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'5'"; I,’ v'iirIl; g'r'rrll’” r "a or: 1 if}! j ”I, .ILIBRA K Y .2 Michigan Sta rt; .. l Univ-1:313; ,4. 1 p ‘.__. 5'3" ~ xv FTrflA V . q “3:; 91’ - ._‘. w This is to certify that the thesis entitled BIOCHEMICAL STUDIES ON THE MEMBRANES OF PEROXISOMES AND GLYOXYSOMES presented by Robert Paul Donaldson has been accepted towards fulfillment of the requirements for 77470 dggreein Ble/IFHH-El 7‘] "27. 5 731m Major professor Date W 6 0-7 639 ABSTRACT BIOCHEMICAL STUDIES ON THE MEMBRANES OF PEROXISOMES AND GLYOXYSOMES By Robert Paul Donaldson Subcellular organelles from Spinach leaves, ' castor bean endosperm, sunflower cotyledons, rat liver and dog kidney were separated using sucrose density grad- ient centrifugation. Microbodies (peroxisomes or glyoxy- somes) were obtained at densities ranging from 1.24 to 1.27 g x cm-3. Etioplasts were found at 1.21 to 1.22, mitochondria at 1.18 to 1.22, chloroplasts at 1.16 to 1.22, microsomes at 1.14 to 1.17, and lysosomes at 1.12 to 1.14 g x cm'3. In each instance there was a peak of NADH- cytochrome c reductase coincident with the peak activity of the microbody marker, catalase. The NADH-cytochrome c reductase of the plant microbodies was similar to the microsomal enzyme from the same tissue, since it was not inhibited by antimycin A and had a similar pH Optimum. The plant mitochondria had an NADH-cytochrome c reductase which was inhibited by antimycin A and had a lower pH Optimum than the enzyme in the microbodies and microsomes. Robert Paul Donaldson Rat liver and dog kidney peroxisomes also had an antimycin A insensitive NADH-cytochrome c reductase. Further studies on the rat liver peroxisomes indicated that the NADH-cytochrome c reductase was a component of the limiting membrane. The phospholipid compositions of microbodies from rat liver and castor bean endosperm were found to be very similar to the microsomes. The most abundant phopho- lipids were phosphatidyl choline, phOSphatidyl ethanol- amine and phosphatidyl inositol. The presence of the microsomal type NADH-cyto- chrome c reductase in the microbody membrane and the similarities in phosPholipid composition suggest that the microbody membrane is derived from the endoplasmic reticulum. BIOCHEMICAL STUDIES ON THE MEMBRANES OF PEROXISOMES AND GLYOXYSOMES BY Robert Paul Donaldson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1971 ACKNOWLEDGMENTS I am very much indebted to Professor N. E. Tolbert who gave me a place in his laboratory after I had failed a preliminary exam. Drs. Steve Aust and L. L. Bieber were especially helpful in my research. Dwayne Rehfeld and Claus Schnarrenberger helped in the assays of some of the sucrose density gradients. I thank Angelika Oeser and Sandy Wardell for excellent technical assistance. ii TABLE OF CONTENTS LIST OF TABLES. LIST OF FIGURES LIST OF ABBREVIATIONS Chapter I. II. Introduction -- Subcellular Organelles A. EQWNUO Microbodies, Peroxisomes, and Glyoxysomes. . l. Morphology . . 2. Biochemical Observations . Endoplasmic Reticulum and Microsomes 1. Morphology . 2. Biochemical Observations Mitochondria . Chloroplasts and Etioplasts. The Golgi. Lysosomes. . . Storage Particles. Nuclear and Plasma Membranes Methods and Materials. A. B. Isolation of Organelles. 1. Spinach. . . 2. Castor Bean Endosperm and Sunflower Cotyledons 3. Animal Tissues Enzyme Assays. l. Catalase . 2. Glycolate Oxidase. 3. Urate Oxidase. 4. Aspartate Aminotransferase iii Page vi .viii 17 17 17 19 19 21 22 22 22 22 Chapter 5. 6 7. 8. 9. 10. 11. 12. 13. 14. 15. Serine-Pyruvate Aminotransferase Glutamate-glyoxylate Aminotrans- ferase . NADP- Isocitrate Dehydrogenase. Malate Dehydrogenase Cytochrome c Oxidase NADPH- -Diaphorase . Triose phosphate isomerase NADH- -cytochrome c Reductase. NADPH-cytochrome c Reductase . NADH- -cytochrome b5 Reductase Phosphatases . C. Chlorophyll and Protein Assays D. Lipid Determinations . . l. Lipid extraction . . 2. Thin Layer Chromatography. 3. Lipid Identification . . 4. Lipid Phosphate Determination. 111. Results -- Tissues Fractionation. A. The Isolation of Spinach Leaf Peroxisomes. . 1. Differential Centrifugation. 2. Grinding Procedures. . . 3. Density Gradient Flotation . 4. Breakage of Spinach Peroxisomes B. Zonal Sucrose Density Gradient Centrifugation . 1. Rat liver. . 2. Castor Bean Endosperm. 3. Spinach. IV. Results -- Membrane Bound Enzymes A. NADH- -Cytochrome c Reductase. 1 Spinach. . . 2. Sunflower Cotyledons 3. Castor Bean Endosperm. 4. Rat Liver. 5. Dog Kidney B. Other Microsomal Enzymes C. Subfractionation . iv Page 23 23 23 23 23 24 24 24 25 25 26 26 26 26 27 28 29 31 32 32 35 4O 41 42 42 43 45 52 52 52 56 6O 69 79 83 86 Chapter Page V. Results -- Phospholipid compositon of Microbodies . . . . . . . . . . . . . . . . 89 A. Rat Liver. . . . . . . . . . . . . . . 89 B. Castor Bean EndosPerm. . . . . . . C. Spinach. . . . . . . . . . . . . . . . 91 VI. Discussion. . . . . . . . . . . . . . . . . 93 A. NADH-cytochrome c Reductase in Peroxisomes and Glyoxysomes. . . . . . 95 B. Plant Microsomes and Endoplasmic Reticulum. . . . . . 97 C. The Relationship of the Microbody Membrane to the Endoplasmic Reticulum. . . . . . . . . . . . . . . 99 BIBLIOGRAPHY. . . . . . . . . . . . . . . . . . . . 103 Table II. III. IV. VI. VII. VIII. IX. LIST OF TABLES Specific activities of marker enzymes in peroxisomes from spinach leaves homog- enized in two different media. Specific activities of marker enzymes in castor bean endosperm fractions from a zonal sucrose density gradient Densities of subcellular organelles from various tissues in sucrose density gradients. Specific activities of marker enzymes in Spinach leaf fractions Specific activities of marker enzymes in organelle fractions from sunflower cotyledons Specific activities of marker enzymes in organelle fractions from castor bean endosperm. Specific activities of marker enzymes in subcellular fractions from rat liver The effects of detergents on the NADH- cytochrome c reductase and oxidase activities of rat liver peroxisomes. Specific activities of marker enzymes in subcellular fractions from dog kidney. Specific activities of microsomal enzymes in subcellular fractions from rat liver, dog kidney, and castor bean. vi Page 39 44 50 51 59 66 73 75 82 84 Table XI. XII. XIII. Subfractionation of spinach leaf peroxisomes. Phospholipid composition of rat liver peroxisomes, mitochondria and microsomes Phospholipid composition of glyoxysomes, mitochondria and microsomes from castor bean endOSperm . vii Page 87 9O 92 Figure 10. LIST OF FIGURES Differential centrifugation of spinach homogenate (10 min). Isolation of spinach leaf organelles from different grinding media . Distribution of subcellular organelles from spinach leaves on a sucrose density gradient PH optima of the NADH-cytochrome c reductase in spinach peroxisomes and mitochondria. Distribution of subcellular organelles from sunflower cotyledons on a sucrose density gradient ' PH optima for microbody and mitochondrial NADH-cytochrome c reductases from sun- flower cotyledons. Distribution of subcellular organelles from castor bean endOSperm on a sucrose density gradient . PH optima for glyoxysomal, mitochondrial, and microsomal NADH-cytochrome c reductase from castor bean endosperm . Distribution of subcellular organelles from rat liver on a zonal sucrose density gradient Isolation of rat liver peroxisomal membranes on a sucrose density gradient. viii Page 33 38 48 55 58 62 64 68 71 78 Figure Page 11. Distribution of subcellular organelles from dog kidney on a zonal sucrose density gradient . . . . . . . . . . . . . . . . . 81 12. Suggested geneology of some subcellular components . . . . . . . . . . . . . . . . 101 ix ATP EDTA ER DNA NAD(H) NADP (H) Tricine Tris UDP v/v w/v w/w LIST OF ABBREVIATIONS adenosine-S’-triphosphate ethylenediamine tetraacetate endoplasmic reticulum deoxyribonucleic acid nicotinamide adenine dinucleotide (reduced) nicotinamide adenine dinucleotide phosphate (reduced) N-tris (hydroxymethyl) methylglycine tris (hydroxymethyl) aminomethane uridine diphOSphate volume per volume weight per volume weight per weight CHAPTER I INTRODUCTION -- SUBCELLULAR ORGANELLES A. Microbodies, Peroxisomes and Glyoxysomes 1. Morphology. Microbodies were first described by electron microscopists studying kidney and liver cells. They have since been observed in many animal (47) and plant tissues (36, 38). Microbodies have diameters ranging from 0.2 to 1.7 u and are often Spherical, but being pliable they may take other shapes. Their single limiting membrane is about the same thickness (60 to 80 A) as the membranes of the endoplasmic reticulum (ER) or mitochondria but thinner than the plasma membrane or the lysosome membrane (47). The membrane has the typical three-layered "unit- membrane” structure (67). Microbodies are distinguished from mitochondria by their single membrane and by the absence of internal membranes or ribosomes. The micro- body matrix usually has a granular appearance and may contain a dense core or crystalloid structure (36, 47). 2 2. Biochemical Observations. Peroxisomes (subcellular particles containing hydrogen peroxide-producing oxidases and Catalase) were first isolated from rat liver and dog kidney and thorough- ly characterized by de Duve's group (7, 23, 24, 24a, 81). Subsequently similar particles were obtained from other organisms and tissues including protozoa (76), yeast (98), and plant tissues (103-106, 116). Baudhin demonstrated that what the biochemists called peroxisomes were iden- tical to what the electron microscopists called micro- bodies (7). Rat liver peroxisomes attain a density of 1.23 3 in sucrose density gradient centrifugation. g x cm' This is because the membrane of the peroxisome is freely permeable to sucrose and other small molecules. Also, for this reason rat liver peroxisomes are not sensitive to osmotic shock (24). Rat liver peroxisomes contain catalase, NADPH- isocitrate dehydrogenase, D-amino acid oxidase, L-a- hydroxy acid oxidase and urate oxidase. The urate oxidase is located in the crystalloid core (24). In addition, they contain serine-glyoxylate aminotransferase and alanine-glyoxylate aminotransferase (85, 108). The compartmentation of the hydrogen peroxide-producing 3 flavin oxidases with catalase has an internal logic; however the physiological function of these organelles in the liver is not clear (24). Spinach leaf peroxisomes, which are isolated at a density of 1.26 to 1.27 g x cm-3 in sucrose gradients, have all of the enzymes present in the liver particles (103), including urate oxidase (100) but excluding the D-amino acid oxidase (104, 114, 116). The catalase of leaf peroxisomes is often located in the crystalloid core, in contrast to liver peroxisomes (37). In addition, Spin- ach peroxisomes contain malate dehydrogenase, hydroxy- pyruvate reductase, and four transaminases (85, 114, 116). This amounts to almost complete compartmentation of the glycolate pathway for metabolism which converts the two carbon by-product of photosynthesis -- glycolate -- to glycine, serine, and glycerate (102, 104). The only com- ponents of the glycolate pathway absent from the peroxisomes are the glycine decarboxylase and serine hydroxymethyl transferase which are located in the mitochondria (52, 53). This pathway is responsible for the release of CO2 during photosynthesis (photorespiration), for the production of glycine and serine which are necessary for protein and porphyrin synthesis, and for the supply of C1 groups needed for cell wall and nucleic acid synthesis (103, 106). 4 Glyoxysomes, which have been isolated in sucrose gradients from castor bean and other fat storing seeds, are clearly reSponSible for the mobilization of the lipid stores during seed germination. These microbodies are in close association with the lipid bodies (spherosomes) .gg.§igg. Glyoxysomes contain all the enzymes necessary for B-oxidation of fatty acids (21) and all the enzymes of the glyoxylate cycle (14, 20). In addition, they con- tain several enzymes known to be in plant and animal peroxisomes including urate oxidase (100), L-d-hydroxy- acid oxidase (l4), and catalase which is located in the crystaloid (110). Furthermore, Schnarrenberger has re- cently shown that castor bean glyoxysomal preparations also contain hydroxypyruvate reductase, serine-glyoxylate aminotransferase and glutamate-glyoxylate aminotransferase, enzymes found in Spinach leaf peroxisomes (91). The cell may synthesize and destroy peroxisomes as whole units rather than dealing with the component parts individually. Once rat liver peroxisomes are synthesized they do not acquire additional protein and they do not grow (82). These peroxisomes have a half life of about 3.5 days. They are destroyed at random as wholes (83). Insect fat body microbodies (61) and castor bean glyoxy- somes (110) may be destroyed as wholes by autophagic vacuole formation. The frequent association of microbodies with glu- coneogenic function is interesting but not completely understood. The glycolate pathway in leaf peroxisomes is certainly gluconeogenic as is the glyoxylate cycle of the glyoxysomes. Liver is a gluconeogenic tissue although it is not known how its peroxisomes could be involved in gluconeogenic metabolism. B. Endoplasmic Reticulum 1. Mbrphology. This network of membranes is seen throughout most cells. Some parts of the ER are covered with ribosomes; other parts are smootho The ER appears to be responsible for the production of other organelles such as the Golgi apparatus and microbodies. In meristematic and differentiating cells, microbodies are closely associated with the ER and in some cases the limiting membrane of the microbody appears to be continuous with the ER, usually the rough ER (38, 47, 61, 110). The catalase which can be identified by Specific staining techniques is apparently synthesized by ribosomes on the ER and then transferred to the microbodies (45, 58). 6 2. Biochemical Observations. Microsomes are small membranous vesicles resulting from the breakage of the endoplasmic reticulum during grinding (19). Because of their small size they sediment more slowly than other organelles and thus can be sep- arated from other components by differential centri- fugation. The final step of isolation usually involves pelleting the microsomes at high speed (eg., 100,000 g). Microsomes bearing ribosomes (i.e., rough or heavy micro- somes) can be separated from smooth microsomes on sucrose density gradients. Smooth microsomes are found at a 3 density of 1.17 g x cm' , while rough microsomes are more .dense (95). Microsomes isolated from rat livers have been studied in detail. They are capable of lipid and protein synthesis. They contain a unique electron transport system, the components of which include cytochrome P450, cytochrome b5, NADH-cytochrome b5 reductase and NADPH- cytochrome c reductase (91, 113). The cytochrome b5 and its flav0protein reductase will function as a NADH- cytochrome c reductase in vitro as follows: NADH Flavoprotein (ox.) Cytochrome b5 (red.x Cytochrome c (ox) NAD AxFlavoprotein (red.) Cytochrome b5 (ox.) Cytochrome c (red.) 7 However, cytochrome c is not the physiological electron acceptor (96, 97). Glucose-6-phOSphatase is also typi- cally associated with rat liver microsomes. The rough ER is the precursor of the smooth ER. This transition involves a change in phospholipid compo- sition, i.e., the proportion of Sphingomyelin increases (54, 70, 112). Although separation and analysis of smooth and rough microsomes revealed subtle differences in enzymatic composition (23), cytochemical studies of the glucose-6-phOSphatase in §i£u_revealed no differences between the smooth and rough ER (60). It has been suge gested that the smooth microsome fraction contains, in addition, fragments of the Golgi and the plasma membrane (23, 69). Microsomes have also been obtained from plant tissues such as swiss chard, cauliflower, bean cotyledons, and etiolated wheat (22, 64, 101). These plant microsomes have NADH- and NADPH cytochrome c reductase activities but contain cytoChrome b3 instead of b5. Microsomes from pea cotyledons or leaves and from.A£gm spadix have cytochrome b5 and cytochrome P450 (16). Bean cotyledon microsomes have glucose-6-phosphatase (101). As will be shown in part III. B., microsomes may also be isolated from Spinach leaves, castor bean endOSperm, and sunflower cotyledons. C. Mitochondria Mitochondria have been isolated from plant and animal tissues by differential and density gradient cen- trifugation. In sucrose density gradients mitochondria from.most tissues band at a density of about 1.2 g x cm’3, and thus may be separated from the heavier peroxisomes and the lighter microsomes. The density of rat liver mitochondria slowly increases on eXposure to concentrated sucrose so that, after longer centrifugation times, mitochondrial contamination of the peroxisomes becomes greater (8, 39, 81). The outer membrane of the mitochondria may be selectively ruptured by osmotic treatment, sonication, or digitonin treatment and then separated from the inner membrane plus matrix by differential or density gradient centrifugation (29, 90, 95). The inner membrane plus 3, while the outer 3 matrix has a density of 1.21 g x cm- membrane has a density of 1.13 g x cm" , near that of the microsomes (29). The inner membranes of the mitochondria bear the components of respiratory electron transport including cytochrome c oxidase and a succinate or NADH-cytochrome c reductase which is distinguished from the microsomal 9 reductase by its sensitivity to inhibitors such as rotenone and antimycin A. The matrix of the mitochondria contains the enzymes of the citric acid cycle and includes some enzymes or isozymes also found in peroxisomes and glyoxysomes such as malate dehydrogenase and aSpartate aminotransferase (29). The outer mitochondrial membranes have much in common with the microsomes and, therefore, with the ER, including the NADH-cytochrome b5 reductase and cytochrome b5. The NADH-cytochrome c reductase of the outer membrane is immunologically similar to that of the microsomes (99). However, the outer membrane lacks the NADPH-cytochrome c reductase, cytochrome P450 and glucose-6-phosphatase (29, 90, 95). Similarities in protein composition have also been observed in electrophoretic analysis of the protein components of the two membrane types (89). The phOSpholipid composition of the outer membrane is similar to the microsomes: phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol are the major species. Compared to the inner mitochondrial membranes, the microsomes and the outer mitochondrial membranes have relatively high amounts of cholesterol. It is for this reason that the outer membrane is sensitive to digitonin. The major phospholipids of the inner membrane are 10 phOSphatidyl choline, phOSphatidyl ethanolamine, and cardiolipin (29). Mitochondria are self replicating; they divide and grow (56). They contain DNA, ribosomes, and are capable of producing some of their own lipids and pro- teins such as the membrane bound cytochromes. However, other mitochondrial components, such as cytochrome c and some lipids, are synthesized by the ER and transported to the mitochondria. The proteins and lipids of the outer membrane are exclusively imported products, probably coming from the ER though not necessarily in direct con- tinuity with it (54, 56, 88). D. Chloroplasts and Etioplasts Chloroplasts, like the mitochondria, are enclosed in a double membrane. The chloroplast contains a network of membranes derived from the inner limiting membrane, the thylakoids, which are arranged in regular stacks, the grana. This membrane system is surrounded by the stroma, which is analogous to the mitochondrial matrix, and enclosed in the double membrane. The thylakoids contain the components of photoelectron transport -- the pigments and cytochromes. Most of the enzymes involved in the photosynthetic carbon dioxide fixation cycle, such 11 as ribulose-1,5-diph08phate carboxylase, triose phOSphate isomerase, and NADP-glyceraldehyde-3-phOSphate dehydro- genase are components of the stroma (51). Like the outer mitochondrial membrane and the peroxisomal membrane, the outer membrane of the chloro- plast is freely permeable to sucrose and other low molecular weight Species, while the inner membrane is the site of selective transport of certain metabolites such as malate and 3-phosphoglycerate (44). The lipid composition of chloroplasts is quite unique; galactolipids such as monogalactosyl diglyceride, digalactosyl diglyceride, and the sulfolipid predominate rather than phOSpholipids. Phosphatidyl glycerol is the major phospholipid (1, 10, 51, 75). Isolated chloroplasts are usually broken -- devoid of the outer membrane and much of the stroma. In sucrose density gradients the broken chloroplasts reach a density of 1.14 to 1.17 g x cm'3. Whole chloroplasts have a density of 1.21, about the same as mitochondria (87, 92). Etioplasts are the precursors of chloroplasts and are present mainly in non-green tissues such as castor bean endosperm and etiolated leaves or cotyledons. The internal membranes of this organelle are arranged in a crystalloid structure. Etioplasts have been isolated by 12 Schnarrenberger, et a1 (92) on sucrose density gradients at a density of 1.26, very near the peroxisomes and glyoxysomes. Etioplasts contain many of the enzymes known in mature chloroplasts: triose phosphate isomerase, NADPH-glyoxylate reductase, Dopa oxidase, phosphoglycolate phOSphatase, and NAB-triose phosphate dehydrogenase. The enzymatic composition of etioplasts may indicate some role for the developing particle in carbohydrate metabolism in the non-green cell (92). E. The Golgi . ‘1 The Golgi consist of dictyosomes which are com- posed of stacks of membranes. These membranes and their enclosed contents may be derived in part from the ER, although dictysomes are capable of dividing and repro- ducing themselves. The Golgi produce other membranous structures such as lysosomes (25), zymogen granules (70), and secretory vesicles (71). Secretory vesicles may contain cell wall components, for example. AS the con- tents of a secretory vesicle are released to the outside of the cell the membrane becomes a part of the plasma membrane (71). The transition of the ER to a lysosome or plasma membrane via the Golgi is accompanied by a thickening of the membrane from 50 A in the ER to 70 R 13 in the Golgi to almost 100 R in the plaSma membrane (74). Golgi have been isolated from onion leaves, cauliflower, beef liver, and rat liver (17, 31, 33, 57, 71, 73). They have a density of 1.13 in a sucrose grad- ient. UDP—galactose-N-acetflglucosamine galactosyl trans— ferase is apparently unique to this organelle, at least in rat liver. Nucleoside diphosphate hydrolases and thiamine pyrophosphorylase are enzymes which have also been detected in some Golgi preparations (18, 31, 71, 73). The biochemical data support the morphological observations of membrane differentiation. In contrast to microsomes, the Golgi lack cytochrome P450, glucose-6- phOSphatase, and have less cytochrome b5 (33). The transformation of the ER to the plasma membrane also involves a change in lipid composition; phosphatidyl choline becomes less prominent and the proportion of Sphingomyelin increases (27, 49, 69, 84). Rm Lysosomes function in the digestion of endogen- ously and exogenously derived materials. Exogenous mater- ial is brought into the cell enclosed in a bit of the plasma membrane. The resulting vesicle is then engulfed by a lysosome. Lysosomes also engulf cellular organelles 14 such as microbodies (25, 30). Thus lysosomes may be re5ponsible for the turnover of microbodies (61, 110). Lysosomes contain a group of acid hydrolases (proteases, lipases, and nucleases) which digest the en- gulfed materials. The hydrolases are synthesized in the ER, transported to the Golgi, and released in membrane bound vesicles, the lysosomes (8, 25). The density of rat liver lysosomes is 1.22 g x '3 in sucrose gradients, intermediate between mito- cm chondria and peroxisomes. However, when rats are in- jected with Triton WR-l339, the detergent accumulates in the lysosomes and their equilibrium density is reduced ‘to 1.12. Acid phOSphatase activity is commonly used to detect lysosomes. Lysosomal enzymes have been found in isolated spherosomes (lipid bodies) and vacuoles from plants (66). G. Storage Particles Lipid storage bodies (spherosomes) are abundant in castor bean endosperm and cotyledons of germinating fatty seeds. They have a very low density and float to the top of a sucrose gradient. The membrane of the lipid body is a single line rather than a three-layered "unit membrane" (107). Two types of lipid vesicles have 15 been isolated from pea and bean cotyledons. One type is found in adherent groups. The other is larger and singu- lar (2, 72) The larger type contains more triglyceride and phospholipid than the adherent type. Both contain phosphatidyl choline, phOSphatidyl ethanolamine and phos- phatidyl inositol. Protein storage bodies (alueron grains) also occur in seeds such as castor bean and sunflower (93, 110). These very dense (greater than 1.3 g x cm‘3) par- ticles have been isolated from germinating sunflower by Schnarrenberger et a1 (93) and contain acid protease activity. H. Nuclear and Plasma Membranes The nuclear membrane and the plasma membrane appear to be at opposite ends of the spectrum of cellular membranes. The nuclear membrane is similar to the ER and the two membrane systems are continuous in places. The nuclear membranes are richer in protein than the microsomes and therefore have a greater density, 1.20 g x cm73. They contain NADPH- and NADH-cytochrome c reduc- tases but lack glucose-6-ph03phatase (35, 117). Nuclear membranes have a lipid composition similar to microsomes (48, 50, 117). The isolated plasma membrane has a density of 16 1.14 to 1.17 in sucrose gradients. It is characterized by a series of phOSphate eSter—splitting enzymes, in- cluding 5’-nucleotidase, acid thSphomonoesterase, glucose-6-phosphatase, and ATPases (23, 28, 55, 69). Compared to the microsomes, the plasma membranes have relatively high amounts of sphingomyelin and cholesterol (23, 27, 70). CHAPTER II METHODS AND MATERIALS A. Isolation of Organelles 1. Spinach Spinacia oleracea L., Longstanding Bloomsdale, was grown in a controlled environment chamber. Deribbed leaves (200 g) were homogenized for 10 sec in a Waring blendor with 200 m1 of a solution of 30% (w/w) sucrose 'and 0.02 M glycyl-glycine, pH 7.5. The homogenate was squeezed through 8 layers of cheese cloth and centrifuged at 650 g for 5 min. Zonal centrifugation techniques, as developed by Anderson (3) were used to separate organelles from the spinach homogenate. A discontinuous sucrose gradient was pumped (Cole-Farmer Masterflex pump, model #7014) into an IEC B-30 zonal rotor at 2500 rpm. The gradient was pumped to the edge of the rotor starting with 20 ml of 25% (w/w) sucrose. This was followed by 20 ml each of 33%, 37%, 40%, 40.5%, 41%, 42%, 43%, 44%, 49%, 50%, 51%, 51.5%, 52%, and enough 56% sucrose to fill the rotor to 17 18 the center. The capacity of the head was about 560 m1. All sucrose solutions were prepared from Schwarz-Mann "density-gradient grade” sucrose in 0.02 M glycyl-glycine, pH 7.5. The spinach supernatant, about 230 ml, was pumped into the center of the rotor, displacing an equal amount of the 56% sucrose from the edge. About 50 m1 of 56% sucrose remained in the rotor. After centrifugation at 30,000 rpm for 2 hr, the rotor was decelerated to 2500 rpm, and cold water was pumped into the rotor to displace the gradient which was collected in 10 ml fractions ’ beginning with the 56% sucrose. Fractions were collected with a rapid (ca. 50 ml per min) continuous flow. Cross- leakage with the incoming water at the rotating seal assembly was likely if the pump were stopped. The material was kept near 50 throughout the procedure. Various other grinding techniques and centrifuga- tion procedures were used in the fractionation of Spinach leaves, and the fractions obtained were further purified by density gradient flotation (see part III). However, the above procedure was found to give excellent yields and reasonable purity in a short time (ca. 3 hr). l9 2. Castor Bean EndOSperm and Sunflower Cotyledons. Castor bean (Ricinus communis L.) endosperm.was obtained from seedlings after 5 day germination in the dark at 30°. Fifteen grams of endOSperm were homogen- ized for 5 sec in a Sorvall Omni-mixer with 22.5 ml of 24% (w/w) sucrose, 0.165 M Tricine, 10 mM KCl, MgCl EDTA, 2, and dithiothreitol (Calbiochem), pH 7.5 (20). About 16 ‘ml of the resulting homogenate was layered on a discontin- uous gradient (5 ml of 60%, 2.5 ml each of 56.5%, 53%, 51.5%, 50%, 47.5%, 45%, 42.5%, 40%, 37.5%, 2 ml each 35%, 32.5%, 30%, 27.5%, 25%, 22.5% and 20% W/w sucrose‘in 10 mM EDTA, pH 7.5). This was centrifuged for 2 hr at 25,000 rpm in a Beckman SW 25.2 rotor. Fractions of 2.5 or 2 ml were collected from the bottom of the tube starting with the most dense sucrose as indicated in Figure 7 (p64). Sunflower (Helianthus annus L.) cotyledons were homogenized and fractionated using procedures similar to those used for Castor bean (91). 3. Animal Tissues. Livers were obtained from three female Sprague- Dawley rats, 190 to 210 g body weight, which had been injected with 1.5 m1 of 10% (w/w) Triton W-l339 (Ruger 20 Chemical Co., Irvington, N.J.) 3.5 days previous and starved overnight. The livers, 20 to 40 g total, were perfused with grinding medium in sign, minced and homog- enized with 7% (w/w) sucrose, 0.02 M glycyl-glycine, pH 7.5, in a Potter-Elvejhem homogenizer with a motor driven teflon pestle (81). The homogenate was filtered through 4 layers of cheese cloth and centrifuged at 2000 rpm for 7 min, including acceleration time. The loaded B-30 zonal rotor contained 295 ml of 3% sucrose, 44 ml of rat liver supernatant, 12 ml each of 15%, 20%, 25%, 30%, 33%, 36%, 38%, 40%, 45%, 46%, 46.5%, 47%, 47.5%, 48%, 49%, and 40 m1 of 56% (w/w) sucrose in 0.02 M glycyl- glycine, pH 7.5. This was centrifuged at 35,000 rpm for 35 min. Fractions were collected in 5 ml volumes. The sedimentation distance in this gradient is only about 1 cm which makes the short centrifugation time possible. To process larger amounts of tissue (eg., 10 livers) it was necessary to pellet the peroxisomes as described by Poole (81). The homogenate was centrifuged at 5000 rpm for 7 min (Beckman 30 rotor, Beckman screw cap tubes) in order to remove cell debris. The resulting pellet was resuSpended in about 20 ml of grinding medium and centrifuged as before. The combined 5000 rpm super- natants were centrifuged at 25,000 rpm for 8 min 20 sec 21 (all centrifugation times include acceleration time). The pellet obtained at 25,000 rpm was resuSpended, using a Potter-Elvejhem homogenizer, and recentrifuged at 25,000 rpm. The final pellet was resuSpended in about 50 ml and pumped onto the zonal sucrose gradient described above. Dog kidneys were homogenized and fractionated in the same manner as rat liver. Details are given in Figure 11. The exact procedures and sucrose gradients used were varied because different types of organelles were encountered in each tissue and because the equilibrium ‘density of a particular organelle varied from tissue to tissue (Table III). B. Enzyme Assays All assays were carried out at 250 except where noted. In assays where NADH or NADPH was involved, a change of one A340 was equivalent to 161 nmoles in a one m1 assay. Components of the enzyme assays that were obtained from Sigma Chemical Co., St. Louis, Mo., were cytochrome c (type II which was usually 75% oxidized), NADH (grade III), NADPH (type II), and antimycin A (type III). Cytochrome b5 from rat liver microsomes 22 was generously supplied by Drs. D. Roerig and S. Aust. 1. Catalase. The rate of disappearance of hydrogen peroxide was measured at 240 nm (12). A change in one A240 was equivalent to 71.5 umoles of hydrogen peroxide in a 3 ml assay. The initial A240 of the peroxide was 0.5 to 0.6. 2. Glycolate Oxidase (L-d-hydroxy acid oxidase). The reduction of the dye, dichlorOphenolindo- phenol, was followed under anaerobic conditions at 600’ nm (105). The maximum dye concentration that could be used in this Spectrophotometeric assay did not saturate the enzyme so that the rates obtained were about one third of the optimum. 3. Uric Acid Oxidase. The absorbance decrease of uric acid was recorded at 293 nm; one A293 was equivalent to 81.2 nmoles in the 1 ml assay. 4. ASPartate Aminotransferase. The production of oxaloacetate was coupled to malate dehydrogenase so that the NADH oxidation could be followed at 340 nm (12, 114). 23 5. Serine-Pyruvate Aminotransferase. Hydroxypyruvate production was linked to hydroxy- pyruvate reductase (glycerate dehydrogenase) and NADH oxidation (116). 6. Glutamate-glyoxylate aminotransferase. The amount of radioactive glycine produced from glyoxylate-1,2-14C during a 15 min incubation at 300 was determined by separating the glycine from the glyoxylate on Dowex 50 H+ and counting the radioactivity in the glycine fraction (53). 7. NADP-Isocitrate Dehydrogenase. NADP reduction was measured at 340 nm (116). 8. Malate Dehydrogenase. With oxaloacetate as substrate NADH oxidation was followed at 340 nm (115). 9. Cytochrome c Oxidase. After incubating an enzyme aliquot with digitonin for a minute, buffer and reduced cytochrome c were added and the disappearance of reduced cytochrome c was followed at 550 nm. The cytochrome c had been reduced by adding a few crystals of sodium dithionite until the ASSO/A565 24 ratio was greater than 6, which was equivalent to 70% re- duction. A ratio of 10 was equivalent to 90% reduction (94). The units of activity are described in the NADH- cytochrome c reductase procedure. 10. NADPH-Diaphorase. The NADPH-dependent reduction of dichlorophenol- indophenol was followed at 600 nm. The assay mixture consisted of 0.5 m1 of 0.1 M Tris, pH 7.5, 0.1 m1 of the dye (0.25 g per ml), 0.35 ml of enzyme plus water, and 0.05 ml of NADPH (2 mg per ml). A change of one A600 was equal to 1.83 nmoles. ll. Triose phosphate isomerase. NADH oxidation, which could be followed at 340 nm, was obtained by coupling the isomerase to glycerol- phosphate dehydrogenase (9, 92). 12. NADH-Cytochrome c Reductase. NADH-cytochrome c reductase was assayed by measur- ing the rate of cytochrome c reduction at 550 nm in a microcuvette containing 0.1 ml of 0.2 M phOSphate or glutamate buffer at the indicated pH, 50 ul of oxidized cytochrome c (5 mg/ml), 5 ul of 10 mM KCN (to inhibit any cytochrome c oxidase activity), 70 ul of organelle 25 suspension plus water (22, 64). Where indicated 2 ul of antimycin A (2 mg/ml in ethanol) was added to the mixture. After obtaining the NADH-independent rate the reaction was initiated with 50 ul of NADH (3 mg/ml). A change of one A550 was equivalent to a change of 12.8 nmoles in the 0.27 ml assay. The extinction coefficient for cytochrome c (reduced minus oxidized) is 21.1 mM'lcm-l (109). 13. NADPH-Cytochrome c Reductase. NADPH-cytochrome c reductase was assayed in a similar way except that 0.15 ml of 0.05 M phosphate, pH 7.3 and 50 ul of organelle suspension plus water were used. Antimycin was included in all assays. The reac- tion was initiated with 2 ul of NADPH (8.6 mg/ml in 0.1 M bicarbonate, pH 10.4) (113). 14. NADH-Cytochrome b5 Reductase. NADH-cytochrome b5 reductase was assayed by measuring the rate of cytochrome b5 reduction at 426 nm in a microcuvette containing 0.1 ml of 0.2 M phosphate, pH 8.0, 50 ul of NADH (3 mg/ml). A change of one A426 was equivalent to a change in concentration of 2.55 nmoles in the 0.255 ml assay. The extinction coefficient of cytochrome b5 (reduced minus oxidized) is 100 mM—lcm-1 26 (97). 15. PhOSphatases. Glucose-6-ph03phatase was assayed by determining inorganic phosphate released after 10 min, at 350 (79). Acid phosphatase was assayed using the chromo- genic substrate, p-nitrophenyl phosPhate. After 10 min incubation at 35°, 0.02 M NaOH was added and the A440 read (15). C. Chlorophyll and Protein Assays Chlorophyll was determined by the method of Arnon (4). The Lowry procedure was used to estimate protein (62). The sample volume was kept Small enough, eg. 50 ul, so that the sucrose content had little effect on the assay (40). D. Lipid Determinations. 1. Lipid Extraction. Fractions of interest from the density gradients were extracted overnight with 10 volumes of 2:1 (v/v) chloroform-methanol. The residue, which usually contained some sucrose crystals, was re-extracted with with 10 volumes chloroform-methanol. The combined extracts were 27 washed with 0.2 volume of 0.1% MgClz. The phases were separated by centrifugation, the upper aqueous phase dis- carded and the wash repeated (34). The extract, now con- siderably reduced in volume by the wash procedure, was evaporated to dryness in yagug_at about 50°. The lipids were then transferred to a screw cap vial (with teflon lined cap). During the transfer the flask was washed at least 5 times with 1 ml portions of chloroform. The chloroform was removed by a stream of nitrogen and the lipids redissolved in a known amount of chloroform. Aliquots were taken for phosphate determinations and for thin layer chromatography. All solvents were redistilled. 2. Thin Layer Chromatography. The plates, coated with silica gel F254, 0.25 mm thick (Brinkman) were prewashed by running them in acetone. Aliquots of the lipid samples containing known amounts of phosPholipids (600 to 1000 pg phospholipid, assay described below) were taken to dryness, redissolved in a small amount of chloroform and applied to the plate, alternating with standards, about 1.5 cm from the edge. The standards, phosphatidyl inositol, phosphatidyl serine, phosphatidyl ethanolamine and its lyso derivative, phos- phatidyl choline and its lyso derivative, phosphatidyl 28 glycerol, cardiolipin, Sphingomyelin, and monogalactosyl diglyceride, were obtained from Applied Science Labora- tories, State College, Pa. The plates were run in chlor- oform, methanol, acetic acid, water (170:25z25z6 v/v) in a chamber lined with filter paper soaked with the solvent (78). The solvent front reached the top edge of the plate in 2 to 3 hr. 3. Lipid Identification. The plates were dried by heating to 1000 for a few min or air dried for a few hours. They were then Sprayed with ninhydrin (0.2% in butanol saturated with water) and heated to 1000 in an oven containing a pan of water. By comparison with the standards it was possible to identify those lipids containing amino groups, phos- phatidyl ethanolamine and its lyso derivative, and phOSphatidyl serine. Following ninhydrin development, the plates were exposed to iodine which gives a temporary yellow-brown color to all lipid Spots. Lipids such as phosphatidyl inositol, choline and glycerol, Sphingomyelin and cardiolipin, were identified by comparison with the respective standards. 29 4. Lipid PhOSphate Determination. The micro-modification of the Bartlett phosphate assay (6) was used to quantitate the lipid phOSphorus in the original lipid sample or in lipid Spots from thin layer plates. The area of the thin layer containing a lipid Spot was removed from the plate with a stiff razor blade cut to 1 cm wide and the silica gel transferred, with the aid of a fine brush and glassine paper, to a 12 ml conical centrifuge tube. Then 0.3 ml of 10 N H2804 was added to each tube and heated to 160° for at least 3 hours, often overnight. The tubes were cooled; 2 drops of phosphate free 30% hydrogen peroxide were added to -each sample; each was vortexed tocompletely stir up the silica gel; and after 1.5 hours at 160° they were in- spected. If brown or yellow color remained in any sample the peroxide treatment was repeated in all. When the digestion process was completed the reagents for color development were added: 0.65 ml water, 0.2 ml of 5% ammonium molybdate, and 0.05 ml of Fiske-Subbarow reagent (0.5 g of l-amino-Z-anaphthol-4- sulfonic acid and l g of sodium sulfite, anhydrous, in 200 ml of 15% sodium bisulfite). The tubes were cooked in a boiling water bath for 10 min, centrifuged for about 5 min to pellet the silica gel and the contents read at 30 830 nm. A series of standards, 5 to 120 nmoles of phos- phate, were also carried through the procedure. The background phOSphate per unit area in each thin layer plate was determined and taken into account when calcu- lating the amount of phosphorus in each lipid Spot. The nmole values obtained were multiplied by the average molecular weight of a phospholipid, 775, to give nano grams of phospholipid. CHAPTER III RESULTS -— TISSUE FRACTIONATION The main purpose of tissue fractionation in these studies was to obtain reasonably pure microbodies in quantities sufficient for analysis of membrane components. The term microbody is used here to include peroxisomes and glyoxysomes. The marker enzymes used to detect microbodies in sucrose density gradient fractions were catalase, glycolate oxidase (L-a-hydroxyacid oxidase), and urate oxidase. The purity of a microbody fraction was estimated from the activities of the markers unique to other organelle fractions. For example, the specific activity of cytochrome c oxidase in the microbody fraction relative to the specific activity in the mitochondria indicated the proportion of mitochondrial contamination in the microbody fraction. Similarly, chloroplast contam- ination was indicated by chlorophyll or NADPH-diaphorase and etioplasts by triose phosphate isomerase. Acid phosphatase was the marker enzyme used to detect lysosomes. NADH-cytochrome c reductase was used to locate the 31 32 microsomes, but it was not unique to this fraction Since it was also found in mitochondria and microbodies (see Part IV. A.). A. The Isolation of Spinach Leaf Peroxisomes 1. Differential Centrifugation. The first approach to the isolation of cellular components usually involves differential centrifugation. Indeed for many years chloroplasts and plant mitochondria were obtained using only this method (13). In view of recent developments these preparationswere impure. For example, catalase, which was thought to reside in the chloroplasts, is actually a component of the peroxisome (104). The sedimentation of organelles from Spinach leaves is shown in Figure l. The chloroplasts, which are large, sediment faster than the other organelles. The initial Steep portion of the chloroplast sedimentation curve probably represents whole chloroplasts while the latter portion corresponds to the broken chloroplasts (87). Spinach peroxisomes, in contrast to liver peroxisomes, sediment more rapidly than the mitochondria. Had the microsomes been assayed for in this experiment they probably would have been least inclined to sediment. 33 Percent of Total Precipetable FIGURE 1. 00 9 m ‘3 43 ‘3 no 9 /I Chloropl . . ._ Oi . '0 Peroxisom 0 _ O . . . ' ' Mitochondria _ . I .’ U . I ‘ l l l l |000 2000 3000 4000 5000 Centrifugal Force (9) Differential centrifugation of spinach homog- enate (10 min). Sixty glof spinach were chopped in a parsley grater with 300 ml of 25% (w/w) sucrose, 0.02 M glycyl-glycine, pH 7.5, and filtered through 8 layers of cheese cloth. Ten ml of the grinding medium were placed in the bottom of 45 ml centrifuge tubes; 15 m1 of the spinach homogenate was layered on top of each; each tube was centrifuged for 10 min at the force indicated; the pellets were resus- pended and assayed for chlorophyll (chloro- plasts), glycolate oxidase (peroxisomes) and cytochrome c oxidase (mitochondria). The values are eXpressed as the percent of the amount of each marker enzyme that had been sedimented at 27,000 g. 34 The purpose of this experiment was to try to select a centrifugation force which would best discrim- inate between peroxisomes, chloroplasts and mitochondria. It is clearly not possible to separate the peroxisomes from the mitochondria by this approach. However, one may remove a great deal of chloroplasts from a peroxisomal preparation by differential centrifugation. A force of 1000 3 applied for 10 min sedimented more than 50% of the chloroplasts but only 15% of the peroxisomes and 10% of the mitochondria. To minimize the losses of peroxi- somes a force of 650 g was selected for routine work. ‘ A second differential centrifugation step has often been employed to concentrate the peroxisomes to be placed on a sucrose gradient. It was found that most of the remaining chloroplasts could be removed by increasing the sucrose concentration to about 40% (w/w) after the first centrifugation and then centrifuging at 27,000 g for 60 min to sediment the peroxisomes. When these peroxisomes were subjected to density gradient centrifu- gation they were obtained relatively free of chlorophyll but the yields were very low. 35 2. Grinding Procedures. An attempt was made to develop grinding procedures which would give better yields of peroxisomes from spinach leaves. Homogenization in the blender results in the breakage of varying amounts (60% to 90%) of the peroxi- somes, assuming that the marker enzymes such as catalase and glycolate oxidase are exclusively confined to the peroxisomes in the cell. Spinach leaves were carefully sliced with a french cookery knife, chopped with razor blades, or shredded in a Mouli parsley grater. Using such methods the percentage yields were greatly improved; up to 60% of the peroxisomes could be recovered.2 However, the absolute yields were no better because fewer cells were being broken, and the chopping procedures were tedious. Thus, the blender remains the method of choice for processing moderate or large amounts of spinach (100 g to 2.5 Kg). Good yields of peroxisomes may also be obtained from Small amounts of leaves using a mortar and pestle. In addition to trying different mechanical tech- niques, the standard sucrose and glycyl-glycine grinding medium was varied. When the sucrose was replaced by sorbitol in the grinding medium and also in the density gradient, good yields were obtained but the separation of 36 peroxisomes from chloroplasts and mitochondria was poor. A medium, containing mannitol, EDTA, bovine serum albumin and cysteine, which has been used to isolate mitochondria from plants (13) was tried. Compared to the usual grinding medium, the yields from this more complex medium were poor. The only medium tested which resulted in better separation_and yields was that used by Beever's group to isolate glyoxysomes from castor bean endOSperm (20) Tigure 2). This medium contained sucrose, tricine buffer, KCl, MgClz, EDTA, and dithiothreitol. The separation bf the chloroplasts (represented by NADH-diaphorase) (5), from the other organelles was better when this medium was used (Figure 2B). The amount of catalase and glycolate oxidase in the peroxisome fraction was somewhat higher. There was less soluble catalase, but this could have been due to inhibition. The specific activities of the peroxisomal enzymes were also higher while that of the contaminating species, the diaphorase, was less (Table I). However, the amount of soluble glycolate oxidase in this gradient could not be determined because dithiothreitol interfered with the assay. Furthermore, something in the grinding medium destroyed most of the cytochrome c oxidase activity. Excluding the dithiothreitol from the FIGURE 2. 37 Isolation of spinach leaf organelles from different grinding media. Forty g of spinach were chopped in the parsley grater with 80 ml of 16% (w/w) sucrose, 0.02 M glycyl-glycine, pH 7.5 (Medium A). Another 40 g_of Spinach were chopped with 80 ml of 16% (w/w) sucrose, 0.165 M Tricine, 0.01 M KCl, 0.01 M MgCl , 0.01M EDTA, 0.01 M dithiothreitol, pH 7.3 (Medium B). The resulting homogenates were filtered through cheese cloth, the pellets, obtained between 480 g (10 min) and 12,000 g (20 min), were each resuSpended in the respective grinding media to make 5 ml and layered on a discontinuous gradient. (5 ml of 56%, 13 ml of 51.5% and 49%, 10 ml of 43%, and 5 ml of 38% w/w sucrose in 0.02 M glycyl- glycine; no EDTA was included in the gradient). The gradients were centrifuged for 3 hr at 25,000 rpm in the SW 25.2 rotor. Percent (w/w) 60 50 4O 3O 20 Grinding Medium A 38 Grinding Medium B r r r Sucrose l l l l Sucrose pmoles x min" x ml"I 2.0 l.5 |.O 0.6 0.4 0.2 NADPH Diaphorase 1 l NADPH Diaphorase I d T r U l U Cytochrome c Oxidase Cytochrome c Oxidase 400 300 200 l 00 I a-i . . r'T'F‘T—t Glycolate Oxidase __ Glycolate Oxidase _ ' t t t t 1‘ t : Catalase .. Catalase .4 l l .MFTL IO 20 30 Volume (ml) 40 IO *20 30 40 Volume (ml) 39 TABLE I. Specific activities of marker enzymes in peroxisomes from spinach leaves homogenized in two different media. Peroxisomes were isolated as shown in Figure 2. Glycolate NADPH- Grinding Medium Catalase Oxidase Diaphorase mmole xmin'1 nmoles x min"1 x mg protein“1 x mg protein" A. Sucrose-glycyl- glycine 1.37 733 184 B. Beever's Medium 1.59 943 123 40 grinding medium did not result in higher cytochrome c oxidase activity. For these reasons Beever's grinding medium was not used for the routine preparation of organ- elles from spinach leaves. 3. Density Gradient Flotation of Spinach Peroxisomes and Mitochondria. Density gradient flotation has been used to further purify glyoxysomes (41) and liver peroxisomes (81). The method was applied to spinach organelles as follows: The peroxisomal fractions and mitochondrial fractions were taken from sucrose density gradients similar to those shown in Figure 2. The sucrose concentration was increased 'to 55% for the peroxisomal fraction and to 51% for the mitochondrial fraction by adding 66% sucrose. The organelle suspension was then placed in the bottom of an ultracentrifuge tube, a linear gradient was constructed over it, and it was centrifuged at 25,000 rpm overnight. The peroxisomes were recovered (50% to 60% recovery) in a white band just above the 55% sucrose; all of the visible contaminating chlorophyll floated to the top of the gradient. The Specific activities of the catalase and glycolate oxidase were consistently increased by at least half. However, the specific activity of cytochrome 41 c oxidase in the mitochondrial fraction was doubled but much of the chlorophyll remained in the mitochondrial band. 4. Breakage of Spinach Peroxisomes. Unlike their counterparts from rat liver, Spinach peroxisomes from density gradients are easily broken by dilution with water. Dilution with one or two volumes of water (or dialysis, see Table XI) resulted in 80% to 90% breakage of the peroxisomes. Centrifugation of a broken peroxisome suSpension at 12,000 g for 10 min sedimented most of the contaminating chlorophyll and cytochrome c oxidase, leaving the soluble peroxisomal enzymes such as catalase and glycolate oxidase in the supernatant. The specific activities of these enzymes were higher in such a supernatant than in the original peroxisome fraction. It is not known to what extent the contaminating mito- chondria were broken by this procedure, since a soluble mitochondrial marker was not assayed. If the peroxisomal membranes behave at all like the outer mitochondrial membranes they should have remained in the supernatant of the 12,000 g_centrifugation (90). This supernatant was centrifuged at 144,000 g for an hour or more to sediment the membranes and the membranes were analyzed 42 for enzyme content (Table XI). B. Egggl Sucrose Density Gradient Centrifugation The zonal type rotor offered several advantages over the swinging bucket type. Because of the large capacity (the B-30 contains 560 ml; the B-29, 1450 ml) several hundred ml of tissue homogenate could be processed and it was not necessary to pellet the particles. Also, gradients having very short sedimentation distances could ~be used in the peripheral part of the rotor. This tech- nique was esPecially successful in the fractionation of C rat liver homogenates. l. Rat Liver. Usually the low speed supernatant of a homogenate from three rat livers was fractionated on a Small (180 ml) gradient in the B-30 rotor (see part II. A. 3.). It was not necessary to pellet the peroxisomes. Because the gradient was only about 1 cm deep the centrifugation time was very short (35 min). This was a rapid procedure, producing reasonably pure particles (Table VII, page 73). It was found that higher Speeds (50,000 rme in the zonal rotor and the use of EDTA in the gradient caused the liver peroxisomes to sediment with the mitochondria. Re-isolation of the peroxisomes from a high Speed gradient 43 revealed that at least 50% were broken, whereas 97% of the peroxisomes were recovered when re-isolated from gradients centrifuged at 30,000 rpm. High centrifugal force or hydrostatic pressure probably damaged the liver peroxisomes (23). 2. Castor Bean EndOSperm. Large quantities of castor bean endosperm can be fractionated by zonal centrifugation without prior differential centrifugation. This method was used to prepare quantities of glyoxysomes sufficient for lipid analysis. The homogenate was pumped directly into the rotor containing a suitable gradient. It was not possible to resolve the organelles from this tissue in a 35 min centrifugation time as it was in the case of rat liver. At least two hours were required to obtain equilibrium. Table II contains the results of zonal sucrose density gradient centrifugation of castor bean endOSperm. The results are comparable to those obtained in the swinging bucket rotor (Table VI, page 66). The amount of contaminating mitochondria (cytochrome c oxidase) and etioplasts (triose phOSphate isomerase) in the glyoxysomes was slightly higher. No significant amount of particulate acid phOSphatase was observed, that is, no lysosomes were TABLE II. Specific activities of marker enzymes in castor bean endosperm fractions from a zonal sucrose density grad- ient. The homogenate from.74 g of endosperm (126 ml) was pumped into the zonal rotor containing a gradient 0f 20 Ml eaCh 0f 15%: 20%! 25%: 30%: 3205%9 35%: 37.5%! 40%. 42.5%. 44%. 45%. 46%. 47%. 48%. 48.5%. 49%. 49.5%. 5 EDTA, pH 7.5. 0%. 51.5%. and 50 ml of 56% (w/w) sucrose in 10 mM Centrifugation'wasat 30,000 rpm.for 3 hr. Fractions of 10 ml were collected from.the edge of the rotor. Ratios are relative to the maximum specific activity for that particular enzyme. Catalase Acid Phosphatase . mmoles x min:% nmoles x min:i Fraction x mg protein Ratio x.mg protein Ratio Glyoxysomes 7.87 1.000 22 0.066 Etioplasts 1 . 96 0. 250 30 0. 090 Mitochondria 0.23 0.029 8 0.024 Microsomes 0.25 0.032 86 0.260 Supernatant 0.13 0.017 335 1.000 Triose Phosphate Cytochrome c Oxidase Isomerase nmoles x min:1 nmoles x min:% Fraction .x mg protein Ratio x.mg protein Ratio Glyoxysomes 100 0.024 1180 0.160 Etioplasts 1600 0.390 7200 1.000 Mitochondria 4150 1.000 1420 0.200 Microsomes 35 0.008 1650 0.230 Supernatant 4 0.001 1260 0.180 45 detected in the gradient. 3. Spinach. It was especially important to eliminate the necessity of pelleting spinach peroxisomes since they are very fragile and many were broken when pelleted. Often after pelleting in the second step of differential centrifugation only about 10% of the total catalase or glycolate oxidase in the original homogenate was re- covered in the peroxisomal fractions from a density gradient. However, 35% recovery was routinely obtained when the peroxisomes were not pelleted. Thus, the zonal rotor was extremely valuable in the isolation of spinach peroxisomes. It was possible to process large amounts of spinach in the B-29 (1450 m1) rotor. The 650 g_superna- tant from 630 g of deribbed leaves, which amounted to 950 m1, could be put over a 500 m1 gradient in this rotor. After centrifuging long enough to move the per- oxisomes into the gradient (about 30 min at 33,000 rpm) the supernatant could be pumped off and a new batch of spinach juice put on. Using this technique, as much as 2.5 kg of Spinach have been processed. Standard peroxisomal preparations were made from 46 200 g of Spinach leaves in the B-30 (560 ml) zonal rotor, as described in the methods section (part II. A. 1.). The typical distribution of marker enzymes in such a zonal sucrose density gradient is shown in Figure 3. Catalase was the peroxisomal marker. About 35% of the total catalase on the gradient was found in a prominant peak in the most dense region of the gradient (56% to 52% sucrose). The rest of the catalase activity was distributed over the gradient with a large amount in the supernatant. Most of the solubilized catalase probably represented broken peroxisomes since other peroxisomal markers such as glycolate oxidase were solubilized to a 'similar extent. Frederick and Newcomb (37) used a cyto- chemical stain for catalase to demonstrate that catalase was confined to the peroxisomes of tobacco leaves. The low catalase level in the other organelle fractions such as the small peak of catalase with the chloroplasts was thought to be artifactitious. This may have been the result of some peroxisomes being trapped in the dense chloroplast band. Most of the chlorophyll, probably broken chloroplasts, accumulated between 40% and 41% sucrose. Approximately 60% of the cytochrome c oxidase, the mitochondrial marker, was located in a peak in the 42% to 49% sucrose region of the gradient. The estimated FIGURE 3. 47 Distribution of subcellular organelles from Spinach leaves on a sucrose density gradient. The gradient was constructed as indicated by the sucrose percentages. The organelles detected were the broken chloroplasts (chloro- phyll) which peaked at 230 ml, the peroxisomes (catalase) at 30 ml, the mitochondria (cytochrome c oxidase and NADH-cytochrome c reductase) at 140 m1, and the microsomes (NADH-cytochrome c reductase and cytochrome c oxidase) at 270 ml. Above 330 ml was the supernatant which extended to 530 ml. The specific activities of the peak fractions are given in Table IV., 50 48 E Sucrose 3 4O '- E 30 *- 3 20 - o 0. I0 .. l — Chlorophyll. '_ l.0- E X E' 0.5 — 2 .. Cytochrome c Oxidase | .- 'i. 5 I000— x 7 Catalase .E E x 500- ._ U) .2 E :1. , L i n NADH Cytochrome c Reductase .04 - .03 - .02 - .Ol - 1 I00 200 300 ' Volume (ml) 49 densities of the peak fractions are given in Table III. The Specific activities of the marker enzymes in the peak fractions are given in Table IV, page 51, along with the ratios of the specific activities in the other organelle fractions relative to the maximum Specific activity. These specific activity ratios are an indi- cation of the amount of cross contamination (17). For example, the specific activity of the cytochrome c oxidase in the peroxisomes was less than 5% of that in the mitochondria. That is, less than 5% of the protein in the peroxisomal fraction was mitochondrial contamini ation. However, the actual specific activityof the mitochondrial cytochrome c oxidase was probably higher since the mitochondria were contaminated with chloroplasts. Therefore, the actual mitochondrial contamination in the peroxisomes was probably lower than estimated. The chlorophyll data indicate than more than 30% of the pro- tein in the mitochondrial fraction was whole chloroplasts. Although, only 2% of the total chlorophyll on the gradient was in the peroxisomal fraction, this amounted to 20% contamination on a protein basis. 50 .mmma-3_aouwue snags omH.H MNHH.H moEomommq m¢H.H 0¢H.H omH.H mHH.H NH.H mQEomOHofiz wma.a wH.H coxoum .mummamouoaso NN.H HN.H among .mummfieoHOHnu an.H qom.H me.H HmH.H NN.H mwhpconoouwz MNN.H NHN.H mummamowum o¢N.H mmN.H omN.H m¢N.H RN.H mmwponouowz hmdex H0>HA GopmH%uoo Ehwmmopcm won umm Hm3ona5m cmmm uoummo somawmm .mucmwpmuw huwmamp mmouosm Eouw maowuomuw Mama can mo mooHpcw m>wuomummu wgu Eoum pofiwahmumn .OOH um mnao N w a“ mofiuwmamn .mucmfipmuw kuamcmp mmouosm cw modmmfiu muowum>_aoum mmHHmewuo HmHDHHoondm mo mowuflmcma .HHH mam000mhxouvhz 0000030 00000000o 00000000 00320 012000009 me x HI pofidom 0:0p0nuoanm 0300 0000 0800 0000 :08 X 00005: 000000 0800 .60 30.0004 00008.00 00.0.0004. 0.008.. 00 008.30 0.00000 0080 0000 00000 0000 0800 :00 000000000000 0:0p0cummdm podaom .2000000 0800 0000.. 000 000 3030000000 0800 0000.. 000. 0000000.0 0.0.. 00 pom.m,ooo.:00 p0 vowzm0upno0 0003 00:0u0au0m00 00:00E00 0&9 :.00000m 000m0 300: 0:» 000m on.m ooo.~0 00 0005000020000 000 00000000 0mm 00 000§0900000 003 000003 0:0000000 000 .0 000.00 00 00.. 00 .80 0000000008 00... 30000000 00.0 428.000 000 000 .0 .0 00 .0009.2 moo.o 00:00m0 00000000 000 00000000 0003 0:00000w 0000090 0 no 0000090 no - 0mm 5000 00000000 020000000 008000N000A 0:9 .008000xonom H000 £00c0m0 no 0000000000000000 .Hx.mqm MICRGBODIES / \-\ OUTER MITOCHONDRIAL MEMBRANE OUTER CHLOROPLAST MEMBRANE GOLGI\ -¢ \LYSOSOMES .SECRETORY ZYMDGEN VESICLES GRANULES \ / BLASMA MEMBRANE FIGURE 12. 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