- Email: [email protected]
A rapid method for preparing insect microsomes
Comp. Biochem. Physiol. Vol. 82B, No. 3, pp. 559--562, 1985 Printed in Great Britain
0305-0491/85 $3.00 + 0.00 ~ 1985 Pergamon Press Ltd
A RAPID METHOD FOR PREPARING INSECT MICROSOMES R. FEYEREISEN*'['~,G. D. BALDRIDGE* and D. E. FARNSWORTH* *Department of Entomology and tDepartment of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331, USA (Received 14 March 1985) AImraet--I. The 1000g supematant of a tissue homogenate is layered on top of a small (< 5 ml) sucrose density gradient and centrifuged for 20 rain at very high centrifugal forces in a vertical rotor. 2. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. 3. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centfifugation method. 4. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.
INTRODUCTION Microsomes are the isolated form of the endoplasmic reticulum when this cytoplasmic constituent is obtained by centrifugation after homogenization of eukaryotic cells (Claude, 1969). Biochemical studies on microsomal enzymes require the preparation of a subcellular fraction which is reasonably enriched in microsomes and whose properties are representative of the organdie from which it derives. Preparation of microsomes from insect tissues is usually done by the technique of differential centrifugation (Wilkinson and Brattsten, 1972; Wilkinson, 1979) which was originally developed with mammalian liver cells (Siekevitz, 1963). Thus, the subcellular topology of many enzyme activities is often assigned solely on the basis of a rather arbitrary set of centrifugation conditions and only in a few cases have other criteria, such as 'marker' enzymes, electron microscopical examination, lipid, protein and R N A content, buoyant density, etc. been used to characterize insect microsomes. The study o f microsomal enzyme systems such as cytochrome P-450 monooxygenase has benefited greatly from the application of the classical differential centrifugation technique, but a number of studies have also documented some limitations of the technique (Wilkinson and Brattsten, 1972; Wilkinson, 1979). A great reduction in the time of preparation may be achieved by rapid centrifugation of the mitochondrial and post-mitochondrial fractions (Keeley, 1973; Brattsten and Gunderson, 1981). However, c o m m o n problems remain, such as the sedimentation of 'microsomal' oxidative enzyme activities at low centrifugal forces (Wilkinson and Brattsten, 1972; Wilkinson, 1979; Benke et al., 1972) or the ambiguity of subcellular localization of some cytochrome P-450 monooxygenases which may be mitochondriai or microsomal (Feyereisen and Durst, 1978). Attempts to solve those problems have in~/AII correspondence to: R. Feyereisen, Department of Entomology, Oregon State University, Corvallis. OR 97331, USA. 559
volved centrifugation on sucrose density steps or gradients (Benke et aL, 1972; Feyereisen and Durst, 1978). We wish to report here our efforts to combine speed of preparation with the use of sucrose density gradients, which have resulted in a technique that may prove particularly useful in invertebrate biochemistry, where the amount of biological material is often limited. MATERIALS AND METHODS Insects The viviparous cockroach Diploptera punctata (Orthoptera: Blaberidae) was reared at 27°C under a 12 hr light: 12 hr dark photoperiod and fed Wayne rodent blox and water ad libitum. An anautogenous strain of the mosquito Culex pipiens (Diptera: Culicidae) was maintained at 26~C under a 16 hr light: 8 hr dark photoperiod. Adults were fed a 10% sucrose solution ad libitum and live quail were used to provide blood meals. Enzyme assays NADPH cytochrome c reductase activity (Williams and Kamin, 1952) was measured spectrophotometrically at 30°C in an Aminco DW2a instrument. Reduction of cytochrome c was measured at 550 nm in the split beam mode in I ml cuvettes each containing 50/aM cytochrome c, 1 mM EDTA, 5raM NaCN and enzyme in 50mM MOPS (morpholinopropanesulfonate) buffer, pH 7.2. Reduction of cytochrome c was started in the sample cuvette by the addition of 50 #1 of an NADPH (final concentration in the assay: 150#M) regenerating system (10/zmol glucose 6-phosphate and 1.5 units glucose 6-phosphate dehydrogenase). Succinate cytochrome c reductase activity was measured by the same procedure, but 10#1 of a I M suecinate solution was used to start the reaction, in the cytochrome c oxidase assay, NaS2 O4-reduced cytochrome c was used as substrate, NaCN was deleted from the reaction mixture, and the reaction was started by the addition of the enzyme. Methoxyresorufin O-demethylation activity was assayed spectroltuorometrically at 30°C in a Perkin-Elmer 650-10S instrument set at 560 and 3 nm slit for excitation, and at 580 and 5 nm slit for emission. The reaction mixture (1 ml) contained enzyme, methoxyresorufin (40 # M, Molecular Probes, Junction City, Oregon, USA), 1 mM EDTA and an NADPH (250#M) regenerating system in 50 mM MOPS buffer, pH 7.2 (Feyereisen and Farnsworth, 1985).
560
R. FEYEREISENCI a/.
Protein was determined according to the Bradford (1976) assay using gamma-globulins (Calbiochem) as standard. Sucrose concentrations were determined by refractometry.
Tissue homogenates Midgut tissue from adult female Diploptera pum'tata v,as dissected in Yeager's cockroach saline, then transferred to 50 mM MOPS buffer, pH 7.2 containing lff!;, sucrose, I mM EDTA and 0.4 mM PMSF (phenylmethylsulfonylfluoride, freshly prepared). The tissue was ground in a motor-driven Teflon/glass homogenizer for 10sec and centrifuged at 1000g for 10min to remove cell debris and nuclei. Abdomens of adult female Culex pipiens were dissected in Lure's mosquito saline and midguts and ovaries were pulled out with forceps. The abdomens were transferred to 50mM MOPS buffer, pH 7.2 containing 0.4 mM PMSF and ground in a motor-driven Teflon/glass homogenizer for 10 sec, then centrifuged at 1000g for 10min. Centr([ugation All low-speed centrifugation (1000g) was done in 1.5 ml Eppendorf tubes in a Beckman Microfuge I I instrument at 5 C. Centrifugation of gradients was done in polyallomcr Quick-seal tubes using a VTi 80 rotor in a Lg-gO ultracentrifuge (all from Beckman) at 5 C. Lahelhng af subcelhdar ./?actions Fifty female mosquitoes (6 days after adult emergence) were injected with 7 x 10~cpm of[3SS]methionine. Six hours after injection, abdomens were removed and homogenized for 30sec in 250,ul MOPS buffer in a 2ml Teflon;glass homogenizer. The homogenate was centrifuged at 1000g for 10 min. The supernatant was loaded on a linear 45 to 1500 sucrose gradient and centrifuged at 65,000 rpm for 20 min in a Beckman VTi 80 rotor. Fifteen fractions of 350pl were collected from the gradient and radioactivity was assayed on 5 pl aliquots of each fraction. RESUI.TS
I. Rationale Separation of mitochondrial from microsomal fractions has been achieved by centrifugation on sucrose density gradients (Feyereisen and Durst, 1978; Feyereisen et al., 1979; Feyereisen, 1983) but the procedure is very long ( > 4 h r ) and therefore cannot be used for routine purposes. The reason for the length of the procedure is that gradients were centrifuged in swinging bucket rotors. The maximum speed is limited for these rotors, and their geometry (horizontal during the run) maximizes the distance particles have to travel in the gradient (length of the tube). Vertical rotors are very stable when spinning and can be designed for high speed operation. Their geometry (reoriented gradient during the run) minimizes the distance particles have to travel (diameter of the tube) (Rickwood, 1982). It was felt that centrifugation in a vertical rotor at high speed might drastically reduce the time of preparation of microsomes while at the same time preserving the advantages of gradient separation of mitochondria from microsomes.
2. Application to Diploptera punctata Midgut The 1000g supernatant of a midgut homogenate was subjected to various centrifugal conditions in a sucrose density gradient run in a vertical rotor for an arbitrarily set time of 20 min. At the end of the run, the time integral of the squared angular velocity (¢o"t in r a d 2 ' s e c ') which represents the cumulative cen-
5o I
-1
"6 25
....
/3
,~
>
/-/
i
.......
~
['l ,
co2t (1010 .rad 2 .sec -1 ) Fig. 1. Cross-contamination of microsomal and mitochondrial enzyme activities as a function of the cumulative centrifugal effect. (A --A)",, of total succinate tyrochrome c reductase activity found in microsomal fractions. (IS] .--U])",, of total NADPH cytochrome c reductase activity found in mitochondrial fractions. (© --©) total volume o f microsomal fractions. trifugal effect was recorded from the instrument's integrator. The centrifuge tube was punctured at the bottom with an 18 gauge needle and after puncturing the top of the tube, a total of 15 fractions (350pl) was collected. The fractions were routinely analyzed for sucrose concentration and marker enzyme activities, succinate cytochrome c reductasc and N A D P H cytochrome c reductasc tbr mitochondria and microsomes respectively. In certain experiments cylochrome c oxidase was also used as mitochondrial marker and aldrin epoxidation or methoxyresorufin O-demethylation as microsomal marker. We first tried linear, then discontinuous sucrose gradients, with three steps at 50. 35 and 25"i, sucrose. Centrifugation speeds ranged from 30,000 to 65,000 rpm. Two peaks of activity were more or less well resolved, a heavy, mitochondrial peak and a lighter microsomal peak. It was found that there was always a slight cross-contamination of microsomal and mitochondrial enzyme activities, and that the total volume of the gradient occupied by microsomal fractions was dependent on thc speed of centrifugation. Figure 1 shows the cross-contamination and microsomal volume as a function of the cumulative centrifugal effect obtained in five different experiments. The amount of N A D P H cytochrome c reductase activity found in the mitochondrial peak was relatively constant around 15'!.. In contrast, the amount of succinate cytochrome c reductase (mitochondrial) activity tbund in the microsomal peak was increased at the higher ¢o2t values. The total volume of the gradient occupied by microsomal fractions was minimal at a ,,-'t value of 2.35 × I0 'n rad 2 s e c ' . Because mitochondrial contamination of the microsomal fractions was small, those conditions were chosen as optimal. Figure 2 shows the profile of the gradient obtained under those conditions. It appears that the steps in the sucrose gradient have been flattened, and that mitochondrial enzyme activities were focussed at the 50/35'~.,', interface, whereas the microsomal enzyme activities were focussed at the 35/2501; interface. The close coincidence of the two marker enzyme activities for each fraction testifies to their usefulness. The majority of the protein (presumably of cytosolic origin) was found in the top three fractions. These experiments led us to adopt the following procedure for the preparation of D. punctata midgut
Rapid preparation of insect microsomes
561 50
T ",.~.
""I"'. 25 ~,~,
•25 ~
I
I
5
I0
. . . . . . . . . . . .5 W)
15
Fraction number
Fig. 2. Separation of Diploptera punctata midgut enzymes on a sucrose density gradient. (A A) succinate cytochrome c reductase activity; (A A) cytochrome c oxidase activity; (© (3) NADPH cytochrome c reductase activity; (Q O) methoxyresorufin O-demethylation activity; (©---©)% sucrose. The 1000g supernatant of a midgut homogenate was loaded on a discontinuous (50, 35 and 25%) sucrose density gradient and centrifuged for 20min at 50,000rpm in a Beckman VTi 80 rotor (to2t = 2.35 × 101°). microsomes: 350/~1 o f a 1000g supernatant of midgut homogenate (the equivalent of 2-4 midguts or up to 2 mg protein) was layered on top of a sucrose gradient which consisted of three layers: 800/~1 of 50%, 2100 td of 35% and 2000/~1 of 25% sucrose in 50mM MOPS buffer, pH 7.2 containing i mM EDTA. The gradient was centrifuged at 50,000 rpm for 20 min (to2t = 2.35 x 10~° rad 2 sec-~). The bottom 2.5ml of the tube were discarded as 'mitochondriar and the next 1.5 ml were collected as 'microsomal' fraction. This fraction had a sucrose concentration of 29% (I. 122 g/cm3). 3. Application to Culex pipiens abdominal tissue A similar approach was used to determine optimal conditions for the preparation of microsomes from abdominal tissues of the mosquito. Linear gradients of sucrose (45-15% in MOPS buffer) were prepared by adding equal volumes (1.25 ml) of 45, 35, 25 and 15% sucrose solutions to a centrifuge tube and placing the tube in the horizontal position for two hours prior to the experiment. Various centrifugation conditions from 30,000 to 75,000 rpm were tried, with a constant 20min centrifugation time (results not shown). The best resolution of mitochondria and microsomes was obtained with a centrifugation at 65,000 rpm for 20 min. Figure 3 shows the separation of succinate cytochrome c reductase and NADPH cytochrome c reductase activity under these conditions. The enzyme activities were much lower than those observed with the cockroach tissue, but we found that only a limited amount of homogenate supernatant could be loaded onto the gradient. Indeed, when total protein loaded exceeded approx. I mg, the separation of the two peaks was not satisfactory, i.e. the mitochondrial peak broadened considerably into the microsomal peak. It was also found that the initial 1000g centrifugation was essential to remove most lipids (floating on top after centrifugation) which, when loaded on the gradient caused erratic results. Presumably, the presence of
] 15
FraelIwm numb4m'
Fig. 3. Separation of Culex pipiens abdominal tissue enzymes on a sucrose density gradient. (A A) succinate cytochrome c reductase activity; ( 0 O) NADPH cytochrome c reductase activity; ( 0 - - - 0 ) % sucrose. The 1000g supernatant of a tissue homogenate was loaded on a linear 15 to 45% sucrose density gradient and centrifuged for 20min at 65,000rpm in a Beckman VTi 80 rotor (to2t = 3.70 x 101°). low-density lipids prevented the smooth reorientation of the gradient at the end of the run. When abdomens were prepared 6 hr following injection of [35S]methionine, and the 1000g supernatant of the abdomen homogenate was analyzed by sucrose gradient centrifugation, the radioactivity profile shown in Fig. 4 was obtained. Three peaks were observed, the lower peak coincided with the succinate cytochrome c reductase peak, and the middle peak coincided with the NADPH cytochrome c reductase peak (see Fig. 3). A very large amount of radioactivity was found at the top of the gradient. This profile confirms that a good resolution of mitochondrial, microsomal and cytosolic proteins can be achieved by this procedure. These experiments led us to adopt the following procedure for the preparation of microsomes from abdominal tissues of C. pipiens: 350~1 (up to I mg protein) ofa 1000g supernatant of tissue homogenate was layered on a linear 45 to 15~ sucrose gradient, and centrifuged at 65,000 rpm for 20 min (w2t = 3.70 x l0 ~°rad 2 sec ~). The bottom 2.0 ml of the tube were discarded as 'mitochondrial' and the next 2. l ml were collected as 'microsomal' fraction. Depending I0-
-50
~-
-25
9
i
I 5
Frll~l~l
I0
15
Fig. 4. Distribution of radioactivity from [35S]methioninein subcellular fractions of Culex pipiens abdominal tissues analyzed by sucrose density gradient centrifugation. Tissues were homogenized 6 hr after injection of [35S]methionine. Radioactivity was assayed on 5/al aliquots of each fraction.
562
R. FEYEREISENet al.
on the age of the mosquito, from 10 to 35 insects could be used for each separation. DISCUSSION The procedures described in this paper have been used extensively in this laboratory (Feyereisen and Farnsworth, 1985) and have been found superior to differential centrifugation for preparation of microsomes with respect to time and with respect to the definition of the subcellular fraction obtained. Using this procedure with D. punctata midgut tissue we routinely lose only 13~o of NADPH cytochrome c reductase activity in mitochondrial fractions, compared to 30% by various differential centrifugation techniques. Sedimentation of 'microsomal' activities at low g forces noted in the past (Wilkinson and Brattsten, 1972: Wilkinson, 1979) found a partial solution in the centrifugation of homogenates over a dense cushion of sucrose (1.6 M). In our hands that technique did not prove satisfactory because the fraction containing microsomes also contained the bulk of the soluble cellular constituents. This is often undesirable for work on microsomal enzymes, especially when soluble inhibitors of monooxygenase activities are present. Soluble proteolytic activity also compounds the problem for certain tissues, and some NADPH cytochrome c activity was always found tailing in lighter fractions (about 20'~oin Culex pipiens preparations, see also Feyereisen, 1983). The part of the gradient collected as 'microsomal" fraction is somewhat arbitrary, because it results from a compromise between greater recovery of microsomal activity and smaller contamination by soluble or mitochondrial elements. Our technique should therefore not bc used without prior verification, and the difference between the protocol for C. pipiens and D. punctata shows that conditions optimal for one insect or tissue are not necessarily (probably not) optimal for others. The profile of the gradients also show that one should not expect to be able to prepare 'pure' subcellular fractions, but only 'defined' fractions. This is often overlooked when using differential centrifugation, because the overlap of physicochemical properties between the various cellular organelles (which dictates their behavior in gradients) is not so obvious with that technique. For many applications the differential centrifugation technique may be adequate, in particular when its conditions are optimized (i.e. when the separation between mitochondrial and post-mitochondrial fractions is made with some justification). Differential centrifugation can be very fast (Brattsten and Gunderson, 1981), when a high-performance rotor is used. Sucrose density gradient centrifugation in a vertical rotor is limited to small quantities of biological material, and therefore is not suited to large-scale preparation of microsomes as is differential centrifugation. The technique described here is however well suited for the preparation of microsomes from very small tissue samples, because microsomes are obtained in suspended form in a known volume of a sucrose/buffer solution. The resuspension of microscopic or invisible pellets obtained by differential centrifugation inevitably leads to erratic and non-
measurable losses of microsomes. Obtaining microsomes in suspended form is also advantageous when many replicates are prepared simultaneously. Centrifugation of corpora allata homogenates from locusts on sucrose density gradients in a swinging bucket rotor led to microsomal preparations which could be used satisfactorily for a biochemical characterization of methyl farnesoate epoxidase (Feyereisen et al.. 1981). It is anticipated that the technique described in this paper will be useful in the study of insect endocrine gland enzymology. With the increased use of vertical rotors in modern molecular biology laboratories, it may be economical to use these instruments for more applications, and the preparation of subcellular organelles is but one such application. Acknowledgements --This work was supported b~r NIH grant AI 19192 to R. F. Oregon Agricultural Experiment Station Technical paper No. 7346. REFERENCE,N
Benke (3. M., Wilkinson ('. F. and Telford J. N. (1972)
Microsomal oxidases in a cockroach. Gromphadorhina portentosa. J. Econ. Entomol. 65, 1221 1229. Bradford M. M. 0976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254. Brattsten L. B. and Gunderson C. A. (1981) Isolation of insect microsoma[ oxidases by rapid centrifugation. Pest. Biochem. Physiol. 16, 187-198. Claude A. (1969) Microsomes, endoplasmic reticulum and interactions of cytoplasmic membranes. In Microsomes and Drug Oxidations. (Edited by Gillette J. R. et al.), pp. 3-39. Academic Press, New York. Feyereisen R. (1983) Polysubstrate monooxygenase (cytochrome P-450) in larvae of susceptible and resistant strains of house flies. Pest. Biochem. Physiol. 19, 262-269. Feyereisen R. and Durst F. (1978) Ecdysterone biosynthesis: a microsomal cytochrome P-450 linked ecdysonc 20-monooxygenasefrom tissues of the African migrator~ locust. Eur. J. Biochem. 88, 37 47. Feyereisen R. and Farnsworth D. E. (1985) Developmental changes of microsomal cytochrome P-450 monooxygenases in larval and adult Diploptera pum'tata, hlsec't Biochem. (in press.) Feyereisen R., Pratt G. E. and Hamnett A. F. (198[) Enzymic synthesis of juvenile hormone in locust corpora allata: evidence for a microsomal cytochrome P-450 linked methyl farnesoate epoxidase. Eur. J. Biochem. 118, 231.-238. Keeley L. L. (1973) Characterization of insect fat body mitochondria isolated by a rapid procedure. Comp. Biochem. Physiol. 46B, 147 151. Rickwood D. (1982) An assessment of vertical rotors. Analyt. Biochern. 122, 33.-40. Siekevitz P. (1963) Protoplasm: endoplasmic reticulum and microsomes and their properties. ,'Inn. Rer. Physiol. 25, 15-40.
Wilkinson C. F. (1979) The use of insect subcellular components for studying the metabolism of xenobiotics. ACS Symposium Series 97, 249..284. Wilkinson C. F. and Brattsten L B. (1972) Microsomal drug metabolizing enzymes in insects. Drug Metab. Rer. I, 153-228. Williams C. H. and Kamin H. (1952) Microsomal triphosphopyridine nucleotide cytochrome c reductase: isolation, characterization and kinetic studies. J. Biol. Chem. 237, 587-595.
0305-0491/85 $3.00 + 0.00 ~ 1985 Pergamon Press Ltd
A RAPID METHOD FOR PREPARING INSECT MICROSOMES R. FEYEREISEN*'['~,G. D. BALDRIDGE* and D. E. FARNSWORTH* *Department of Entomology and tDepartment of Agricultural Chemistry, Oregon State University, Corvallis, OR 97331, USA (Received 14 March 1985) AImraet--I. The 1000g supematant of a tissue homogenate is layered on top of a small (< 5 ml) sucrose density gradient and centrifuged for 20 rain at very high centrifugal forces in a vertical rotor. 2. Microsomes can be recovered rapidly in suspended form from the middle of the gradient, well separated from mitochondria and soluble (cytosolic) components. 3. Applications to cockroach midgut microsomes and mosquito abdominal tissue microsomes are described, and the method is compared to the classical differential centfifugation method. 4. Cytochrome P-450 monooxygenase activities can be measured on microsomes prepared from midgut tissue of 2-3 Diploptera punctata using this method.
INTRODUCTION Microsomes are the isolated form of the endoplasmic reticulum when this cytoplasmic constituent is obtained by centrifugation after homogenization of eukaryotic cells (Claude, 1969). Biochemical studies on microsomal enzymes require the preparation of a subcellular fraction which is reasonably enriched in microsomes and whose properties are representative of the organdie from which it derives. Preparation of microsomes from insect tissues is usually done by the technique of differential centrifugation (Wilkinson and Brattsten, 1972; Wilkinson, 1979) which was originally developed with mammalian liver cells (Siekevitz, 1963). Thus, the subcellular topology of many enzyme activities is often assigned solely on the basis of a rather arbitrary set of centrifugation conditions and only in a few cases have other criteria, such as 'marker' enzymes, electron microscopical examination, lipid, protein and R N A content, buoyant density, etc. been used to characterize insect microsomes. The study o f microsomal enzyme systems such as cytochrome P-450 monooxygenase has benefited greatly from the application of the classical differential centrifugation technique, but a number of studies have also documented some limitations of the technique (Wilkinson and Brattsten, 1972; Wilkinson, 1979). A great reduction in the time of preparation may be achieved by rapid centrifugation of the mitochondrial and post-mitochondrial fractions (Keeley, 1973; Brattsten and Gunderson, 1981). However, c o m m o n problems remain, such as the sedimentation of 'microsomal' oxidative enzyme activities at low centrifugal forces (Wilkinson and Brattsten, 1972; Wilkinson, 1979; Benke et al., 1972) or the ambiguity of subcellular localization of some cytochrome P-450 monooxygenases which may be mitochondriai or microsomal (Feyereisen and Durst, 1978). Attempts to solve those problems have in~/AII correspondence to: R. Feyereisen, Department of Entomology, Oregon State University, Corvallis. OR 97331, USA. 559
volved centrifugation on sucrose density steps or gradients (Benke et aL, 1972; Feyereisen and Durst, 1978). We wish to report here our efforts to combine speed of preparation with the use of sucrose density gradients, which have resulted in a technique that may prove particularly useful in invertebrate biochemistry, where the amount of biological material is often limited. MATERIALS AND METHODS Insects The viviparous cockroach Diploptera punctata (Orthoptera: Blaberidae) was reared at 27°C under a 12 hr light: 12 hr dark photoperiod and fed Wayne rodent blox and water ad libitum. An anautogenous strain of the mosquito Culex pipiens (Diptera: Culicidae) was maintained at 26~C under a 16 hr light: 8 hr dark photoperiod. Adults were fed a 10% sucrose solution ad libitum and live quail were used to provide blood meals. Enzyme assays NADPH cytochrome c reductase activity (Williams and Kamin, 1952) was measured spectrophotometrically at 30°C in an Aminco DW2a instrument. Reduction of cytochrome c was measured at 550 nm in the split beam mode in I ml cuvettes each containing 50/aM cytochrome c, 1 mM EDTA, 5raM NaCN and enzyme in 50mM MOPS (morpholinopropanesulfonate) buffer, pH 7.2. Reduction of cytochrome c was started in the sample cuvette by the addition of 50 #1 of an NADPH (final concentration in the assay: 150#M) regenerating system (10/zmol glucose 6-phosphate and 1.5 units glucose 6-phosphate dehydrogenase). Succinate cytochrome c reductase activity was measured by the same procedure, but 10#1 of a I M suecinate solution was used to start the reaction, in the cytochrome c oxidase assay, NaS2 O4-reduced cytochrome c was used as substrate, NaCN was deleted from the reaction mixture, and the reaction was started by the addition of the enzyme. Methoxyresorufin O-demethylation activity was assayed spectroltuorometrically at 30°C in a Perkin-Elmer 650-10S instrument set at 560 and 3 nm slit for excitation, and at 580 and 5 nm slit for emission. The reaction mixture (1 ml) contained enzyme, methoxyresorufin (40 # M, Molecular Probes, Junction City, Oregon, USA), 1 mM EDTA and an NADPH (250#M) regenerating system in 50 mM MOPS buffer, pH 7.2 (Feyereisen and Farnsworth, 1985).
560
R. FEYEREISENCI a/.
Protein was determined according to the Bradford (1976) assay using gamma-globulins (Calbiochem) as standard. Sucrose concentrations were determined by refractometry.
Tissue homogenates Midgut tissue from adult female Diploptera pum'tata v,as dissected in Yeager's cockroach saline, then transferred to 50 mM MOPS buffer, pH 7.2 containing lff!;, sucrose, I mM EDTA and 0.4 mM PMSF (phenylmethylsulfonylfluoride, freshly prepared). The tissue was ground in a motor-driven Teflon/glass homogenizer for 10sec and centrifuged at 1000g for 10min to remove cell debris and nuclei. Abdomens of adult female Culex pipiens were dissected in Lure's mosquito saline and midguts and ovaries were pulled out with forceps. The abdomens were transferred to 50mM MOPS buffer, pH 7.2 containing 0.4 mM PMSF and ground in a motor-driven Teflon/glass homogenizer for 10 sec, then centrifuged at 1000g for 10min. Centr([ugation All low-speed centrifugation (1000g) was done in 1.5 ml Eppendorf tubes in a Beckman Microfuge I I instrument at 5 C. Centrifugation of gradients was done in polyallomcr Quick-seal tubes using a VTi 80 rotor in a Lg-gO ultracentrifuge (all from Beckman) at 5 C. Lahelhng af subcelhdar ./?actions Fifty female mosquitoes (6 days after adult emergence) were injected with 7 x 10~cpm of[3SS]methionine. Six hours after injection, abdomens were removed and homogenized for 30sec in 250,ul MOPS buffer in a 2ml Teflon;glass homogenizer. The homogenate was centrifuged at 1000g for 10 min. The supernatant was loaded on a linear 45 to 1500 sucrose gradient and centrifuged at 65,000 rpm for 20 min in a Beckman VTi 80 rotor. Fifteen fractions of 350pl were collected from the gradient and radioactivity was assayed on 5 pl aliquots of each fraction. RESUI.TS
I. Rationale Separation of mitochondrial from microsomal fractions has been achieved by centrifugation on sucrose density gradients (Feyereisen and Durst, 1978; Feyereisen et al., 1979; Feyereisen, 1983) but the procedure is very long ( > 4 h r ) and therefore cannot be used for routine purposes. The reason for the length of the procedure is that gradients were centrifuged in swinging bucket rotors. The maximum speed is limited for these rotors, and their geometry (horizontal during the run) maximizes the distance particles have to travel in the gradient (length of the tube). Vertical rotors are very stable when spinning and can be designed for high speed operation. Their geometry (reoriented gradient during the run) minimizes the distance particles have to travel (diameter of the tube) (Rickwood, 1982). It was felt that centrifugation in a vertical rotor at high speed might drastically reduce the time of preparation of microsomes while at the same time preserving the advantages of gradient separation of mitochondria from microsomes.
2. Application to Diploptera punctata Midgut The 1000g supernatant of a midgut homogenate was subjected to various centrifugal conditions in a sucrose density gradient run in a vertical rotor for an arbitrarily set time of 20 min. At the end of the run, the time integral of the squared angular velocity (¢o"t in r a d 2 ' s e c ') which represents the cumulative cen-
5o I
-1
"6 25
....
/3
,~
>
/-/
i
.......
~
['l ,
co2t (1010 .rad 2 .sec -1 ) Fig. 1. Cross-contamination of microsomal and mitochondrial enzyme activities as a function of the cumulative centrifugal effect. (A --A)",, of total succinate tyrochrome c reductase activity found in microsomal fractions. (IS] .--U])",, of total NADPH cytochrome c reductase activity found in mitochondrial fractions. (© --©) total volume o f microsomal fractions. trifugal effect was recorded from the instrument's integrator. The centrifuge tube was punctured at the bottom with an 18 gauge needle and after puncturing the top of the tube, a total of 15 fractions (350pl) was collected. The fractions were routinely analyzed for sucrose concentration and marker enzyme activities, succinate cytochrome c reductasc and N A D P H cytochrome c reductasc tbr mitochondria and microsomes respectively. In certain experiments cylochrome c oxidase was also used as mitochondrial marker and aldrin epoxidation or methoxyresorufin O-demethylation as microsomal marker. We first tried linear, then discontinuous sucrose gradients, with three steps at 50. 35 and 25"i, sucrose. Centrifugation speeds ranged from 30,000 to 65,000 rpm. Two peaks of activity were more or less well resolved, a heavy, mitochondrial peak and a lighter microsomal peak. It was found that there was always a slight cross-contamination of microsomal and mitochondrial enzyme activities, and that the total volume of the gradient occupied by microsomal fractions was dependent on thc speed of centrifugation. Figure 1 shows the cross-contamination and microsomal volume as a function of the cumulative centrifugal effect obtained in five different experiments. The amount of N A D P H cytochrome c reductase activity found in the mitochondrial peak was relatively constant around 15'!.. In contrast, the amount of succinate cytochrome c reductase (mitochondrial) activity tbund in the microsomal peak was increased at the higher ¢o2t values. The total volume of the gradient occupied by microsomal fractions was minimal at a ,,-'t value of 2.35 × I0 'n rad 2 s e c ' . Because mitochondrial contamination of the microsomal fractions was small, those conditions were chosen as optimal. Figure 2 shows the profile of the gradient obtained under those conditions. It appears that the steps in the sucrose gradient have been flattened, and that mitochondrial enzyme activities were focussed at the 50/35'~.,', interface, whereas the microsomal enzyme activities were focussed at the 35/2501; interface. The close coincidence of the two marker enzyme activities for each fraction testifies to their usefulness. The majority of the protein (presumably of cytosolic origin) was found in the top three fractions. These experiments led us to adopt the following procedure for the preparation of D. punctata midgut
Rapid preparation of insect microsomes
561 50
T ",.~.
""I"'. 25 ~,~,
•25 ~
I
I
5
I0
. . . . . . . . . . . .5 W)
15
Fraction number
Fig. 2. Separation of Diploptera punctata midgut enzymes on a sucrose density gradient. (A A) succinate cytochrome c reductase activity; (A A) cytochrome c oxidase activity; (© (3) NADPH cytochrome c reductase activity; (Q O) methoxyresorufin O-demethylation activity; (©---©)% sucrose. The 1000g supernatant of a midgut homogenate was loaded on a discontinuous (50, 35 and 25%) sucrose density gradient and centrifuged for 20min at 50,000rpm in a Beckman VTi 80 rotor (to2t = 2.35 × 101°). microsomes: 350/~1 o f a 1000g supernatant of midgut homogenate (the equivalent of 2-4 midguts or up to 2 mg protein) was layered on top of a sucrose gradient which consisted of three layers: 800/~1 of 50%, 2100 td of 35% and 2000/~1 of 25% sucrose in 50mM MOPS buffer, pH 7.2 containing i mM EDTA. The gradient was centrifuged at 50,000 rpm for 20 min (to2t = 2.35 x 10~° rad 2 sec-~). The bottom 2.5ml of the tube were discarded as 'mitochondriar and the next 1.5 ml were collected as 'microsomal' fraction. This fraction had a sucrose concentration of 29% (I. 122 g/cm3). 3. Application to Culex pipiens abdominal tissue A similar approach was used to determine optimal conditions for the preparation of microsomes from abdominal tissues of the mosquito. Linear gradients of sucrose (45-15% in MOPS buffer) were prepared by adding equal volumes (1.25 ml) of 45, 35, 25 and 15% sucrose solutions to a centrifuge tube and placing the tube in the horizontal position for two hours prior to the experiment. Various centrifugation conditions from 30,000 to 75,000 rpm were tried, with a constant 20min centrifugation time (results not shown). The best resolution of mitochondria and microsomes was obtained with a centrifugation at 65,000 rpm for 20 min. Figure 3 shows the separation of succinate cytochrome c reductase and NADPH cytochrome c reductase activity under these conditions. The enzyme activities were much lower than those observed with the cockroach tissue, but we found that only a limited amount of homogenate supernatant could be loaded onto the gradient. Indeed, when total protein loaded exceeded approx. I mg, the separation of the two peaks was not satisfactory, i.e. the mitochondrial peak broadened considerably into the microsomal peak. It was also found that the initial 1000g centrifugation was essential to remove most lipids (floating on top after centrifugation) which, when loaded on the gradient caused erratic results. Presumably, the presence of
] 15
FraelIwm numb4m'
Fig. 3. Separation of Culex pipiens abdominal tissue enzymes on a sucrose density gradient. (A A) succinate cytochrome c reductase activity; ( 0 O) NADPH cytochrome c reductase activity; ( 0 - - - 0 ) % sucrose. The 1000g supernatant of a tissue homogenate was loaded on a linear 15 to 45% sucrose density gradient and centrifuged for 20min at 65,000rpm in a Beckman VTi 80 rotor (to2t = 3.70 x 101°). low-density lipids prevented the smooth reorientation of the gradient at the end of the run. When abdomens were prepared 6 hr following injection of [35S]methionine, and the 1000g supernatant of the abdomen homogenate was analyzed by sucrose gradient centrifugation, the radioactivity profile shown in Fig. 4 was obtained. Three peaks were observed, the lower peak coincided with the succinate cytochrome c reductase peak, and the middle peak coincided with the NADPH cytochrome c reductase peak (see Fig. 3). A very large amount of radioactivity was found at the top of the gradient. This profile confirms that a good resolution of mitochondrial, microsomal and cytosolic proteins can be achieved by this procedure. These experiments led us to adopt the following procedure for the preparation of microsomes from abdominal tissues of C. pipiens: 350~1 (up to I mg protein) ofa 1000g supernatant of tissue homogenate was layered on a linear 45 to 15~ sucrose gradient, and centrifuged at 65,000 rpm for 20 min (w2t = 3.70 x l0 ~°rad 2 sec ~). The bottom 2.0 ml of the tube were discarded as 'mitochondrial' and the next 2. l ml were collected as 'microsomal' fraction. Depending I0-
-50
~-
-25
9
i
I 5
Frll~l~l
I0
15
Fig. 4. Distribution of radioactivity from [35S]methioninein subcellular fractions of Culex pipiens abdominal tissues analyzed by sucrose density gradient centrifugation. Tissues were homogenized 6 hr after injection of [35S]methionine. Radioactivity was assayed on 5/al aliquots of each fraction.
562
R. FEYEREISENet al.
on the age of the mosquito, from 10 to 35 insects could be used for each separation. DISCUSSION The procedures described in this paper have been used extensively in this laboratory (Feyereisen and Farnsworth, 1985) and have been found superior to differential centrifugation for preparation of microsomes with respect to time and with respect to the definition of the subcellular fraction obtained. Using this procedure with D. punctata midgut tissue we routinely lose only 13~o of NADPH cytochrome c reductase activity in mitochondrial fractions, compared to 30% by various differential centrifugation techniques. Sedimentation of 'microsomal' activities at low g forces noted in the past (Wilkinson and Brattsten, 1972: Wilkinson, 1979) found a partial solution in the centrifugation of homogenates over a dense cushion of sucrose (1.6 M). In our hands that technique did not prove satisfactory because the fraction containing microsomes also contained the bulk of the soluble cellular constituents. This is often undesirable for work on microsomal enzymes, especially when soluble inhibitors of monooxygenase activities are present. Soluble proteolytic activity also compounds the problem for certain tissues, and some NADPH cytochrome c activity was always found tailing in lighter fractions (about 20'~oin Culex pipiens preparations, see also Feyereisen, 1983). The part of the gradient collected as 'microsomal" fraction is somewhat arbitrary, because it results from a compromise between greater recovery of microsomal activity and smaller contamination by soluble or mitochondrial elements. Our technique should therefore not bc used without prior verification, and the difference between the protocol for C. pipiens and D. punctata shows that conditions optimal for one insect or tissue are not necessarily (probably not) optimal for others. The profile of the gradients also show that one should not expect to be able to prepare 'pure' subcellular fractions, but only 'defined' fractions. This is often overlooked when using differential centrifugation, because the overlap of physicochemical properties between the various cellular organelles (which dictates their behavior in gradients) is not so obvious with that technique. For many applications the differential centrifugation technique may be adequate, in particular when its conditions are optimized (i.e. when the separation between mitochondrial and post-mitochondrial fractions is made with some justification). Differential centrifugation can be very fast (Brattsten and Gunderson, 1981), when a high-performance rotor is used. Sucrose density gradient centrifugation in a vertical rotor is limited to small quantities of biological material, and therefore is not suited to large-scale preparation of microsomes as is differential centrifugation. The technique described here is however well suited for the preparation of microsomes from very small tissue samples, because microsomes are obtained in suspended form in a known volume of a sucrose/buffer solution. The resuspension of microscopic or invisible pellets obtained by differential centrifugation inevitably leads to erratic and non-
measurable losses of microsomes. Obtaining microsomes in suspended form is also advantageous when many replicates are prepared simultaneously. Centrifugation of corpora allata homogenates from locusts on sucrose density gradients in a swinging bucket rotor led to microsomal preparations which could be used satisfactorily for a biochemical characterization of methyl farnesoate epoxidase (Feyereisen et al.. 1981). It is anticipated that the technique described in this paper will be useful in the study of insect endocrine gland enzymology. With the increased use of vertical rotors in modern molecular biology laboratories, it may be economical to use these instruments for more applications, and the preparation of subcellular organelles is but one such application. Acknowledgements --This work was supported b~r NIH grant AI 19192 to R. F. Oregon Agricultural Experiment Station Technical paper No. 7346. REFERENCE,N
Benke (3. M., Wilkinson ('. F. and Telford J. N. (1972)
Microsomal oxidases in a cockroach. Gromphadorhina portentosa. J. Econ. Entomol. 65, 1221 1229. Bradford M. M. 0976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248 254. Brattsten L. B. and Gunderson C. A. (1981) Isolation of insect microsoma[ oxidases by rapid centrifugation. Pest. Biochem. Physiol. 16, 187-198. Claude A. (1969) Microsomes, endoplasmic reticulum and interactions of cytoplasmic membranes. In Microsomes and Drug Oxidations. (Edited by Gillette J. R. et al.), pp. 3-39. Academic Press, New York. Feyereisen R. (1983) Polysubstrate monooxygenase (cytochrome P-450) in larvae of susceptible and resistant strains of house flies. Pest. Biochem. Physiol. 19, 262-269. Feyereisen R. and Durst F. (1978) Ecdysterone biosynthesis: a microsomal cytochrome P-450 linked ecdysonc 20-monooxygenasefrom tissues of the African migrator~ locust. Eur. J. Biochem. 88, 37 47. Feyereisen R. and Farnsworth D. E. (1985) Developmental changes of microsomal cytochrome P-450 monooxygenases in larval and adult Diploptera pum'tata, hlsec't Biochem. (in press.) Feyereisen R., Pratt G. E. and Hamnett A. F. (198[) Enzymic synthesis of juvenile hormone in locust corpora allata: evidence for a microsomal cytochrome P-450 linked methyl farnesoate epoxidase. Eur. J. Biochem. 118, 231.-238. Keeley L. L. (1973) Characterization of insect fat body mitochondria isolated by a rapid procedure. Comp. Biochem. Physiol. 46B, 147 151. Rickwood D. (1982) An assessment of vertical rotors. Analyt. Biochern. 122, 33.-40. Siekevitz P. (1963) Protoplasm: endoplasmic reticulum and microsomes and their properties. ,'Inn. Rer. Physiol. 25, 15-40.
Wilkinson C. F. (1979) The use of insect subcellular components for studying the metabolism of xenobiotics. ACS Symposium Series 97, 249..284. Wilkinson C. F. and Brattsten L B. (1972) Microsomal drug metabolizing enzymes in insects. Drug Metab. Rer. I, 153-228. Williams C. H. and Kamin H. (1952) Microsomal triphosphopyridine nucleotide cytochrome c reductase: isolation, characterization and kinetic studies. J. Biol. Chem. 237, 587-595.