Neurochemical Reasearch (4) 25-35 (1979)
M A C R O M O L E C U L A R SYNTHESIS IN MITOCHONDRIA ISOLATED FROM DIFFERENT REGIONS OF DEVELOPING RAT BRAIN MARIA N. GADALETA, ~ ANNA M. GIUFFRIDA, 2 M A R C E L L A R E N I S , a I D A SERRA, 2 G I O V A N N A D E L P R E T E , t E R N E S T O G E R E M I A , 2 A N D C E C I L I A SACCONE 1 Institutes of Biological Chemistry, Universities oft~JBari and ~Catania, and aMental Hospital of Bisceglie, Italy
Accepted June 2, 1978
DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-oldrats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.
INTRODUCTION The essential role of mitochondria as energy suppliers during e m b r y o g e nesis is largely recognized, whereas v e r y little information about mitochondrial biogenesis and/or differentiation during early or late stages of animal d e v e l o p m e n t is so far available (1, 2). Indeed, only recently have the main characteristics of the mitochondrial genetic s y s t e m b e e n clarified, thus making a m o r e detailed study of these problems at a molecular level possible. It is now well k n o w n that the products of the mitochondrial g e n o m e are the two ribosomal R N A species, about 20 different t R N A species, and a limited n u m b e r o f proteins that are part o f the inner mitochondrial
25 0364-3190/79/0200-0025503.00/0 9 1979Plenum PublishingCorporation
26
GADALETA ET AL.
membrane (3). Furthermore, it is known that all the remaining mitochondrial proteins are coded for by nuclear DNA, translated at the level of cytoplasmic ribosomes, and then imported into the mitochondrion. It is therefore clear that the interrelationships between the mitochondrial and the nucleocytoplasmic systems are very important for the regulation of growth and the division of the mitochondria (biogenesis) and their final size, structure, and enzyme composition (differentiation) as well. A suitable model for studying the biogenesis and/or the differentiation of mitochondria during late organogenesis is the developing rat brain. Indeed, at birth the rat brain is in a very immature state, acquiring during the first three weeks of life approximately 50% and 97% of the final number of cells in, respectively, the forebrain and the cerebellum (4). Furthermore, in this period of life prenatally and postnatally formed cells increase in size and differentiate; they form dendritic arborization and synaptic connections, and begin to show electrical activity and higher oxygen requirement. Until now the data available on macromolecular mitochondrial metabolism referred only to adult animals (5-8). A study on extramitochondrial DNA and RNA synthesis in cells from different brain regions during postnatal development has already been reported by some of us (9, 10). The aim of the present research was to study macromolecular synthesis in brain mitochondria isolated from 10- and 30day-old rats. Experiments were performed using three different brain areas, namely cerebral hemispheres, cerebellum, and brain stem. Isolated mitochondria were used as an in vitro system, because with purified organelles it is possible to measure the level of enzyme activities without interference from the endogenous pool of substrates; moreover, their ability to reproduce processes occurring in vivo with fidelity, in shortterm experiments, has been repeatedly demonstrated (11).
EXPERIMENTAL PROCEDURE Animals, Wistar rats, 10 and 30 days old, bred in the institute under standard conditions were used. The number of animals in each experiment ranged from twenty 30-day-old animals to forty 10-day-old animals. Rats were killed by decapitation. All further operations were carried out at 4~ using sterile solutions and glassware. The brains were rapidly removed and dissected into three regions: cerebral hemispheres, cerebellum, and brain stem (the last was dissected excluding the medulla oblongata). Isolation of mitochondria. Free (nonsynaptic) brain mitochondria were prepared according to Goldberg (12) with some modifications. The different regions were minced, washed repeatedly, and homogenized manually 1:20 (w/v) in 0.32 M sucrose + 1 mM EDTA + 10raM Tris HCI, pH 7.4, with 10 up-and-down strokes in a Thomas Teflon-glass homogenizer. The homogenate was centrifuged at 1000 g for 15 min. The nuclear pellet was washed once, and the combined supernatants were centrifuged at 1500 g for 15 min. The precipitate
MITOCHONDRIAL MACROMOLECULAR SYNTHESIS IN BRAIN
27
was discarded, and the supernatant was centrifuged at 12,000 g for 30 rain to yield the crude mitochondrial pellet containing myelin and synaptosomes. Purification of Mitochondria. After careful removal of the fluffy layer, the mitochondrial pellet was resuspended with 0.32 M sucrose containing 1 mM EDTA and 10 mM Tris HC1, pH 7.4 (medium A), 1 mug of fresh tissue. A 2- to 3-ml portion of this suspension was layered onto 24 ml of a discontinuous gradient of Ficoll (5%, 8%, 10%, 12%) in medium A and centrifuged in a Spinco ultracentrifuge at 65,000 g for 1 hr with an SW 27 rotor. The pellet was washed once, centrifuged at 15,000 g for 15 rain, and resuspended in medium A. Enzyme Assays. All assays were carried out 1 hr after mitochondrial preparation. All solutions were freshly prepared or kept frozen until use. The incubation medium for DNA polymerase activity (total volume of 0.5 ml) was 50 mM Tris HC1 buffer, pH 8.0; 4 mM KC1; 7 mM MgCI~; 20 mM NaH2PO4; 0.6 mM ATP; 20 mM Na succinate; 15/zM each of dGTP, dCTP, and dATP; and 4 / z M [3H]TTP (specific activity 1 mCi//zmoI). Incubation was carried out at 37~ for 10 rain. The reaction was started by adding 0.75 mg of mitochondrial protein and stopped by adding 4 ml of ice-cold 5% (w/v) trichloroacetic acid. The precipitate was collected on membrane filters, washed seven times with 7 ml of icecold 5% trichloroacetic acid, and dried. The acid-insoluble radioactivity was solubilized by RNAse-free DNAse treatment. The incubation medium for RNA polymerase (total volume of 0.1 m!) was 3 mM MgC12; 60 mM KCI; 100 mM Tris HCI, pH 7.4; 3 mM MnC12; 0.l mM each of CTP, GTP, and ATP; 4/zg pyruvate kinase; 4 rnM phosphoenolpyruvate; and 50/zM [aH]UTP (specific activity 1 mCi//zmol). Incubation was carried out at 30~ for 15 rain. The reaction was started by adding 0.15 mg mitochondrial protein and stopped by adding 5 ml ice-cold trichloroacetic acid (5% w/v) containing 30% saturated pyrophosphate solution. The precipitate was collected on filters, washed seven times with 7 ml ice-cold 5% trichloroacetic acid, and dried. Acid-insoluble radioactivity was solubilized by alkali and RNAse treatment. The incubation medium for the protein synthesis (total volume of 0.1 ml) was 100 mM Tris HC1, pH 7.4; 20 mM KC1; 6 mM MgC12; 1.5 mM EDTA; 15 mM potassium phosphate buffer; 30 mM NH4C1; 2 mM ADP; 30 mM Na succinate; 50/xg/ml of a synthetic amino acid mixture, minus leucine (13); and 45 or 135/zM[14C]leucine (specific activity 125 t~Ci/ p.mol). Incubation was carried out at 37~ for 60 rain. The reaction was started by adding 0.2 mg mitochondrial protein, stopped by adding 5 ml ice-cold 5% (w/v) triehloroaeetic acid. After trichloroacetic acid addition, samples were kept at 70~ for 15 rain. The hot acid-insoluble radioactive material was collected on membrane filters, washed seven times with 7 ml ice-cold 5% trichloroacetic acid, and dried. Radioactivity was counted in a solution of toluene containing PPO (5 rag/liter) and POPOP (0.3 rag/liter) in a Packard Tri-Carb Scintillator. When "inhibitors were used, the various drugs were dissolved in redistilled water and used at the final concentrations reported in Table I. The proteins were measured according to Waddel (14), with serum albumin as standard.
RESULTS
Properties of the Incorporation Systems. D N A , R N A , a n d p r o t e i n synthesis in brain mitochondria was studied following the incorporation of the labeled precursors into acid-precipitable material as reported in Experimental Procedure.
28
GADALETA ET AL.
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M A C R O M O L E C U L A R SYNTHESIS IN MITOCHONDRIA ISOLATED FROM DIFFERENT REGIONS OF DEVELOPING RAT BRAIN MARIA N. GADALETA, ~ ANNA M. GIUFFRIDA, 2 M A R C E L L A R E N I S , a I D A SERRA, 2 G I O V A N N A D E L P R E T E , t E R N E S T O G E R E M I A , 2 A N D C E C I L I A SACCONE 1 Institutes of Biological Chemistry, Universities oft~JBari and ~Catania, and aMental Hospital of Bisceglie, Italy
Accepted June 2, 1978
DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-oldrats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.
INTRODUCTION The essential role of mitochondria as energy suppliers during e m b r y o g e nesis is largely recognized, whereas v e r y little information about mitochondrial biogenesis and/or differentiation during early or late stages of animal d e v e l o p m e n t is so far available (1, 2). Indeed, only recently have the main characteristics of the mitochondrial genetic s y s t e m b e e n clarified, thus making a m o r e detailed study of these problems at a molecular level possible. It is now well k n o w n that the products of the mitochondrial g e n o m e are the two ribosomal R N A species, about 20 different t R N A species, and a limited n u m b e r o f proteins that are part o f the inner mitochondrial
25 0364-3190/79/0200-0025503.00/0 9 1979Plenum PublishingCorporation
26
GADALETA ET AL.
membrane (3). Furthermore, it is known that all the remaining mitochondrial proteins are coded for by nuclear DNA, translated at the level of cytoplasmic ribosomes, and then imported into the mitochondrion. It is therefore clear that the interrelationships between the mitochondrial and the nucleocytoplasmic systems are very important for the regulation of growth and the division of the mitochondria (biogenesis) and their final size, structure, and enzyme composition (differentiation) as well. A suitable model for studying the biogenesis and/or the differentiation of mitochondria during late organogenesis is the developing rat brain. Indeed, at birth the rat brain is in a very immature state, acquiring during the first three weeks of life approximately 50% and 97% of the final number of cells in, respectively, the forebrain and the cerebellum (4). Furthermore, in this period of life prenatally and postnatally formed cells increase in size and differentiate; they form dendritic arborization and synaptic connections, and begin to show electrical activity and higher oxygen requirement. Until now the data available on macromolecular mitochondrial metabolism referred only to adult animals (5-8). A study on extramitochondrial DNA and RNA synthesis in cells from different brain regions during postnatal development has already been reported by some of us (9, 10). The aim of the present research was to study macromolecular synthesis in brain mitochondria isolated from 10- and 30day-old rats. Experiments were performed using three different brain areas, namely cerebral hemispheres, cerebellum, and brain stem. Isolated mitochondria were used as an in vitro system, because with purified organelles it is possible to measure the level of enzyme activities without interference from the endogenous pool of substrates; moreover, their ability to reproduce processes occurring in vivo with fidelity, in shortterm experiments, has been repeatedly demonstrated (11).
EXPERIMENTAL PROCEDURE Animals, Wistar rats, 10 and 30 days old, bred in the institute under standard conditions were used. The number of animals in each experiment ranged from twenty 30-day-old animals to forty 10-day-old animals. Rats were killed by decapitation. All further operations were carried out at 4~ using sterile solutions and glassware. The brains were rapidly removed and dissected into three regions: cerebral hemispheres, cerebellum, and brain stem (the last was dissected excluding the medulla oblongata). Isolation of mitochondria. Free (nonsynaptic) brain mitochondria were prepared according to Goldberg (12) with some modifications. The different regions were minced, washed repeatedly, and homogenized manually 1:20 (w/v) in 0.32 M sucrose + 1 mM EDTA + 10raM Tris HCI, pH 7.4, with 10 up-and-down strokes in a Thomas Teflon-glass homogenizer. The homogenate was centrifuged at 1000 g for 15 min. The nuclear pellet was washed once, and the combined supernatants were centrifuged at 1500 g for 15 min. The precipitate
MITOCHONDRIAL MACROMOLECULAR SYNTHESIS IN BRAIN
27
was discarded, and the supernatant was centrifuged at 12,000 g for 30 rain to yield the crude mitochondrial pellet containing myelin and synaptosomes. Purification of Mitochondria. After careful removal of the fluffy layer, the mitochondrial pellet was resuspended with 0.32 M sucrose containing 1 mM EDTA and 10 mM Tris HC1, pH 7.4 (medium A), 1 mug of fresh tissue. A 2- to 3-ml portion of this suspension was layered onto 24 ml of a discontinuous gradient of Ficoll (5%, 8%, 10%, 12%) in medium A and centrifuged in a Spinco ultracentrifuge at 65,000 g for 1 hr with an SW 27 rotor. The pellet was washed once, centrifuged at 15,000 g for 15 rain, and resuspended in medium A. Enzyme Assays. All assays were carried out 1 hr after mitochondrial preparation. All solutions were freshly prepared or kept frozen until use. The incubation medium for DNA polymerase activity (total volume of 0.5 ml) was 50 mM Tris HC1 buffer, pH 8.0; 4 mM KC1; 7 mM MgCI~; 20 mM NaH2PO4; 0.6 mM ATP; 20 mM Na succinate; 15/zM each of dGTP, dCTP, and dATP; and 4 / z M [3H]TTP (specific activity 1 mCi//zmoI). Incubation was carried out at 37~ for 10 rain. The reaction was started by adding 0.75 mg of mitochondrial protein and stopped by adding 4 ml of ice-cold 5% (w/v) trichloroacetic acid. The precipitate was collected on membrane filters, washed seven times with 7 ml of icecold 5% trichloroacetic acid, and dried. The acid-insoluble radioactivity was solubilized by RNAse-free DNAse treatment. The incubation medium for RNA polymerase (total volume of 0.1 m!) was 3 mM MgC12; 60 mM KCI; 100 mM Tris HCI, pH 7.4; 3 mM MnC12; 0.l mM each of CTP, GTP, and ATP; 4/zg pyruvate kinase; 4 rnM phosphoenolpyruvate; and 50/zM [aH]UTP (specific activity 1 mCi//zmol). Incubation was carried out at 30~ for 15 rain. The reaction was started by adding 0.15 mg mitochondrial protein and stopped by adding 5 ml ice-cold trichloroacetic acid (5% w/v) containing 30% saturated pyrophosphate solution. The precipitate was collected on filters, washed seven times with 7 ml ice-cold 5% trichloroacetic acid, and dried. Acid-insoluble radioactivity was solubilized by alkali and RNAse treatment. The incubation medium for the protein synthesis (total volume of 0.1 ml) was 100 mM Tris HC1, pH 7.4; 20 mM KC1; 6 mM MgC12; 1.5 mM EDTA; 15 mM potassium phosphate buffer; 30 mM NH4C1; 2 mM ADP; 30 mM Na succinate; 50/xg/ml of a synthetic amino acid mixture, minus leucine (13); and 45 or 135/zM[14C]leucine (specific activity 125 t~Ci/ p.mol). Incubation was carried out at 37~ for 60 rain. The reaction was started by adding 0.2 mg mitochondrial protein, stopped by adding 5 ml ice-cold 5% (w/v) triehloroaeetic acid. After trichloroacetic acid addition, samples were kept at 70~ for 15 rain. The hot acid-insoluble radioactive material was collected on membrane filters, washed seven times with 7 ml ice-cold 5% trichloroacetic acid, and dried. Radioactivity was counted in a solution of toluene containing PPO (5 rag/liter) and POPOP (0.3 rag/liter) in a Packard Tri-Carb Scintillator. When "inhibitors were used, the various drugs were dissolved in redistilled water and used at the final concentrations reported in Table I. The proteins were measured according to Waddel (14), with serum albumin as standard.
RESULTS
Properties of the Incorporation Systems. D N A , R N A , a n d p r o t e i n synthesis in brain mitochondria was studied following the incorporation of the labeled precursors into acid-precipitable material as reported in Experimental Procedure.
28
GADALETA ET AL.
~
~
OX
O
-6
Z~ O [-
o e~ o o
t~
