kerkut et al., 1965 ; Usherwood et al., 1968). It was thought worth while to investigate the biochemical processes involved in excitatory neuromuscular transmission in insects by attempting to isolate the nerve-muscle junctions from fleshfly fltght muscle. Such a subcellular fraction would be expected to be enriched in the enzymes responsible for both the synthesis and degradation of the putative transmitter in the synaptic terminals in vivo. The technology employed was largely based on the methods described for the isolation of nerve terminals (synaptosomes) from mammalian cerebral cortex and other brain sources (De Robertis et al., 1961 ; Gray and Whittaker, 1962; Whittaker and Greengard, i97i ). These workers have shown that, when nervous tissue is homogenized under the appropriate conditions, the nerve terminals 'pinch off' and reseal to retain their original contents so that the nerve endings can be separated by various density gradient centrifugation procedures. It was hoped that the insect neuromuscular junctions would behave in a similar manner so that the resultant synaptosomes could be differentiated from the remainder of the insect muscle constituents. Electron microscopy has been employed as the primary method for the identification of muscle synaptosomes in subcellular fractions obtained by density gradient centrifugation of flight muscle homogenates. These cell fractions have also been examined for their content of a number of enzymes likely to be involved in glutamate metabolism in both the synaptic terminals and in the muscle cell in general. In particular it was thought that such an approach would indicate whether biochemical

244

DONNELLAN AND OTHERS

Insect Biochem,

parameters could serve as a useful adjunct to electron microscopy in aiding the isolation of a discrete synaptosomal fraction f r o m insect muscle. T h e enzymes studied were L-glutamate: N A D ( P ) oxidoreductase (E.C. 1.4.1.3. ) ( G D H ) , L-alanine: 2-oxoglutarate aminotransferase (E.C. 2.6.1.2.) ( G P T ) , L-aspartate: 2-oxoglutarate aminotransferase (E.C. 2.6.I.I.) ( G O T ) , L-glutamate: i-carboxylase (E.C. 4 . I . r . I 5 ) (GAD), and L-glutamate: ammonia ligase (ADP) (E.C. 6.3.1.2. ) (GS), and it was necessary to examine the properties and o p t i m u m assay conditions of these enzymes present in fleshfly flight muscle. T h e intramitochondrial location of G D H , G P T , and G O T has also been established by the selective disruption of flight muscle mitochondria by either digitonin (Schnaitman and Greenawalt, 1968 ) or ultrasonic disintegration treatments (Donnellan and Beechey, 1969). Similar studies on G A D are reported elsewhere (Langcake and Clements, 1974). MATERIALS AND METHODS

Subcellular fractions were prepared by three techniques using muscle from adult fleshflies (Sarcophaga barbata Thomson) which were fed on sugar solution and used i to 7 days after emergence. All manipulations were carried out at 4 ° C. a.

DENSITY GRADIENT FRACTIONS

Flies (15o-5oo) were immobilizedby chilling on ice before the abdomens were cut off. The heads, plus the residue of the alimentarycanals, were then pulled away from the thoraces. To obtain muscle essentially free from ganglionic tissues a transverse cut was then made in the thoraces such that only the dorsal third was retained. This tissue consistsof six pairs of giant cells of the dorsal longtitudinalmuscles, some direct fight muscles, tracheoles, the dorsal blood vessel, and some lipid droplets. It was scraped or squeezed from the exoskeleton and placed in 0"45 M sucrose

•

Muscle (approximately 50 mg. wet weight per ml.) was dispersed by homogenization (20 plunges) using a glass-Teflon homogenizer before being filtered through nylon net (159/zm. mesh) to remove myofibrils and tracheolar fragments. The filtrate obtained was then fractionated by centrifuging (I 5 ml. per tube) for 2 to 3 hr at 67,000 g into a discontinuous sucrose density gradient (sp. gr. I'O8-1"2o) constructed in polycarbonate tubes suitable for the 3 x 65 ml. head of an MSE Superspeed 65 centrifuge. Fractions were removed by suction using Pasteur pipettes and the sucrose concentrations adjusted to 0"45 M by the careful addition of water. Particulate fractions were then resedimented by centrifugation for I hr at 63,000 g and the pellets were suspended in 0"45 M sucrose. b. PREPARATIONOFTHORACICMUSCLEMITOCHONDRIA Mitochondria were prepared from the thoracic muscle of fleshflies by the Nagarse proteinase method described by Donnellan and Beechey (1969). The isolated mitochondria were finally suspended in an ice-cold solution containing 0"25 M sucrose, 5 mM Tris chloride, and I mM EGTA adjusted to pH 7"4 with 4 M KOH. The final protein concentrationwas approximately IO mg. per ml. C. PREPARATION OF SARCOPLASM

The soluble component of the thoracic muscle tissue was prepared by differential centrifugation of muscle homogenates whereby muscle from 50 fleshflies was suspended in IO ml. of the medium described above and homogenized in a Tefon-glass homogenizer (20 passes). After filtration through nylon net (159/zm. aperture) to remove myofibrils and tracheolar fragments, the homogenate was centrifuged for IO rain at 6000 g. The supernatant was carefully removed and centrifuged for 30 rain at 80,000 g. Studies on the enzymes present in the sarcoplasm were performed with the supernatant from this step.

1974, 4

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

24.5

DIOITONIN FRACTIONATIONOF THORACICMUSCLE MITOCHONDRIA Preliminary experiments established that the optimum concentration of digitonin for the disruption of the outer membrane of thoracic mitochondria was 2"5 rag. of digitonin per IO mg. mitochondrial protein under conditions essentially as described for rat liver mitochondria by Schnaitman and Greenawalt (1968). Aliquots of ice-cold digitonin solution (2 per cent w/v in the mitochondrial isolation medium) were added with continuous stirring to lO ml. of a mitochondrial suspension (90 rag. of protein) to give the required digitonin/protein ratio. After stirring for I5 rain the suspension was diluted to 30 ml. with isolation medium, homogenized gently by hand, and centrifuged for S min at 9ooo g. The supernatant was carefully drawn off before suspending the pellet in 1S ml. of isolation medium prior to centrifugation again for S rain at 9ooo g. This washed' low-speed' pellet was suspended in isolation medium and represents the inner membrane plus matrix components of the thoracic mitochondria. Centrifugation of the combined supernatants from the low speed spins for i hr at 8o,ooo g yielded a 'high-speed' supernatant derived from the intermembrane contents of the mitochondria and a 'high-speed' pellet composed primarily of outer membrane fragments. Electron micrographs of the pellet fractions substantiated their expected composition, though a few small mitochondria were observed in the outer membrane fraction of the 'high-speed' pellet. ULTRASONIC DISINTEGRATIONOF THORACICMUSCLE MITOCHONDRIA Mitochondrial suspensions (5 rag. of protein per ml.) were subjected to the output of a IooW M S E ultrasonicator for 2 min before centrifugation for 5 rain at 900o g to sediment the ' damaged mitochondria', which were suspended in the isolation medium. T h e supernatant was further centrifuged for I hr at lOO,OOOg to give a pellet of 'submitochondrial particles' which was suspended in the isolation medium. The supernatant, designated as the 'sonic soluble fraction', conrains the contents of the matrix and the space enclosed by the two mitochondrial membranes, together with the remnants of the outer membrane (Donnellan and Beechey, 1969). PREPARATIONOF SAMPLESFOR ELECTRON MICROSCOPY Subcellular fractions were fixed as suspensions by the dropwise addition of glutaraldehyde in 9o m M cacodylate buffer, pH 7"2, and o'15 M sucrose until a concentration of 2"5 per cent (v/v) glutaraldehyde was attained. After standing for i hr the fixed material was sedimented by centrifugation for 3° m i n at 67,0o0 g. T h e pellets were then washed overnight in the cacodylate/sucrose buffer and then prepared for electron microscopic examination by the procedures described by Clements and Potter (I967). ENZYME AssAYS The spectrophotometric assays were performed at 25 ° C with a Unicam SP 800 using silica cuvettes of $ mm. light path and with a final reaction volume of 1"5 ml. Substrates (of A.R. grade) were adjusted to the pH of the assay buffer and low levels of Triton X-Ioo (o'1 per cent w/v) were included in most assay systems to ensure that occluded enzymic activity in particulate fractions was determined. This level of Triton X-Ioo did not inhibit enzymic activity and also eliminated any problems caused by mitochonclrial swelling. T o minimize the possible inhibitory effects of sucrose on enzyme activity (Hinton et al., I969) the sucrose content in any set of reaction mixtures was always equalized. The activities of the commercial enzymes (Sigma Chemical Co., London) used in the coupled aminotransferase assays were not affected by the various experimental conditions used to optimize the assay conditions. All recorded enzyme activities (expressed, unless stated, in the units m o l e s m i n - l m g . - 1 protein) were the means of duplicate assays and were corrected for any endogenous activity. GDH Activity was assayed by following the reduetive amination of 2-oxoglutarate. Extracts were added to reaction mixtures containing 5o m M Tris adjusted to pH 7"9 with 2 M HCI, IOO m M ammonium acetate, 1 m M ADP, o'22 m M N A D H , and o.i per cent (w/v) Triton X-too. N A D H oxidation was monitored at 340 nm. after the addition of 5 m M 2-oxoglutarate.

246

DONNELLAN AND OTHERS

Insect Biochem.

GPT Activity was assayed by coupling pyruvate production to the oxidation of N A D H by lactic dehydrogenase. Extracts were added to reaction mixtures containing 5o m M KH~PO4 adjusted to pH 6"9 with 2 M NaOH, 40 m M L-alanine, lactic dehydrogenase (5 i.u.), o'22 m M N A D H , and o.i per cent (w/v) Triton X-I oo. N A D H oxidation was monitored at 34o nm. after the addition of 5 m M 2-oxoglutarate. GOT Activity was assayed by coupling oxaloacetate production to the oxidation of N A D H by malate dehydrogenase. Extracts were added to reaction mixtures containing 5o m M Tris adjusted to pH 7"9 with 2 M HCI, 4o m M L-aspartate, malate dehydrogenase (5 i.u.), o'22 m M NADH, and o'x per cent (w/v) Triton X-Ioo. N A D H oxidation was monitored at 34o nm. after the addition of 5 m M 2-oxoglutarate. G P D H (L-3-glycerophosphate : NAD oxidoreductase, E.C. 1.1.1.8) Activity was assayed in the direction of L-3-glycerophosphate formation. Extracts were added to reaction mixtures containing 5o m M KH~PO4 adjusted to pH 6"9 with 2 M NaOH, o'22 m M NADH, and o'I per cent.(w/v) Triton X-ioo. N A D H oxidation was monitored at 34o nm. after the addition of o'36 m M dihydroxyacetone phosphate. GPox (g-3-glycerophosphate : flavoprotein oxidoreductase, E.C.I.I.99.5) Activity was assayed by coupling the oxidation of L-3-glycerophosphate to the reduction of ferricyanide to ferrocyanide. Extracts were added to reaction mixtures containing I3o m M KC1, IO m M KH~PO4, and I m M E G T A , adjusted to pH 7"1 with 2 M NaOH, I m M K C N and I m M potassium ferricyanide. The decrease in extinction at 425 nm. due to ferricyanide reduction was then followed after the addition of 66"7 m M VL-3-glycerophosphate. M D H (L-malate : NAD oxidoreductase, E.C. 1.1.1.37) Activity was assayed in the direction of malate formation. Extracts were added to reaction mixtures containing 5o m M glycine and 5 m M E D T A adjusted to pH 8"8 with 4 M NaOH, o'25 m M NADH, and o'I per cent (w/v) Triton X-Ioo. N A D H oxidation was monitored at 34o nm. after the addition of o'3o m M oxaloacetate. A K (ATP : AMP phosphotransferase, E.C. 2.7.4-3) Activity was assayed essentially as described by Schnaitman and Greenawalt (I968) by following the conversion of ADP to A T P and AMP. The production of A T P was monitored by using hexokinase and glucose-6-phosphate dehydrogenase to reduce N A D P under the assay conditions described by Langcake and Clements (i974). GS Activity was measured by substituting hydroxylamine for NH4 + in the reaction mixture to enable the production of 7-glutamyl hydroxamate to be assayed (Berl, I966). Extracts were added to reaction mixtures containing 25 m M Tris adjusted to pH 7"2 with 2 M HCI, IO m M MgCI~, 50 m M hydroxylamine, 5o m M potassium glutamate, io m M phosphoenolpyruvate, and pyruvate kinase (2 i.u.) in a final volume of 0'2 ml. After a I5-min incubation at 25 ° C on a shaking water bath the reaction was terminated by the addition of a solution (o. 4 ml.) containing 8 per cent (w/v) trichloroacetic acid and IO per cent (w/v) ferric chloride in o. 5 M HCI. The precipitated protein was sedimented by centrifugation after the further addition of i ml. of water and the extinction of the supernatant at 5oo nm. was measured against a substrate blank with a Unicam SP 6o0 spectrophotometer. Comparison of the extinction values with a standard curve constructed with authentic 7-glutamyl hydroxamate enabled the amount of product to be estimated. GAD Activity was assayed by measuring the production of t4CO2 from [I-14C]glutamate as described by Langcake and Clements (I974).

1974, 4

247

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

PROTEIN T h e protein content of the sucrose gradient fractions was assayed by a modified Folin-Cio-

calteau procedure (Miller, 1959). Mitochondrial and sarcoplasmic protein concentrations were determined by the Biuret method described by Gornall et al. (z949). Bovine plasma albumin was used as the standard for both methods. RESULTS ISOLATION OF NEUROMUSCULAR]UNCTIONS (SYNAPTOSOMES) T h e appearance in situ of a neuromuscular junction in the flight muscle of S. barbata closely resembles that of other chemically-mediated synapses in having a characteristic content of synaptic vesicles (Fig. i). Our attempts to isolate the nerve terminals were based on the assumption that these vesicles would be retained when the synaptosomes were formed during homogenization of the flight muscle. It was anticipated that the recognition of synaptosomal profiles from electron micrographs of sucrose gradient fractions would be difficult as such fractions would be contaminated by a variety of membrane vesicles that had originated from the sarcolemma and sarcoplasmic reticulum. Consequently a synaptosome was defined by the relatively strict criterion that it would be a profile containing at least four vesicles of approximately 0.o 4/~m. diameter within a limiting m e m b r a n e Confirmatory features would be densely stained postsynaptic Fraction

Sucrose Molarity

' t l tilt'~1,

,

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Faint whitish haze

c. (sp. gr. 1.17)

Strong whitish haze

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d. (sp. gr. 1.174)

Light brown

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Dark brown

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FIG. 2.--Macroscopic appearance of gradient fractions taken for electron microscopic analysis. Muscle from 285 flesbaqies (z days old) was homogenized in 0'45 M sucrose and filtered through nylon net. The filtrate (2 x z5 ml.; 23° nag. protein) was applied to sucrose gradients constructed of xo-ml, layers of 0"8 M, 1"o M, 1"3 M, and z"4 M sucrose. The appearance of the gradient after centrifugation for 2 hr at 67,000 g is illustrated above. The indicated fractions were then processed for electron microscopy as described in the Methods section.

248

DONNELLANAND OTHERS

Insect Biochem.

membrane attachments and also some small mitochondria enclosed along with the synaptic vesicles. It was anticipated that if muscle synaptosomes were similar in their equilibrium density to those isolated from mammalian brain, then the appropriate gradient fractions should contain up to a one-hundredfold enrichment of synaptosomes as less than 2 per cent of the applied protein sedimented at this density. This, of course, assumes that the synaptic terminals produce a homogeneous population of synaptosomes. Under these conditions it was thought feasible to detect the presence of muscle synaptosomes with reasonable certainty on electron micrographs of such gradient fractions. The macroscopic appearance of a typical sucrose gradient after the fractionatiou of flight muscle (285 flies, 2 days old) is shown in Fig. 2. The gradient fractions indicated were then processed for electron microscopic analysis. Membrane vesicles were present in fractions a, b, and c in a wide variety of forms and sizes (up to 3/zm. diameter). The most common form had a single limiting membrane and its contents, if any, were unstained. Larger vesicles sometimes contained heavily stained material or small mitochondria as well as smaller vesicles. A common feature of these micrographs of the membrane fractions was the ubiquitous presence of granular material, presumed to be glycogen, which was often enclosed, making recognition of synaptosomes even more difficult. Incubation of muscle homogenates with m-amylase prior to the gradient fractionation produced no obvious improvement in the clarity of electron micrographs of these fractions. Mitochondria (mean cross-section up to length 2.o × breadth 1. 4/~m.) were the main components of the lower gradient fractions (d, e, and f). No material of synaptic origin could be clearly observed in these mitochondrial fractions. The basic isolation procedure was modified by varying the age of the flies, the conditions of homogenization and also the composition of the sucrose gradients. Many profiles were observed in the appropriate membrane fractions that could have been synaptosomes, but were stained too densely or lightly to enable positive identification. It became clear that distinguishing synaptosomes, even if they were formed in good yield, from artefacts was more difficult than would be predicted from reported work with brain tissue. Close examination of from lO4 to lO5 vesicular profiles revealed only 3 or 4 that conformed to the criteria laid down for the recognition of synaptosomes. These profiles occurred in the i-o M to 1.2 M sucrose bands where mammalian brain synaptosomes would be expected to sediment. Fig. 3 shows two micrographs of a gradient fraction (sp.gr.I.17) containing profiles which satisfied our criteria for the recognition of synaptosomes. The possibility that the synaptosomes were predominantely associated with the myofibril fraction was also investigated by examination of the material of flight muscle homogenates that did not filter through the nylon mesh step of the fractionation procedure. Electron micrographs of this material revealed myofibrils, tracheolar fragments, and some mitochondria together with a few nuclei and ribosomes but no obvious synaptosomal profiles were observed in this myofibril fraction. Another source of insect muscle was also used in case the neuromuscular junctions of the indirect flight muscle of the fleshffies were either less abundant or more fragile than could be predicted. The entire hind leg muscle of up to 2o adult locusts (2 weeks old, Schistocerca gregaria) was fractionated on three occasions by the procedures employed for flight muscle. The membrane fractions derived from this muscle appeared to contain relatively low levels of glycogen and this was reflected in the greater clarity of the micrographs compared to those obtained from the fly muscle. However, no readily recognisable synaptosomes were observed in the locust muscle fractions.

i.

FIG. I.-Indirect

I

flight muscle

O.lpm -

6%

of S. barbata

showing

a neuromuscular

junction

FPIG. 3.-Electron micrographs of synaptosomes isolated from S. bavbata flight muscle. Thl e micrographs show the appearance of two synaptosomes observed in a gradient fraction of S. barbata (4 days old) flight muscle had been @P. gr. 1.17). In this run a homogenate frac :tionated on sucrose density gradients by the procedures described in Fig. z.

1974, 4

249

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

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Insect Biochem.

DONNELLAN AND OTHERS

SUBCELLULARLOCALIZATIONOF GLUTAMATEMETABOLIZINGENZYMES In conjunction with our attempts to isolate a synaptosomal preparation we have also examined density gradient fractions of flight muscle for the presence of a number of glutamate-metabolizing enzymes in order to establish their subcellular location. It was also thought that such an exercise might lead to the detection of membrane-bound enzymes that had originated from neuromuscular junctions which appear predominantly to disrupt when the muscle was homogenized prior to fractionation. Ten such density gradient fractionations of fleshfly flight muscle were carried out with slight variations in gradient composition, in the fractions taken from the gradients and also in the age of the flies from which the muscle was obtained. The gradient fractions were screened for the presence of the indicated glutamate enzymes, with GPDH and GPox respectively serving as marker enzymes for the sarcoplasm and mitoehondria. A consistent pattern of enzyme localization on the gradients emerged, which is illustrated by the density gradient fractionation of the homogenate of flight muscle obtained from 475 fleshflies. Table 1 records the enzyme activities and protein contents of the filtered homogenate and of the nine fractions (FI-F9) taken from the sucrose gradient. It also shows the percentage recoveries of enzyme activity and protein from the gradient. Table 2 shows the relative specific activities (RSA) of the individual enzymes in each fraction. Values greater than I indicate a concentration of enzyme in a particular fraction compared with the whole gradient and hence show where the enzyme is predominantly located on the gradient. ENZYME DISTRIBUTION

GDPH This enzyme served as the marker for the soluble components of the muscle cell (Zebe and McShan, 1957; Bticher and Klingenberg, 1958). Activity was almost entirely (99"5 per cent, RSA 4.1o) in FI at the top of the gradient where sarcoplasm would be expected to be located. GPox The enzyme has been shown to be firmly bound to the inner membrane of flight muscle T a b l e 2.--RELATIVE SPECIFIC ACTIVITIES OF GRADIENT FRACTIONS RELATIVE SPECIFIC ACTIVITY FRACTION

I 2 3 4 5 6 7 8 9

GPDH

GPox

GDH

GOT

GPT

GAD

GS

4.io o'18 o"15 0"27 0"03 o'o5 o'o7 o o

o o o 0"26 o o'19 o'57 1"38 1"39

0"08 0"64 0"64 0"62 o'46 o'55 I'II I'75 I'25

2"48 o'81 0"50 o o o o'87 o'42 o'56

o o o o o o o I'57 I'28

o'I 3 0"50 0"56 0"49 0"50 o'34 o'63 I'38 I'35

1"45 0"55 o'z8 1.18 0"34 o'23 o'9z o'66 o'9o

D a t a d e r i v e d f r o m T a b l e 1. Relative specific activity ( R S A ) is defined for each f r a c t i o n as R S A = ( P e r c e n t a g e total e n z y m e activity r e c o v e r e d f r o m g r a d i e n t ) / ( P e r c e n t a g e total p r o t e i n r e c o v e r e d f r o m gradient).

1974, 4

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

251

mitochondria (Chance and Sacktor, 1958; Donnellan et al., 197o). Its location in F8 (RSA 1.38) and F 9 (RSA 1.39) at the bottom of the gradient is consistent with the appearance of electron micrographs of similar gradient fractions which are almost entirely composed of mitochondria (Fig. 2d, e, f). The low levels of enzyme activities ( < 0. 5 per cent, RSA < i) present in the membrane fractions (F 4 and F6) are attributed to the small-scale disruption of mitochondria during the preparation of the fractions and also to the presence in these fractions of a few small mitochondria (mean cross-section, length 0"3/~m. x breadth 0.2/~m.).

GDH RSA values greater than I are observed in F7, F8, and F9, in which the mitochondrial marker, GPox, is located. Such a mitochondrial localization for GDH has been reported for housefly (Van den Bergh and Slater, 1962) and L. migratoria (Delbrfick et aL, 1959) flight muscle mitochondria. The distribution of GDH in the other gradient fractions again is a reflection of the mitochondria observed in membrane vesicle fractions and also of the disruption of some mitochondria which would release the enzyme from the mitochondrial matrix. GOT Enzyme activity occurs mainly in the soluble fraction (FI, RSA 2.48 ) but the levels of the enzyme in F8 and F 9 also suggests a mitochondrial location for the enzyme, although the RSA values were less than i in this particular gradient run. Further studies described later in this report suggest that distinct GOT isoenzymes are present in the flight muscle mitochondria and sarcoplasm. Such compartmentalization of GOT activities has been reported in L. migratoria flight muscle mitochondria (Pette and Luh, 1962) and is well documented in mammalian tissues (Nisselbaum and Bodansky, I964). GPT A purely mitochondrial location for this enzyme is indicated by its appearance only in F8 (RSA 1.57) and F 9 (RSA 1.28). The low recovery of GPT from this gradient was a consistent feature of these localization studies and suggests that GPT, unlike the other enzymes investigated, may be labile under the conditions employed. Pette and Luh (i962) reported that 85 per cent of the GPT activity in L. migratoria flight muscle was present in the nfitochondria. GS Enzyme activity was distributed throughout the gradient although it appears to be a soluble enzyme (FI, RSA 1.45) with significant activity also in a membrane vesicle fraction, F4 (RSA i.x8). This suggests the enzyme may be loosely bound to cell membranes, as solubilization of GS from membranes of mammalian brain tissue has been reported to be dependent on the isolation medium employed (Waelsch, 1959). Although GS has not previously been reported in insect muscle, the enzyme has been demonstrated in the fat body of S. gregaria (Kilby and Neville, 1957) and in Prodenia eridania larvae (Levenbook and Kuhn, i962 ).

Z52

DONNELLAN AND OTHERS

Insect Biochem.

GAD Enzyme activity was predominantly in the mitochondrial fractions of the gradient (F8, RSA 1.38; F9, RSA 1.35), and the intramitochondrial location of the enzyme has been established (Langcake and Clements, 1974). GAD activity has previously been demonstrated in brain homogenates of adult Apis mellifera (Frontali, i961 ) and in the larval Drosophila melanogaster(Chen and Widner, I958), while the GAD activity present in homogenates of the supraoesophageal ganglion of Apis meUifera is associated with cellular particulate fractions, not all mitoehondrial, sedimenting in a wide range of centrifugal forces (Fox and Larsen, 1972 ). PROTEIN

The distribution of protein on the gradient shows the majority to be present in either the mitochondria (F8 and F9, 71.5 per cent) or the soluble fraction (FI, 24. 3 per cent). Only 4.2 per cent of the total protein on the gradient was present in the remaining membrane fractions in which synaptosome or synaptosome fragments would be expected to be found, together with the sarcolemma and sarcoplasmic reticulum vesicles. One of the objectives of the described localization studies was to determine whether the membrane fractions, in which synaptosomes had been detected albeit very infrequently by electron microscopy, contained any immediately obvious content of the enzymes investigated. It is evident, however, that synaptosomes or fragments thereof could not be readily recognized on the basis of an enrichment in glutamate-metabolizing enzymes which could be involved in the synthesis or degradation of the putative neuromuscular transmitter. PROPERTIESOF SOME GLUTAMATE-METABOLIZINGENZYMES The evident levels of GDH, GPT, and G O T present in distinct cellular fractions prompted further study into their characteristics and locale as such information could contribute to the overall picture of glutamate metabolism in the flight muscle cell.

Glutamate Dehydrogenase (GDH) Enzymic activity, when measured in the direction of glutamate formation, was barely measurable in mitochondrial extracts unless ADP (I mM) was present in the reaction mixtures. This observation indicates that the insect muscle enzyme resembles the majority of animal glutamate dehydrogenases in being a polymeric molecule able to undergo reversible dissociation/association reactions and possessing a purine nucleotide site at which ADP can exert a regulatory effect (Stadtman, I966 ). Preliminary studies established that the level of G D H activity observed in mitochondrial extracts depended on the coenzyme used. Maximum activity, in 5o mM Trischloride, was observed at pH 7"9 with NADH and at pH 6. 9 with NADPH when assayed in the direction of glutamate formation. Under these optimum pH conditions it was noted that the reaction rate with NADP was six times faster than that seen with the same concentration of NADPH. Similar differences in coenzyme specificities have been reported for the mitochondrial glutamate dehydrogenase of Drosophila larvae (Bond and Sang, 1966 ). The insect muscle enzyme appears to have a relatively low affinity for ammonium ions which is markedly increased by ADP, as illustrated in Fig. 4,

I974, 4

FLIGHT

253

MUSCLE SUBCELLULAR COMPONENTS

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50 100 150 Conch. Ammonium Acetate (mM)

200

Fio. 4.--GDH. Effect of ADP on the affinity for ammonium acetate. Plot of enzymic velocity (23o pg. mitochondrial protein) versus concentration of ammonium acetate in the presence of xo mM 2-oxoglutarate with (f-q)o'I mM, (0) o'5 raM, and (O) i.o mM ADP. which shows the velocity versus substrate plots in the presence of io mM 2-oxoglutarate at various concentrations of the nucleotide. The sigmoidal curves are replaced by normal Michaelis-Menten kinetics as the level of ADP in the reaction mixture is raised to I raM. The concentration of ammonium ions giving 5o per cent of the maximum velocity at this level of nucleotide is 37 mM, which is considerably higher than the values recorded for glutamate dehydrogenases isolated from other animal sources, e.g. beef liver, 3.2 mM; frog liver, o. 5 mM (Fahien et al., 1965). Bond and Sang (1968) report, however, that the Drosophila larval enzyme's affinity for ammonium ions varies markedly with the coenzyme employed even in the presence of ADP--with NADH the Km value is very high (o'z M to 0"4 M), whereas with NADPH the K~, is zo mM. ADP also affects the muscle enzyme's affinity for 2-oxoglutarate, although it does not appear to behave as a classic allosteric effector as manifested in the lowering of the enzyme's Km for ammonium ions. This is illustrated in Fig. 5, which shows that maximum activity is observed, in the presence of 200 mM ammonium acetate, with I mM ADP. At lower concentrations of the nucleotide the plots are still normal hyperbolae, although the Vma= values are considerably reduced. The Michaelis constant for 2-oxoglutarate (2 mM) is in the range reported for the enzyme isolated from Drosophila larvae (Bond and Sang, 1968 ) and other animal mitochondria (Fahien et al., 1965). In common with other animal glutamate dehydrogenases, the insect muscle enzyme's activity is extremely sensitive to GTP (Fig. 6, I60 = 2"5/,M) and to a lesser extent to the presence of other nucleotides (Table 3)- Such experiments do not indicate whether the ATP- and GTPbinding sites on the insect mitochondrial enzyme are identical or merely situated in close proximity to each other (De Prisco, 1971).

254

Insect Biochem.

DONNELLANAND OTHERS

60

[vl-'

30

I

!

/

200

3: Q < Z

I

!

2.5

5

l/2--oxoglutarate (mM -1 )

! < -e o

100

-0--

0

0

"

I

5

|

10

Conch, 2-oxoglutarate (mM)

FIG. 5 . - - G D H , Effect of ADP on the affinity for 2-oxoglutarate. Plot of enzymic velocity (z2o/zg. mitochondrial protein) versus concentrations of 2-oxoglutarate in the presence of 20o m M ammonium acetate with (&) 0"2 raM, (O) 0"5 raM, and (Q) i.o m M ADP. The inset shows the double-reciprocal plot of [v] -1 versus [s] -1 in the presence of I m M ADP (0) from which the apparent Michaelis constant can be estimated for 2-oxoglutarate.

L-Alanine: 2-Oxoglutarate Aminotransferase (GPT) The properties of the enzyme were examined in mitochondrial extracts with maximum enzymic activity being observed at pH 6. 9 in 5° mM KH2PO4. This pH optimum is lower than the values (pH 7.4-8.2) reported for mammalian mitochondrial aminotransferases (Hopper and Segal, x964; Swick et al., i965; Saier and Jenkins, r967). The absolute Michaelis constants for L-alanine and 2-oxoglutarate were determined by following enzymic velocity at varying concentrations of one substrate in the presence of three saturating concentrations of the cosubstrate as described by Hopper and Segal

1974, 4

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

255

(1964). This enables the apparent Michaelis constants (K'm) to be determined from conventional double-reciprocal plots, while a secondary plot of the reciprocals of K',. against the reciprocals of the three cosubstrate concentrations employed permits the estimation of the absolute Michaelis constant (Kin) for the substrate at an infinite concentration of the cosubstrate. The Michaelis constants derived in this manner for 2-

100

so 0

o

lO Conch. GTP (VM)

20

FIG. 6.--Effect of G T P on glutamate dehydrogenase activity. Standard reaction mixtures containing 200 fig. mitochondrial protein were preincubated for I rain with the indicated concentrations of G T P before the addition of Io m M 2-oxoglutarate to initiate the reaction. Table 3.--EFFECT OF NUCLEOTIDES ON GLUTAMATE DEHYDROCENASEACTIVITY ADDED NUCLEOTIDE

Control G T P (o'x raM) G D P (I m M ) C D P (I m M ) C T P (I m M ) 5 ' - A M P (i raM) 3 ' , 5 ' - A M P (1 m M ) A T P (1 raM)

PERCENTAGEACTIVITY

ioo o x2"8 84"0 47"5 74"5 83"6 4I"2

All assays contained x o m M 2-0xoglutarate, 200 m M ammonium acetate, 200 pg mitochondrial protein, and I m M - A D P . Nucleotides were preincubated with the reaction mixture for I min before adding the 2-0xoglutarate to initiate the enzymic reaction.

256

DONNELLAN AND OTHERS

Insect Biochem.

oxoglutarate and L-alanine are shown in Table 4 and these values can be compared to those reported for the rat liver mitochondrial enzyme - - K m (al~nine) IX'9 raM, K m (2-oxoglutarate) 1"9 mM (Swick et al., I965). L-Aspartate : 2-Oxoglutarate Aminotransferase (GOT) As described earlier significant levels of aspartate aminotransferase activity were present in both the mitochondrial and sarcoplasmic fractions of flight muscle. Preliminary studies established that the mitochondrial and sarcoplasmic enzymes exhibited maximum activities at pH 7"9 and pH 7"4 respectively when assayed in 5° mM Tris-chloride buffer. The affinities of the aminotransferases for L-aspartate and 2-oxoglutarate in the mitochondria and sarcoplasm were determined at these pH optima by the procedures described for GPT. The Michaelis constants thus obtained are shown in Table 4 and show that the extracts have markedly different affinities for the two substrates. The mitochondrial enzyme has a lower Km for L-aspartate and a higher Km for 2-oxoglutarate than the sarcoplasmic enzyme, which is strong evidence that distinct isoenzymes are present in these muscle cell compartments. Similar differences in K mvalues for the GOT isoenzymes present in the mitochondrial and cytoplasmic fractions of mammalian heart, brain, and liver have been recorded (Fleischer et al., 196o; Boyd, x96x ; Wada and Morino, 1964). The isoenzymes also have radically different electrophoretic and immunological properties (Magee and Phillips, x97i ). Table 4.--MICHAELIS CONSTANTS FORL-ALANINE: ANDL-AsPARTATE: 2-OxoGLUTARATE AMINOTRANSFERASES

ENZYME

GPT

SOURCE

Mitochondria

COSUBSTRATE APPARENT SUBSTRATE ABSOLUTESUBSTRATE CONCENTRATION(mM) K'm (mM) K,~ (at infinite cosubstrate conc.) (mM) 20~ 40 L-Alanine 60]

5~

IO 2-Oxoglutarate 153

GOTm Mitochondria

GOTs

Sarcoplasm

2"13 2"56 2'94

} 2-Oxoglutarate

18"2t L-Alanine

22"2 24"4

3"33 2-Oxoglutarate

27 L-Alanine

4~ 20 L-Aspartate 40)

0"91 ) i'oo 2-Oxoglutarate i'o3

1"o5 2-Oxoglutarate

2"5~ 5 2-Oxoglutarate io)

0"35 ) 0"48 L-Aspartate 0"59

o'71 L-Aspartate

o'o97 ~- 2-Oxoglutarate

o"11 2-Oxoglutarate

20 L-Aspartate 4oJ

0"085] o'1o6J 2"22

2"i} 2-Oxoglutarate

2.44 L-Aspartate 2"50 J

2"56 L-Aspartate

I974, 4

FLIGHT MUSCLE SUBCELLULAR COMPONENTS

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257

258

DONNELLANAND OTHERS

Insect Biochem.

INTRAMITOCHONDRIAL LOCALIZATIONOF GPT, GDH, ANDGOT The location of the three enzymes in flight muscle mitochondria was investigated by examining the fractions obtained when the mitoehondria were disrupted by either ultrasonic or digitonin treatment. In conjunction with the assays for the glutamate enzymes, the fractions were also examined for their content of marker enzymes generally accepted to originate from distinct mitochondrial compartments (Lardy and Ferguson, 1969). In these studies malate dehydrogenase (MDH) was used as a marker for the enzymes present in the mitochondrial matrix. Investigation of the fleshfly mitochondrial enzyme showed that maximum activity was observed at pH 8.8 in 5° m M glycine buffer when assayed in the direction of malate formation. The K m for oxaloacetate was determined to be 5°/zM with substantial substrate inhibition occurring at a concentration of oxaloacetate greater than 400/~M. These values are similar to those reported for the mitochondrial M D H isolated from the muscle of Locusta migratoria (Delbriick et al., I959) and also from adult Drosophila virilis (McReynolds and Kitto, x97o). The presence of adenylate kinase (AK) in extracts of fleshfly mitochondria was taken to indicate the enzyme's suitability as a marker for the space between the inner and outer mitochondrial membranes (the intermembrane space) as described for mammalian mitochondria (Klingenberg and Pfaff, i966; Schnaitman and Greenawalt, 1968 ). The inner membrane marker was chosen to be GPox as this enzyme remains firmly bound to the membrane when fleshfly mitochondria are disrupted (Donnellan and Beechey, 1969). Unfortunately we were unable to find a satisfactory marker for the outer mitochondrial membrane as the presence of kynurenine hydroxylase, monoamine oxidase, or rotenone-insensitive NADH-cytochrome c reductase could not be detected in fleshfly mitochondria. These enzymes have been successfully used as markers for the outer membranes of rat liver mitochondria by Schnaitman and Greenawalt (1968). ULTRASONICDISINTEGRATIONOF FLIGHTMUSCLEMITOCHONDRIA Ultrasonic treatment of mitochondria causes the inner mitochondrial membrane to vesiculate forming submitochondrial particles and releasing the bulk of the matrix enzymes. Table 5 shows the distribution and specific activities of both the glutamatemetabolizing enzymes and the marker enzymes in the three fractions obtained when insect flight muscle mitochondria were subjected to the ultrasonic treatment. The release of the matrix enzymes is reflected in the appearance of malate dehydrogenase primarily in the 'sonic soluble' fraction, which was also enriched in AK, the intermembrane space marker, as would be predicted. GDH, GPT, and G O T m all appear in the soluble enzyme fraction, suggesting that they originate from either the matrix or the intermembrane spaces. The inner membrane marker, GPox, is located almost exclusively in the submitochondrial particles. This fraction also contained low levels of the enzymes predominantly present in the 'sonic soluble' fraction and this observation probably reflects the trapping of the enzymes within the particles when the inner membrane vesiculates owing to the ultrasonic treatment. The specific activity values of the enzymes also reflect the distribution of the enzymes in the mitochondrial fractions, in that purification occurred in the fractions where the bulk of the enzyme activity was located. The relatively poor recovery of G P T from the mitochondrial fractions can be compared with similar observations made on the recovery of this enzyme from sucrose gradients (Table x). Low levels of all the enzymes studied were present in the 'lowspeed' pellet, which consists mainly of damaged mitochondria.

x974, 4

FLIGHT MUSCLE 8UBCELLULAR COMPONENTS

~6~;~o 0

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259

260

DONNELLANAND OTHERS

Insect Biochem.

DIGITONIN FRACTIONATION The mitochondrial location of the enzymes appearing in the soluble fraction obtained by the sonication fractionation procedure was resolved by treating mitochondria with digitonin. This treatment selectively removes the outer mitochondrial membrane and releases the contents of the intermembrane space, while the matrix enzymes remain occluded by the inner membrane. Table 6 shows the distribution and specific activities of the enzymes in the three fractions obtained from the digitonin treatment. M D H (79"9 per cent) and GPox (93"5 per cent) were located primarily in the 'low-speed' pellet, which represents the inner membrane plus matrix fraction of the mitochondria. The levels of the extremely active M D H in the other fractions indicate that some leakage of the matrix contents had occurred during the fractionation procedure. The appearance of GPox in t h e ' high-speed' pellet is attributed to the presence of some inner membrane fragments as contaminants of the outer membrane vesicle fraction. AK, however, was concentrated (79"5 per cent) in the 'high-speed' supernatant, which is taken to contain the intermembrane contents of the mitochondria. Activity observed in the 'low-speed' pellet is possibly due to the presence of intact mitochondria in this fraction. The glutamate-metabolizing enzymes (GDH, GPT, and GOT) all appeared primarily in the 'low-speed' pellet with increased specific activity values compared to the untreated mitochondria. This indicates that the glutamate enzymes are constituents of the mitochondrial matrix of flight muscle mitochondria. Of interest was the high recovery of GPT activity after the digitonin treatment, which contrasted with the poor recovery observed after the sonication procedure (Table 5). This may be a reflection of an inherent instability of the aminotransferase when it is released from its environment in the matrix by the ultrasonic treatment. DISCUSSION Our attempts to isolate neuromuscular junctions from flight muscle proved unsuccessful in that synaptosomes could only be detected at marginal frequencies on electron micrographs of the subceUular fractions of muscle fractionated by the sucrose density gradient technique. This low detection rate could be explained in a number of ways. I. The frequency of axon terminals in the asynchronous muscle used in these studies is not known and may be considerably lower than that estimated from the reported innervation pattern of about 26 nerve endings on a 50o/zm. length 5°/zm. diameter portion of the metathoracic depressor tibia muscle fibre of the locust (Usherwood, ~967). Assuming that the flight muscle is similarly innervated and that the synaptosomes form a homogeneous population with an average diameter of I/zm., the minimum yield of synaptosomes should be in the region of IO-4 of the tissue by volume. Such a concentration should have been discernable by electron microscopy if the synaptosomes sedimented to a discrete region of the gradient. 2. The terminal frequency occurred at the expected rate in the starting material but only a small proportion formed synaptosomes when the muscle was homogenized. 3- Terminals formed synaptosomes in good yield but only a few then survived the subsequent handling procedures. The experiences of workers with mammalian smooth muscle (Austin et al., x967) and the torpedo electric tissue (Sheridan, et al., i966; IsraSl et al., i968 ) lead us to favour 2 as the main cause of our failure to isolate a muscle fraction enriched in synaptosomes.

1974, 4

FLIGHT MUSCLESUBCELLULARCOMPONENTS

261

If this conclusion is correct it indicates that the mechanical conditions necessary for the formation of synaptosomes are not met when the insect muscle is dispersed, because the neuromuscular junctions do not have a greater resistance than the surrounding tissues to shear forces. This may be because the nerve terminals tend to be located in indentations in the muscle cell membrance (Tiegs, 1955) so that the terminals were not rubbed off by the homogenization step as was hoped at the outset of these studies. Attempts to correlate the appearance of the muscle fractions with a biochemical parameter have also proved unsuccessful, in that no enrichment of a number of glutamate-metabolizing enzymes could be detected in gradient fractions expected to contain synaptosomes or fragments thereof (Table 1). The enzymes studied were chosen on the basis that they might be involved in the metabolism of the transmitter at the neuromuscular junction. Glutamate decarboxylase (GAD) was of particular interest because it is essentially irreversible in catalyzing the decarboxylation of glutamate to 7-aminobutyrate and CO 2. The mitochondrial location of GAD appears to rule out the premise that the enzyme might be involved in transmitter inactivation at the neuromuscular junction in an analogous manner to acetylcholinesterase at cholinergic synapses. Another approach has been to examine gradient fractions for their content of glutamate as the measurement of occluded acetylcholine has been successfully used in the isolation of mammalian brain synaptosomes (Gray and Whittaker, 1962). However, glutamate could only be detected in the soluble fraction of flight muscle (FI, Table 2) precluding the use of this method as an aid in the isolation of muscle synaptosomes. SUBCELLULARLOCALIZATIONOF GLUTAMATEENZYMES

Enzymes involved in glutamate metabolism have been reported in a variety of insects (Delbriick et al., i959; Brosemer et al., I963 ; Chefurka, i965; Crabtree and Newsholme, 197o). The studies described on a number of glutamate-metabolizing enzymes present in the flight muscle of S. barbata show that GDH, GPT, and GAD are mitochondrial enzymes while GS appears to be predominately a soluble enzyme. GOT is, however, distinctly bimodal in being distributed in the mitochondrial and soluble fractions. The role of these enzymes in the general metabolism of the flight muscle cell must be viewed in relation to the process whereby the mitochondria are able to generate the ATP necessary for flight. Dipteran muscle mitochondria are peculiar in that they appear only to couple the oxidation of L-3-glycerophosphate, pyruvate, and L-proline to the synthesis of ATP (Van den Bergh and Slater, 1962; Sacktor and Childress, 1967; Donnellan et al., 197o). It is relevant that the oxidation of proline leads to the iutramitochondrial formation of glutamate via I-pyrolline-5-carboxylate, and that the synergistic effect of proline on pyruvate oxidation by flight muscle mitochondria is attributed to the further metabolism of glutmate, within the mitochondria, to metabolites such as 2-oxoglutarate and oxaloacetate that are capable of priming the Krebs' cycle to permit maximum rates of pyruvate oxidation (Sacktor and Childress, 1967). The location of GDH, GPT, and GOT m within the mitochondrial matrix along with the majority of the Krebs' cycle enzymes underlines the expected physiological involvement of these glutamate enzymes in linking amino-acid and carbohydrate metabolism in the flight muscle mitochondria. It should be noted that the substrate affinities of GDH, GPT, and GOT were derived by measuring the enzymes' activities in the direction most amenable to assay, which necessarily makes any detailed conclusions as to the precise physiological role of the enzymes difficult, as all three enzymes are essentially fully reversible. It is apparent,

262

DONNELLANAND OTHERS

Insect Biochem.

however, that the mitochondrial and sarcoplasmic aspartate aminotransferases (GOT m and GOTs) have markedly different affinities for their substrates. This we attribute to the presence of isoenzymes which may well be related to the pattern of amino-acid metabolism in these cellular compartments. The reported properties of the flight muscle glutamate dehydrogenase (GDH) are similar to those described for the Drosophila larval enzyme (Bond and Sang, 1968) and suggest that the muscle enzyme resembles other glutamate dehydrogenases in being an allosteric enzyme with ADP exerting its regulatory effect by increasing the affinity of the dehydrogenase for both 2-oxoglutarate and ammonia. The observation that relatively low levels of ADP can cause such a dramatic increase in the affinity of GDH for ammonia may possibly reflect a scavenging role for the enzyme in vivo. Another enzyme associated with the removal of ammonia is glutamine synthetase (GS), which has been shown to be principally a constituent of the sarcoplasm in the flight muscle cell. GLUTAMATEAS THE EXCITATORYTRANSMITTER

At the outset of these studies we had hoped to isolate a preparation of synaptosomes from flight muscle which would have been used to examine the biochemistry behind the process of excitatory neurotransmission. In particular it was anticipated that such a preparation might be able to resolve how glutamate could act as a transmitter in the presence of high levels of this amino-acid in the haemolymph bathing the muscle tissue. In this laboratory the enzymatically-determined concentration of glutamate in locust blood is in the region of o-2 mM, which is sufficient to cause the desensitization of isolated nervemuscle preparations (Usherwood and Machili, 1968). If glutamate is the transmitter then the glutamate receptor on the postsynaptic membrance must somehow be protected in situ from the glutamate present in the blood as well as from the neurally-released glutamate, which would otherwise accumulate in the synaptic cleft. The work of Faeder and Salpeter (197o) suggests that an active transport process, located primarily in the sheath cells, is one mechanism concerned in the protection of the glutamate receptor at the neuromuscular junctions of a cockroach nerve-muscle preparation. A similar resorption process for glutamate has been reported in lobster nerve-muscle preparations (Iversen and Kravitz, i968), while a synaptosomal fraction isolated from brain tissue has been demonstrated to accumulate specifically glutamic and aspartic acids (Wolsey et al., 1971 ) . There is increasing evidence that such transport processes are commonly employed to shield receptor molecules from excessive concentrations of the transmitter and that the enzymatic degradation of transmitters, as observed at cholinergic synapses, is the exception rather than the rule (Iversen, 1971). SUMMARY I. Homogenates of the flight muscles of Sarcophaga barbata have been fractionated on sucrose density gradients in attempts to isolate a synaptosomal fraction. Electron microscopy has been used to examine the resultant subcellular fractions for their content of synaptic terminals. The marginal frequency of synaptosomal recognition is attributed primarily to the disruption of the neuromuscular junctions when the muscle was dispersed even under the relatively mild conditions employed. 2. The subcellular fractions obtained from such gradients have also been screened for their content of a number of enzymes likely to be involved in the metabolism of glutamate, the putative excitatory neurotransmitter, both in the synaptic terminals and in the

1974, 4

FLIGHT MUSCLE SUBCELLULARCOMPONENTS

263

muscle cell in general. No enrichment in the enzymes studied could be detected in the membrane fractions in which synaptosomes, or fragments thereof, would be expected to be located. Glutamate dehydrogenase, glutamate decarboxylase, and the alanine aminotransferase were shown to be mitochondrial enzymes, while glutamine synthetase was a constituent of the sarcoplasm. Aspartate aminotransferase was found in both the mitochondrial and sarcoplasmic fractions of the muscle cell. 3. Disruption of flight muscle mitochondria by digitonin and sonication treatments has established that glutamate dehydrogenase and the alanine/aspartate aminotransferases are situated in the mitochondrial matrix. 4. T h e observed difference in substrate affinities of the mitochondrial and sarcoplasmic aspartate aminotransferases suggest that isoenzymes are present in these cellular compartments. ACKNOWLEDGEMENTS

We thank Miss V. M. Forster, Mrs. P. R. Langcake, and Mr. B. G. Wallace for their contributions to the work described in this paper. REFERENCES AUSTIN L., CHUBBI. W., and LIW'TT B. G. (x967) The subcellular localization of catecholamines in nerve terminals in smooth muscle tissue. ~7. Neurochem. I4, 473-478. BERANEKR. and MILLERP. L. (I968) The action of iontophoreticaUy applied glutamate in insect muscle fibres. ~t. exp. Biol. 49, 83-93. BERL S. (I966) Glutamine synthetase. Determination of its distribution in brain during development. Biochemistry 5, 916-922. BOND P. A. and SANO J. H. (x968) Glutamate dehydrogenase in Drosophila larvae. J. Insect. Physiol. I4, 34x-359. BOYD J. W. (I96I) The intraceUular distribution, latency and electrophoretie mobility of Lglutamate-oxaloacetate transaminase from rat liver. Biochem. J. 8x, 434-44I. BROSEMEaR. W., VOGELLW., and BOCHEaTH. (I963) Morphologische und enzymatische Muster bei der Entwicklung indirekter Flugmuskeln yon Locusta migratorla. Biochem. Z. 338, 854-91o. B0cn~R TH. and KLINGEN~ERGM. (I958) Wege des Wasserstoffs in der lebendigen Organisation. Angew. Chem. 70, 552-57 o. CHANCEB. and SACKTORB. (1958) Respiratory metabolism of insect flight muscle. Archs Biochem. Biophys. 76~ 5o9-53L CHEFURKAW. (i965) Intermediary metabolism of nitrogenous and lipid compounds in insects. in The Physiology of lnsecta (ed. Rockstein) vol. 2, pp. 483-579. Academic Press, New York. CHEN P. S. and WXDN~RB. (I968) Content and synthesis of 7-amino butyric acid in the larval brain of Drosophila melanogaster. Experientia 24, 5 I6-5 I7. CLEMENTSA. N. and POTTERS. A. (I967) The fine structure of the spermathecae and their ducts in the mosquito Aedes aegypti, ft. Insect Physiol. x3, 1825-36. CRABTe.EEB. and NEWSHOLM~E. A. (I97o) The activities of proline dehydrogenase, glutamate dehydrogenase, aspartate-oxoglutarate aminotransferase and alanine-oxoglutarate aminotransferases in some insect flight muscles. Biochem. ~t. Ix7, IoI9-IO2I. DELBR0CK A., Z~BE E., and B 0 c n ~ TH. (I959) Ober Verteilungsmuster yon Enzymen des Energie liefernden Stoffwechsels in Flugmuskel, Sprungmuskel und Fettk6rper yon Locusta migratoria und ihre cytologisehe Zuordnung. Biochem. Z. 33x~ 273-296. DE PRISCO G. (t97I) Tyrosyl and lysyl residues involved in the reactivity of catalytic and regulatory sites of crystalline beef liver glutamate dehydrogenase. Biochemistry, zo, 585-589. DE ROBEaTISE., DE I~LDI A. P., RODalGUEZG., and GOMEZJ. (1961) On the isolation of nerve endings and synaptic vesicles. ~. biophys, bioehem. Cytol. 9, 229-235. DONNELLANJ. F., BAm~a M. D., WOODJ., and BEECHmrR. B. (197o) Specificity and locale of the L-3-glycerophosphate-flavoprotein oxidoreductase of mitochondria isolated from the flight muscle of Sarcophaga barbata Thorns. Biochem. J. x2o, 467-478.

264

DONNELLANAND OTHERS

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DONNELLAN J. F. and BEECHEY R. B. (I969) Factors affecting the oxidation of glycerol-x-phosphate

by insect flight-muscle mitochondria, y. Insect Physiol. x$, 367-372. F^EDER I. R. and SALPETERM. M. (197o) Glutamate uptake by a stimulated insect nerve muscle preparation, jr. Cell Biol., 46, 3oo-3o7. FAHIEN L. A., WmOERT B. O., and COHENP. O. (1965) Crystallization and kinetic properties of glutamate dehydrogenase from frog liver. J. biol. Chem. 240 , lO83-1o9o. FLEISHERG. A., POTTERC. S., and WAKIMK. G. (196o) Separation of 2 glutamic-oxaloacetic transaminases by paper electrophoresis. Proc. Soc. exp. Biol. Med. IO3, 229-231. Fox P. M. and LARSENJ. R. (1972) Glutamic acid decarboxylase and the GABA shunt in the supraoesophageal ganglion of the honey-bee, Apis meUifera. J. Insect Physiol. I8, 439-457. FRONTAL1N. (1960 Activity of glutamic acid decarboxylase in insect nerve tissue. Nature, Lond. I9I, 178-179. GORNALLA. G., BARDAWILLC. J., and DAVIDM. M. (1949) Determination of serum proteins by means of the Biuret reaction, y. biol. Chem. 17% 751-766. GRAYE. G. and WHITTAKERV. P. (1962) The isolation of nerve endings from brain; an electron microscopic study of cell fragments derived by homogenisation and centrifugation, y. Anat. 96, 79-88. HINTON R. H., BURGEM. L. E., and HARTMANNG. C. (1969), Sucrose interference in the assay of enzymes and protein. Analyt. Biochem. 29, 248-256. HOPPER S. and SEGALH. L. (1964) Comparative properties of glutamic-alanine transaminase from several sources. Archs Biochem. Biophys. xos, 5Ol-5O5. ISRA'~LM., GAUTRONJ., and L~SBATSB. (1968) Isolement des vesicules synaptique de l'organe 61ectrique de la Torpille et localisation de l'acetylcholine a leur niveau. C.R. Acad. Sci., Paris 266, 273-275. IVERSEN L. L. (1971) Role of transmitter uptake mechanisms in synaptic neurotransmission. Br.J. Pharmae. 4I, 571-59 I. IV~SF~t L. L. and K~VlTZ E. A. (1968) The metabolism of y-aminobutyric (GABA) in the lobster nervous system--uptake of GABA in nerve-muscle preparations..7. Neurochem. xs, 609620. KERKUT G. A., L m I ~ L. D., SHAPIRAA., COWANS., and WALKERR. J. (I965) The presence of glutamate in nerve-muscle perfusate of Helix, Cardnus and Periplaneta. Comp. Biochem. Physiol. 15, 485-502. KILEY B. A. and NEVILLEE. (1957) Amino acid metabolism in locust tissues. ~. exp. Biol. 34, 276--289. KLINGENBERCM. and PFAFF E. (1966) Regulation of Metabolic Processes in Mitochondria. BBA Library, vol. 7. P. I8O, Amsterdam: Elsevier. LANGCAKEP. R. and CLEMENTSA. N. (I 974) L-Glutamic acid decarboxylase of fleshfly flight muscle, its properties and subceUular location. Insect Biochem. 4* 225-241. LARDY H. A. and FEROUSONS. M. (1969) Oxidative phosphorylation in mitochondria. A. Re*;. Biochem., 38, 991-1o34. LEVENBROOKL. and KUHN J. (1962) Properties and distribution of glutamine synthetase in the southern armyworm, Prodenia eridania. Biochim. biophys. A a a 65, 219-232. MCRm~OLDS M. S. and KITTO G. B. (197o) Purification and properties of Drosophila malate dehydrogenases. Biochim. biophys. Acta I98, 165-175. MAGE~ S. C. and PHILLIPSA. T. (1971) Molecular properties of the multiple aspartate aminotransferases purified from rat brain. Biocheraistry, IO, 3397-3405. MILLERG. L. (1959) Protein determination for large numbers of samples. Analyt. Chem. 3x, 964. NISSELBAUMJ. S. and BODANSKYJ. (1964) Immunochemical and kinetic properties of anionic and cationic glutamic-oxaloacetic transaminases separated from human heart and human liver. J. biol. Chem. 239, 4232-4236. PETTE D. and LUH W. (1962) Constant-proportion groups of multilocated enzymes. Biochem. biophys. Res. Commun. 8, 283-287. SACKTOR B. and CHILDRESSC. C. (1967) Metabolism of proline in insect flight muscle and its significance in stimulating the oxidation of pyruvate. Archs Bioehem. Biophys. x2o, 585-588. SAIm~M, H. and JENKINSW. T. (1967) Alanine aminotransferase. I. Purification and properties. .7. biol. Chem. 24z, 91-1oo.

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FLIGHT MUSCLE SUBCELLULARCOMPONENTS

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