Glyoxylate cycle in the epiphyseal growth plateIsocitrate lyase and malate synthase identified in mammalian cartilage.код для вставкиСкачать
THE ANATOMICAL RECORD 223:357-362 (1989) Glyoxylate Cycle in the Epiphyseal Growth Plate: lsocitrate Lyase and Malate Synthase Identified in Mammalian CartiI age WALTER L. DAVIS, RUTH G. JONES, GENE R. FARMER, J.L. MATTHEWS, AND DAVID B.P. GOODMAN The Department of Anatomy, Baylor College of Dentistry, Dallas, Texas 75246 (W.L.D., R.G.J., G.R.F.); The Baylor Research Foundation, Baylor University Medical Center, Dallas, Texas 75246 1J.L.M.I; The Department of Pathology and Laboratory Medicine, The University of Pennsylvania Medical School, Philadelphia, Pennsylvania 19104 (D.B.P.G.) ABSTRACT Peroxisomes were identified in chondrocytes from all zones of the mammalian epiphyseal growth plate by using light microscopic techniques for the cytochemical demonstration of catalase, the marker enzyme for these organelles. Additional cytochemistry showed the presence of malate-synthase-positive structures within the chondrocytes. The latter enzyme, also associated with peroxisomes, is unique to the glyoxylate shunt, a metabolic pathway thought to be absent in vertebrate tissues. The glyoxylate cycle allows the net conversion of lipid to carbohydrate, i.e., gluconeogenesis. Biochemical studies on growth plate cartilage indicate that this tissue has the capacity to carry out cyanide-insensitive B-oxidation of fatty acids. The latter takes place in a nonmitochondrial compartment, most likely the peroxisomal compartment. Additionally, both of the unique enzymes associated with the glyoxylate cycle, i.e., isocitrate lyase and malate synthase, were also identified in a cell-free homogenate of this cartilage. These studies indicate that cartilage, a poorly vascularized tissue characterized by its low oxygen tension and anaerobic glycolysis, may have the capacity to convert lipid to carbohydrate, i.e., gluconeogenesis via the glyoxylate pathway. In this way, cartilage may be unique among mammalian tissues. Rat liver peroxisomes are capable of cyanide-insensitive B-oxidation of fatty acids (Lazarow, 1978). In plants, catalase-positive specialized peroxisomes known a s glyoxysomes contain enzymes that not only catalyze B-oxidation via a cyanide-insensitive process but also function in the glyoxylate cycle or shunt (Canvin and Beevers, 1961; Cooper and Beevers, 1969a,b; Beevers, 1980, 1982). The two enzymes unique to the glyoxylate cycle permit the net conversion of acetyl-CoA derived from lipid to hexose (Kornberg and Krebs, 1957; Kornberg and Beevers, 1957). These enzymes are 1) isocitrate lyase (threo-D:]-isocitrate glyoxylate-lyase, EC 22.214.171.124) and 2) malate synthase (L-malate glyoxylate lyase (CoA acetylating, EC 126.96.36.199). It is via the glyoxylate cycle that the two decarboxylative steps in the tricarboxylic acid cycle (isocitrate dehydrogenase and a-ketoglutarate dehydrogenase) can be bypassed allowing the conversion of two-carbon units (acetyl-CoA) to four-carbon acids (such as succinate) instead of the oxidation of 2C-units to COa. Prior to 1980, it had been thought that this metabolic pathway was absent from animal cells, being found only in higher plants, certain unicellular organisms, a few nematodes, and some fungi. However, in 1980, we reported the presence of isocitrate lyase and malate synthase, the enzymes unique to the glyoxylate shunt, in a vertebrate system, the epithelium of the amphibian toad urinary bladder (Goodman et al., 1980; ic! 1989 ALAN R. LISS, INC. Jones et al., 1981,1982).Since this time we have identified malate synthase in other vertebrate tissues by using purely cytochemical techniques (Davis et al., 1986a,b). Such morphologic results suggest that this metabolic pathway may be present in other vertebrate tissues. Cartilage is characterized by a low oxygen consumption (Laskin et al., 1952) and a preponderant reliance on anaerobic glycolysis (Picard and Cartier, 1960). In the epiphyseal growth plate, in the regions closest to a n oxygen supply, i.e., the hypertrophic and calcified zones, gluconeogenesis can apparently take place resulting in a n increase in glycogen (Fitton-Jackson, 1964) and a n associated concomitant decrease in mono, di-, and triglycerides (Irving and Wuthier, 1968).This type of activity is suggestive of the transformation of lipid to carbohydrate and may thus require a n active glyoxylate shunt. Such observations prompted the present biochemical and cytochemical studies on the mammalian epiphyseal growth plate cartilage. MATERIALS AND METHODS A total of 12 weanling 125-g Sprague-Dawley rats were used for this study. Each animal was singly Received September 28, 1987; accepted September 8, 1988. Address reprint requests to Prof. Walter L. Davis, Department of Anatomy, Baylor College of Dentistry, Dallas, TX 75246. 358 W.L. DAVIS E T AL. Fig. 1. Light microscopic section from t h e r a t epiphyseal growth plate. Tissue section was incubated in t h e complete medium for t h e demonstration of t h e peroxisomal enzyme catalase. The dark cytoplasmic granules (arrowheads) indicate the presence of catalase-positive peroxisomes in the chondrocytes. Section unstained. X 400. Fig. 2. Section of mammalian growth plate cartilage prepared for cytochemistry a s in Figure 1 above. The arrowheads again indicate catalase-positive peroxisomes within the cytoplasm of t h e chondrocytes. Section unstained. x 400. Fig. 3. Light photomicrograph of growth plate cartilage from a section incubated in a n incomplete catalase medium, devoid of hydrogen peroxide (cytochemical control). No dense granules a r e seen within the chondrocytes. Section stained with toluidine blue. x 600. Fig. 4. Section of the mammalian growth plate from a specimen incubated in the complete medium for t h e demonstration of t h e enzyme malate synthase. Numerous dark granules a r e seen in the cytoplasm of the chondrocytes (arrowheads). Such structures are indicative of peroxisomes which contain t h e glyoxylate shuttle enzyme malate synthase. Section unstained. x 400. Fig. 5. Section of r a t growth plate prepared a s in Figure 4 above. Malate-synthase-positive granules (arrowheads) a r e again seen in the chondrocytes. Section unstained. x 400. Fig. 6. Section of t h e epiphyseal growth plate from a tissue sample incubated i n a n incomplete malate synthase medium devoid of t h e substrate glyoxylate. No granular structures a r e seen. Section unstained. x 400. GLYOXYLATE CYCLE IN EPIPHYSEAL GROWTH PLATE 359 housed a t 72°F with a 12-hrl12-hr day-night cycle and tilage and the metaphysis were removed. Twenty approximately 0.5-mm-thick slices of the growth plate allowed food and water ad libitum. For cytochemical studies animals were sacrificed by were prepared from each femur and maintained in cervical dislocation and then perfused (intracardiac) chilled mammalian Ringer’s solution. To prepare a tiswith the following freshly prepared fixative: 5.25% ex- sue extract tissues were frozen and thawed five times tensively purified glutaraldehyde, 0.1 M cacodylate in a calcium-free Ringer’s solution. A 33% (wtivol) hobuffer (pH = 7.41, 0.5% calcium chloride, and 4.0% mogenate was prepared in a Potter-Elvehjem homogesucrose. Following perfusion, the proximal epiphysis nizer. The homogenate was then centrifuged a t 3,OOOg from each femur was dissected free, removed, and for 20 min a t 4°C. The resulting supernatant was used stripped of attached soft tissues. Next, the head of each as the tissue extract. The oxidation of palmitoyl-CoA was assayed by the femur was split longitudinally and the resulting sections were placed under the fixative described above. spectrophotometric reduction of NAD a t 340 nm (LazWhile under fixative, the articular cartilage and the arow and DeDuve, 1976). The reaction cuvette conmetaphysis of each sample were removed. The remain- tained, in a total volume of 3.0 ml, the following (final ing growth plates were then sectioned, with a fresh concentrations indicated): 1) 30 mM potassium phosrazor blade, into thin slices each approximately 0.5 mm phate, pH = 7.4; 2) 1.0 mM KCN; 3) 1.0 mM MgC12; 4) thick. These were then allowed additional fixation time 0.1 mM NAD; 5 ) 6.0 mM dithiothreitol; and 6) 10 pM (in the same fixative), usually a total of 2-4 hr, before palmitoyl-CoA. This assay was carried out in a Gilford transfer to a n overnight wash in chilled ( P C ) , freshly model 2000 recording spectrophotometer a t room temprepared 0.1 M cacodylate buffer, pH 7.4, containing perature. Two methods were used to measure isocitrate lyase 0.05% calcium chloride and 5.0% sucrose. This procedure was utilized for the cytochemical demonstration of activity (Dixon and Kornberg, 1959; Cook and Carver, catalase activity. Growth plates from two rats were 1966). In the first procedure, the reaction medium contained the following (final concentrations indicated) in used. For the localization of malate synthase activity the a total volume of 3.0 ml: 1)66 mM imidazole, pH 6.2; 2) above procedure was used for fixation except the per- 12 mM MgC12; 3) 2.0 mM dithiothreitol; 4) 20 mM of fused fixative was freshly prepared cacodylate-buffered freshly neutralized semicarbazide hydrochloride; and formaldehyde-glutaraldehyde (Trelease et al., 1974) 5 ) 3.3 mM isocitrate. The reaction was initiated by the rather than glutaraldehyde. Two rats were also uti- addition of glyoxylate. The formation of glyoxylate semicarbazone was monitored at 252 nm. The molar lized for this cytochemical study. Following the refrigerated-overnight buffer wash extinction coefficient of glyoxylate semicarbazone was and three additional chilled buffer washes, the glutar- then taken as 12,400 at 252 nm (Olson, 1968). aldehyde-fixed growth plate “slices” were quick frozen For the second procedure, the total volume of the in a cryostat (IEC, model CTI, Needham, MA). To dem- final reaction medium was again 3.0 ml. The latter onstrate peroxisome catalase activity with the light contained (final concentrations indicated) 1)66 mM pomicroscope, 50pm sections were cut with the cryostat. tassium phosphate, pH 6.85; 2) 5.0 mM MgCI2; 3) These sections were then incubated according to estab- 3.3 mM phenylhydrazine-HC1; 4) 2.0 mM cysteine-HC1; lished procedures (Novikoff et al., 1972; Jones et al., and 5 ) 3.3 mM isocitrate. The reaction was started by 1981). For cytochemical controls, several sections were the addition of isocitrate and monitored a t 324 nm by incubated in a n incomplete reaction medium devoid of the formation of glyoxylate phenylhydrazone. either hydrogen peroxide or diaminobenzidine (DAB). Malate synthase activity was measured by the proTo demonstrate malate synthase activity with the cedure of Cook (1970). The reaction medium contained light microscope the procedure of Trelease (Trelease et (final concentrations indicated), in a total volume of al., 1974; Trelease, 1975) was employed, as previously 3.0 ml, the following: 1)80 mM Tris-HC1, pH = 8.0; 2) reported (Goodman et al., 1980; Jones et al., 1982; 6.6 mM MgC12; 3) 24 pM acetyl-CoA; and 4) 1.5 mM Davis e t al., 1986a). Sections (50 pm thick) of the form- glyoxylate. The reaction was initiated by the addition aldehyde-glutaraldehyde-fixedtissues were prepared of glyoxylate. The disappearance of acetyl-CoA was foland subsequently incubated in the complete medium. lowed a t 232 nm. For this assay the reaction mixture For cytochemical controls, either acetyl-CoA or gly- was first preincubated in the absence of glyoxylate unoxylate was deleted from the incubation medium. In- til a stable absorbance at 232 nm was observed. This cubation in the absence of glyoxylate is especially nec- allowed for the completion of any acetyl-CoA cleavage essary in order to show the absence of nonspecific due to the presence of enzymes such a s thiolase or transacetylase in the homogenate. Following this, malate deacylase activity. Postincubation, all tissue sections were washed three synthase activity was monitored as a further decrease times in deionized water, osmicated, and dehydrated in absorbance at 232 nm caused by the addition of glyprior to flat embedment in low-viscosity resin. Plastic oxylate to the reaction medium. Each of the enzyme tissue sections 0.5-1.0 pm in thickness were cut with assays was carried out a t three concentrations of tissue freshly prepared glass knives on a Porter-Blum MT-2B extract (100, 200, and 400 pl). However, the results ultramicrotome. Sections were studied and photo- presented are based on a volume of 100 pl of tissue extract. graphed in a Zeiss Photomicroscope 111. As a further assessment of the presence of glyoxylate For the biochemical analyses of the enzymes isocitrate lyase and malate synthase in epiphyseal cartilage, cycle enzymes, the conversion of 1 I4C I-isocitrate to animals were sacrificed by cervical dislocation. The [ 14CI-glyoxylate and I l4C: I-glyoxylate t o I “C I-malate, proximal femoral heads were dissected free, removed, respectively, by the tissue homogenate was determined and cleaned of adhering soft tissues. The articular car- (Jones, 1980). 360 W.L. DAVIS ET AL. To measure the conversion of isocitrate to glyoxylate, the reaction mixture contained the following: 1)75 mM Tris-HC1 (pH 7.0); 2) 4.0 mM MgC12; 3) 0.2 mM dithiothreitol; 4) 5.0 mM of [l,5-'4C1-isocitrate; and 5 ) 0.1 ml of the cartilage homogenate. The total volume of this reaction system was 1.0 ml. The reaction was initiated by the addition of isocitrate. The incubation was carried out a t 30°C and was stopped by the addition of 0.4 M HC104. For controls, the above incubation medium contained either a n additional 2.0 mM acetylCoA or a n additional 10 mM EGTA. For the conversion of glyoxylate to malate, the reaction mixture contained the following: 1) 100 mM Tris-HC1 (pH 7.7); 2) 12.0 mM MgC12; 3) 2.0 mM [l-14C1-glyoxylate; 4) 0.2 mM acetyl-CoA; 5) 1.0 mM acetylphosphate; 6 ) 2 units of phosphotransacetylase; and 7) 0.5 ml of the cartilage homogenate. The final volume was 1.0 ml. The conversion was started by the addition of glyoxylate and allowed to continue at 30°C. As above, the reaction was stopped by the addition of 0.4 M HC104. The control systems were either devoid of acetyl-CoA or contained an additional 10 mM EGTA. (14C1-glyoxylateand malate were extracted according to the following procedure. The HCIO, extracts were first neutralized with KOH. Next, the organic acids were separated on Dowex 1-X8 (Cl-1, 200-400 mesh. The later was previously washed with water to remove impurities and acids. Both the malate and glyoxylate fractions were treated with 0.1 ml of 50 mM p-nitrophenylhydrazine in 10 ml of H2S04.After shaking for 60 min a t room temperature, the glyoxylatep-nitrophenylhydrazone was extracted 3 x with ethylacetate. The aqueous sample was neutralized with KOH. Both the aqueous and ethylacetate fractions were placed in Aquasol and subsequently counted for [14C]-glyoxylateand [14C]-malate in a Packard model 3385 liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL). sues reacted in an incomplete malate synthase medium, devoid of either acetyl-CoA or glyoxylate, no reaction product, and thus no dense granules, were seen in the chondrocytes (Fig. 6). Biochemical Analyses An extract prepared from epiphyseal cartilage possesses the ability to reduce NAD in the presence of cyanide and upon the addition of palmitoyl-CoA, i.e., cyanide-insensitive fatty acid oxidation. The rate of this reaction was 0.41 nmol of NADH formediminilO0 p.1 of cartilage extract. The glyoxylate shuttle contains two characteristic or unique enzymes: 1)isocitrate lyase and 2) and malate synthase. The former was measured by the formation of glyoxylate semicarbazone (assay I) upon the addition of the substrate isocitrate. Since it is possible that this assay for isocitrate lyase activity could be interfered with by isocitrate dehydrogenase activity-i.e., isocitrate addition could give rise to a-ketoglutarate which also forms a semicarbazone-a second method method was employed to measure glyoxylate activity (assay 11). The latter involved the formation of glyoxylate phenylhydrazone. Under the reaction conditions used, the reactivity of glyoxylate was considerably greater than that of a-ketoglutarate. Using these two different derivatization procedures to quantitate glyoxylate formation, the observed rates of isocitrate lyase activity were 0.14 and 0.15 nmoliminilO0 p+lcartilage extract for assay I and assay 11, respectively. In addition, when NADH was added to the reaction mixture in order to inhibit a-ketoglutarate dehydrogenase activity, the rate of rate of phenylhydrazone formation was comparable to the rate of formation of this derivative in the absence of added NADH. This would appear to indicate that the activity observed was due to the presence of the enzyme isocitrate lyase. Malate synthase activity was measured by the disappearance of acetyl-CoA. The activity of this enzyme was monitored as the decrease in absorbance a t 232 nm RESULTS caused by the addition of glyoxylate to the reaction Cyfochemistry mixture. Under the conditions employed, 2.7 nmol of In tissue sections incubated in the standard DAB acetyl-CoA was cleavediminilO0 p.1 of tissue extract. Homogenates of rat epiphyseal rowth plate cartimedium for the localization of catalase activity, numerous dense granules were seen in the cytoplasm lage were able to convert 11,51F14C)-isocitrate into of the chondrocytes (Figs. 1, 2). Such granules were 14C-glyoxylate.This conversion was depressed 67% by seen in both the flattened chondrocytes of the upper the addition of acetyl-CoA and 89% by the addition epiphyseal plate as well as in the ovoid (hypertro- of EGTA to the incubation medium. Cartilage homophied) chondrocytes of the lower epiphyseal plate. genates also converted [ l-14C]-glyoxylate into When the tissues were incubated in a n incomplete I4C-malate. This conversion required acetyl-CoA and medium devoid of either hydrogen peroxide or DAB, was also inhibited by EGTA. The rate of this converno dense granules were seen in the chondrocytes from sion was decreased by 75% when acetyl-CoA was not all zones (Fig. 3). Such cytochemical data indicate that present in the incubation medium. In the presence of the dense granules are cytoplasmic organelles known EGTA, the rate of conversion was depressed by 92%. as peroxisomes. The latter are characterized by their DISCUSSION catalase activity. When cartilage sections were incubated in the comThe cytochemical data in the current study show plete medium for the cytochemical localization of the that the chondrocytes of the mammalian epiphyseal glyoxylate cycle enzyme malate synthase, dense gran- growth plate contain numerous ovoid organelles ules were again seen in the cytoplasm of growth plate known as peroxisomes. These organelles are characterchondrocytes (Figs. 4, 5). As above, these structures ized by their catalase activity. The latter enzyme, genwere identified in both the flattened chondrocytes of erally regarded as the marker enzyme for these orthe upper epiphyseal plate and in the rounded chon- ganelles, is responsible for the metabolism of hydrogen drocytes of the lower growth plate (Figs. 4, 5). In tis- peroxide (DeDuve, 1969; DeDuve and Baudhuin, 1966). GLYOXYLATE CYCLE IN EPIPHYSEAL GROWTH PLATE 361 Peroxisomes also contain hydrogen-peroxide-generating oxidases such a s uricase (urate oxidase), D-amino acid oxidase, and L-a-hydroxy acid oxidase iDeDuve, 1969; DeDuve and Baudhuin, 1966). In a previous cytochemical study, we have shown the presence of uricase in growth plate chondrocytes (Davis et al., 1986a,b) by using a coupled peroxidatic method. The presence of peroxisomes in mammalian chondrocytes should not be surprising since these organelles are thought to be ubiquitous throughout the animal kingdom (Novikoff e t al., 1973; Novikoff and Novikoff, 1973). The cytochemical demonstration of malate synthase activity in chondrocytes, using the copper ferrocyanide procedure, represents, to our knowledge, the first description of this enzyme in mammalian cells. Malate synthase is unique to the glyoxylate cycle (Beevers, 1980,1982). For many years this biochemical pathway was thought to be absent from vertebrates, being found primarily in higher plants in association with specialized peroxisomes known as glyoxysomes. However, beginning in 1980 and thereafter, we showed the presence of the glyoxylate cycle enzymes (isocitrate lyase and malate synthase) in vertebrate (amphibian) urinary bladder epithelial cells (Goodman e t al., 1980; Jones e t al., 1981, 1982). More recently, malate synthase activity has also been localized cytochemically in the microperoxisomes from the adipocytes of the amphibian fat body (Davis e t al., 1986a,b).In the present study, both isocitrate lyase and malate synthase activity were identified in the cartilage homogenate. Isocitrate lyase, plus malate synthase, allow the carbons derived from acetyl-CoA to bypass the decarboxylative steps within the tricarboxylic acid cycle (TCA cycle). In this way, acetyl-CoA, derived from fatty acid oxidation, can be converted to carbohydrate (Kornberg and Krebs, 1957; Kornberg and Beevers, 1957; Beevers, 1969, 1980, 1982). In this shuttle, glyoxylate, formed a s a product of the isocitrate lyase reaction, functions as a carbon carrier. Glyoxylate also serves as the substrate for the malate synthase reaction. In the present communication, we have demonstrated the presence of peroxisomes in the chondrocytes of the mammalian epiphyseal cartilage. These organelles were identified in the chondrocytes from all zones of the growth plate. With this in mind, i t is necessary to identify a function or functions for these organelles in the cells of this specialized connective tissue. To begin with, there remains much speculation regarding the rolels) of the peroxisome in cellular physiology and biochemistry (Beaufay et al., 1959, 1964; DeDuve et al., 1960; DeDuve and Baudhuin, 1966; DeDuve, 1969; Hogg, 1969; Kind1 and Lazarow, 1982). Multiple functions have been advanced for these organelles. Some of these will now be briefly described. may provide oxygen for cell respiration. This may be especially important in epiphyseal cartilage since this tissue is characterized by a low exogenous oxygen consumption (Laskin et al., 1952) and a reliance upon anaerobic glycolysis for its energy supply (Picard and Cartier, 1960). The fact that oxygen tension levels can affect various biochemical processes in cartilage, including collagen synthesis and osteogenesis, has been reported (Stern et al., 1966; Brighton e t al., 1969). 3 . More recently, it has been shown that fatty acid oxidation to acetyl-CoA can take place in rat liver peroxisomes (Lazarow, 1978). This activity is insensitive to cyanide and thus represents a potential pathway for the B-oxidation of fatty acids that does not require mitochondrial metabolism. As demonstrated in the present study, mammalian cartilage also seems to possess this same capability. Thus mammalian cartilage is capable of carrying out cyanide-insensitive fatty acid oxidation. To our knowledge, this ability has not been previously reported for cartilage. 4. In plants, specialized peroxisomes referred to as glyoxysomes not only possess the ability to catalyze the B-oxidation of fatty acids but also utilize the glyoxylate cycle for the net conversion of lipid to carbohydrate and thus contain the unique enzymes isocitrate lyase and malate synthase described above. Previously, these two enzymes were thought to be present only in unicellular animals, certain nematodes, and higher plants (Hogg, 1969; Tomlinson, 1967; Rothstein and Mayoh, 1965, 1966; Beevers, 1969,1980,1982).In 1980, however, we showed the presence of the glyoxylate cycle enzymes in a vertebrate system-the amphibian urinary bladder (Goodman et al., 1980; Jones et al., 1981, 1982). Here, we describe the presence of these enzymes in a higher vertebrate tissue, mammalian cartilage. Thus, cartilage peroxisomes may be involved in gluconeogenesis directly from fatty acids, i.e., the conversion of lipid to carbohydrate, a capability thought for many years to be absent from vertebrate tissues. Such a n observation may explain the fact that in the epiphyseal growth plate there appears to be a n increase in glycogen and a decrease in lipid in both the hypertrophic and calcified zones (Fitton-Jackson, 1964; Irving and Wuthier, 1968). Additionally, it should be mentioned that the enzyme catalase, which composes the major protein component of peroxisomes, is characterized by its peroxidatic activity. Interestingly, i t has been shown that peroxidase, when injected into experimental animals, has a hypolipidemic effect (Caravaca and May, 1964; Caravaca e t al., 1967). Thus, cartilage peroxisomes may possess a second mechanism, involving peroxidase, for the net conversion of lipid to carbohydrate, i.e., gluconeogenesis. Electron microscopic studies by our group have shown chondrocytes to contain considerable lipid (Carson et al., 1978). 1. Since various hydrogen-peroxide-generating oxidases are compartmentalized within the peroxisomes (see above) and since hydrogen peroxide is highly toxic to cells, the segregation of hydrogen-peroxide-generating oxidases with a n enzyme that disposes of hydrogen peroxide (catalase) might be highly advantageous to the cell. 2. The catalytic reaction of peroxisomal catalase In summary, from the present investigation it is apparent t h a t mammalian epiphyseal cartilage may represent a n ideal tissue with which to study the effects of oxygen tension, hydrogen peroxide formation, and gluconeogenesis on the differentiative events that take place between the resting cell zone and the zone of calcification. Obviously, additional experimentation is required. 362 W.L. DAVIS E T AL. ACKNOWLEDGMENTS This research was supported in part by funds allocated by Mr. and Mrs. Thomas Bedford of Fort Worth, Texas, and also by a grant from the General Dynamics Corporation of Fort Worth, Texas. The excellent technical assistance by K. Shibata, T. Kurokawa, and R. Evers is acknowledged. LITERATURE CITED Beaufay, H.. D.S. Bendall, P. Baudhuin, and C. DeDuve 1959 Tissue fractionation studies. 12. Intracellular distribution of some dehydrogenases, alkaline deoxyribonucleasc and iron in r a t liver tissue. Biochem. J., 73:623-628. Beaufay, H., P. Jacques, P. Baudhuin, O.Z. Sellinger, J. Berthet. and C. DeDuve 1964 Tissue fractionation studies. 18.Resolution of mitochondria1 fraction from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients. Biochem. J., 92:184-205. Beevers, H. 1969 Glyoxysomes of castor bean endosperm and thcir relationship to gluconeogenesis. Ann. NY Acad. Sci. USA, 168.313 -324. 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