030S.().I91 ;80,090 1-0327502.00,0 (jllt,r.. Bj(J(-ht'm.Php.ld/.. Vol. 61~ pp-.327 to 332. . rcrj;:l. moo Press Ltd 1980. Prmted In Great Bnt.11n 0

SUBCELLULAR LOCALIZATION OF MONOGLYCERIDE ACYLTRANSFERASE,. XANTHINE OXIDATION, NADP: ISOCITRATE DEHYDROGENASE AND ALKALINE IN THE MUCOSA OF THE GUINEA-PIG SMALL INTESTINE

p, A, MARTIN, N, J. TDIPLE and M. 1. CO:-:KOCK Department of Biological Scienccs, The Polytechnic, Wolverhampton WVI IL Y, England

(Receil"ed II Jallt/aT)' 1980)

Abstract-I. Rate dependent and isopycnic banding in a zonal rotor were used to analyse the subcellular sites of enzymcs in homogenates of guinea-pig small intestinal mucosa. 2, The results demonstrate the following localizations: monoglyceride acyltransferase-microsomal; xanthine oxidase and dehydrogenase-soluble phase, and NADP: isocitrate dehydrogenase-soluble phase and mitochondrial. 3, is confined to brush borders and is absent from the basolateral plasma membrane. A variable proportion of the activity, up to 40%, is on brush borders which during homogen- ization break up into particles of reduced density and slow sedimentation rate.

INTRODUcnO;,\ :\IATERIALS AND METHODS

Microperoxisomes are assumed to be a special type of Chemicals ami peroxisome and are apparently ubiquitous in mam- These were mostly bought or prepared as described pre- malian tissues (NovikolT et a1., 1973; Hruban et a1., viously (Martin er al., 1979). [9,1O-3H]palmityl-( -)- 1972). In contrast to the majority of the peroxisomes carnitine was prepared using [9,1O.3H]palmitic acid. Before use it was diluted with palmityl-( of the liver and kidney, microperoxisomes have no - }-carnitine, simi- larly prepared (Sanchez er ai, 1973). Alumina and DL-Iactic core, are almost invariably smaller (NovikolT et a1., acid were purchased from BDH Chemicals Ltd, Poole, 1973) and, in addition, have been much less exten- Dorset, U.K. Cytochrome c and NADP were from Bohr- sively investigated. inger, (BCL). Bell Lane, Lewes, East Sussex, U.K. Antimy- It is possible that in some tissues enzymes origi- cin A, DL-isocitric acid (sodium salt, type I), homovanillie nally reported as "microsomal" or "mitochondrial" acid, I-monopalmitylglycerol, p-nitrophenyl phosphate are in reality partially or wholly microperoxisomal or (ditris salt), peroxidase (typc II from horseradish) and xan- peroxisomal. Recent studies of rat liver provide thine were from Sigma Chemical Co" Ltd. Glycollic acid several instances of this including palmitoyl-CoA syn- was purchased from Hopkin and Williams, P.O. Box I, thetase (Lippel et a1., 1970; Krisans & Lazarow, 1978), Romford, Essex, U.K. carnitine acetyltransferase (Norum & Bremer, 1967; P;epararion of homogenate and subcellular fracrionation Markwell et a1., 1977), dihydroxyacetone phosphate The procedures employed were as described previously acyltransferase (La Belle & Hajra, 1972; Jones & (Temple et al., 1976). Subcellular fractions were brought to Hajra, 1977), and those enzymes responsible for the the same sucrose concentration before analysis. P-oxidation of palmitoyl-CoA (Lazarow, 1977). Most previously reported fractionation studies of Biochemical allalyses the mammalian intestine have ignored the microper- The following were assayed as described pre\'iously: oxisomes. Conscious of possible erroneous localiz- alkaline phosphatase and (substrate for ations resulting from this and also of the need to both. I-naphthyl phosphate) (Connock et al., 1974), identify peroxisomal enzymes in tissues additional to glueose-6-phosphatase (Connock er 01., 1972), I-naphthyl acetate esterase (non-specific esterase) and succinate de- mammalian liver, we have determined the localization hydrogenase (Temple et 01., 1976~ NADP: isocitrate de- of possible microperoxisomal enzymes in the mucosa hydrogenase (with 6.6mM isocitrate and 0.071% wlv Tri. of both the guinea-pig 'small intestine (Temple et aI" ton X-lOO) and NAD: xanthine dehydrogenase (with 1976; Martin et a1., 1979) and the goldfish intestine 0.26 mM xanthine and no Triton X-lOO) (Temple et al., (Temple et a1., 1979)- In the goldfish intestine 1{}-20% 1979).Other assays were as follows: K + (potassium stimu- of the carnitine acetyltransferase is microperoxisomal lated) p-nitrophenyl phosphatase (Garrahan et al., t969; but the other enzymes studied (including xanthine Muree et 01.,1976), catalase (Baudhuin et 01.,1964), NADH oxidase, xanthine dehydrogenase and NADP linked cytochrome c reductase ( sample preincubated isocitrate dehydrogenase) are apparently absent from 5 min at O'C with 321,g/ml antimycin A where indicated) these organelles. We now report the localization of (Donaldson et aI., 1972), protein (Lowry et al., 1951) and DNA (Burton, 1956). L-:x-hydroxyacid oxidase (12.5 and the latter three enzymes in the guinea-pig small intes- 52 mM DL-laetate, and 5.5 and 24 mM glycollate as sub- tinal mucosa. We also studied monoglyceride aeyl- strates) and xanthine oxidase (with 0.74 mM xanthine) were . .None of these enzymes were found to assayed by methods dependent on measuring the produc- have a microperoxisomal component. tion of hydrogen peroxide (Guilbault et al., 1968; Gaunt & 327 328 P. A. MARTI);, N. J. TEMPLE and M. J. CO~IWCK

De Duve, I 976}. Incubations were for 60 min and allow- dehydrogenase for mitochondria, alkaline phosPha. ances were made for the production of hydrogen peroxide tase for brush borders, acid phosphatase for Iyso- independent of exogenous substrate. Xanthine oxidase only somes, I-naphthyl acetate esterase, NADH cyto. was detectable. Monoglyceride acyl transferase was assayed chrome c reductase and gJucose 6-phosphatase for essentially according to Short et af. (1974). The incubation microsomes (ER component), DNA for nuclei and was for 6 to 10 min in a medium which included 2 mM K+.p-nitrophenyl phosphatase for basolateral plasma I-monopalmitylglycerol, 70 JIM CoA, 100 JIM membranes of enterocytes. Microperoxisomes (9,IO.JH]palmityl-( - }-carnitine (0.11 JlCilJlmol) and up to have 60 JIg sample protein. To reduce quenching due to chloro- equilibrated in region "d" at a lower density than the form the final column eluate was evaporated to dryness nuclei (region "e") but higher than the mitochondria and then redissolved in a small volume of 5% wlv Triton (region "c"). The microsomes span regions "c" and X-loo. An aliquot was measured by liquid scintillation "d" and the more dense part of region "b". The distri- counting in 10 ml of Triton X-loo: xylene scintillant (Mar- bution of monoglyceride acyltransferase resembles tin et al., 1979). Incubations for all enzyme assays were at that of the microsomal markers. Xanthine oxidase 37'C except NADH cytochrome c reductase (20'C) and and dehydrogenase and most of the isocitrate de- catalase (O'C). hydrogenasehave remainedin the sample layerindio Presentation of results cating that they are soluble phase. Some of the iso- Data for distribution of enzymes in subcellular fractions citrate dehydrogenase is distributed similarly to the are presented in the form of histograms as described by De mitochondrial marker. Duve (1967). The ordinates represent the relative concen- Briefcentrifugationof homogenateinto a shaUow tration of the enzyme in the fraction. This is defined as the sucrose gradient (Fig. 2) gave a separation of organ- concentration of the biochemical in the fraction divided by eUes dependent on their rate of sedimentation. the concentration if it were distributed uniformly through. NADH cytochrome c reductase revealed some mito- out the total analysed volume unloaded from the rotor. chondrial activity (discontinuous line) which was The ordinate for density is expressed as glm!. The abscissae mostly inhibited by preincubation of each sample are divided according to 'the percentage \'olume of the frac- tions. with antimycin A (continuous line). The distributions of monoglyceride acyltransferase and of isocitrate de- RESULTS hydrogenase confirm that the former is microsomal and the latter mainly soluble phase and partially Figure I depicts the distribulion of enzymes after mitochondrial. isopycnic fractionation of homogenate in a sucrose The plasma membranemarker used here was K+ gradient contained in a BX1V zonal rotor. Markers stimulated p-nitrophenyl phosphatase. This enzyme used were catalase for microperoxisomes, succinate carries out the second part of the Na + -K+ stimulated

2

Fig. 1. Distribution of markers and enzymes after isopYI:nic banding. 23 ml of homogenate was loaded onto 450 m) of a 15-50% (wjw) linear sucrose gradient. Centrifugation was for 13.5 hr at 25,000 rpm in a BXIV aluminium zona) rotor. Twenty fractions were unloaded and analysed for the following: (A) alkaline phosphatase (94%); (B) K + p-nitrophenyl phosphatase (79%); (C) acid phosphatase (98%); (D) xanthine oxidase (continuous line, 120%) and xanthine dehydrogenase (discontinuous line, 168%); (E) protein (1I5%); (f) ).naphthyl acetate esterase (85%); (G) glucose-6-phosphatase (76%); (H) NADH cytochrome c reductase (102%); (I) monoglyceride acyllransferase (102%); (J) catalase (l28%); (K) den. sity; (L) succinate dehydrogenase (95%); (M) isocitrate dehydrogenase (98%); (N) DNA (138%). Vertical columns labelled "a" to "en represent regions of the gradient discussed in the text. Percentage-figures represent the recovery of activity from the rotor relative to that loaded. . E) 2 \.2 2J 1.1 . 100 1.0 100 ~ 100 Fig. 2. Distribution of markers and enzymes after rate-dependent banding. IS ml of homogenate was loaded onto 350 ml of 13-30% (w/w) linear sucrose gradient resting on 100 ml of 40-50% (w/w) linear sucrose gradient. Centrifugation was for 45 min at 16,000 rpm in an aluminium BXIV 20nal rotor. Twenty fractions were unloaded and analysed for the following: (A) alkaline phosphatase (85%); (B) K + p-nitrophenyl phosphatase (108%); (C) acid phosphatase (95%); (D) protein (97%); (E) density; (F) I-naphthyl acetate esterase (60%); (GJ NADH cytochrome c reductase. Analyses included (continuous line, 107%) or excluded (discontinuous line, 90%) antimycin A. (H) monoglyceride acyltransferase (64%); (I) succinate dehydrogenase (90%); (1) isocilrale dehydrogenase (101%); (K) catalase (111%).

ATPase reaction (Garrahan er 01., 1969). A reason for including K+ p-nitrophenylphosphatase, was assaying K + p-nitrophenyl phosphatase rather than obtained (selected distributions shown in Fig. 3, Na + -K+ -ATPase is that p-nitrophenyl phosphate is' column a). The distributions of xanthine oxidase and more resistant than ATP to hydrolysis by non-specific dehydrogenase confirmed that both have a soluble . phase localization. Two new samples were then pre- In both experiments(Figs I and 2) K + p-nitro- pared by pooling aliquots of several fractions. Both phenyl phosphatase revealed a complex distribution samples were subjected to isopycnic centrifugation suggesting a large contribution from the subceUular (Fig. 3, columns b and c). Each gradient partially fragments containing the brush borders plus another resolvedK+ p-nitrophenylphosphataseand alkaline carrier of the enzyme. The latter migrates slowly caus- phosphatase into separate peaks with median ing it to be spread across the long, shaUowgradient in densities of 1.155and 1.183(sample i, Fig. 3b) or 1.173 Fig. 2 but equilibrates at a density of 1.155gjml Gunc- and 1.195g/ml. respectively (sample ii, Fig. 3e). This tion of regions "b" and "c", Fig. 1). This carrier is indicates that the slow moving carriers of both therefore a light membrane, presumably basolateral enzymes 'have separate identities whose range of plasma membranes unattached to brush borders. The densities overlap to only a minor degree. The distri- possibility of a minoi-Iysosomal component cannot butions of catalase and alkaline phosphatase are be discounted. largely coincident in these isopycnic gradients. How- After rate-dependent banding a substantial part of ever, the fact that these activities are in separate com- the alkaline phosphatase resembled the slowest mov- partments is indicated by the distributions shown in ing peak of K+ p-nitrophenyl phosphatase (Fig. 2). Fig. 1 and in the gradient from which the samples Conceivably, theref9re, basolateral plasma mem- were obtained (column a, Fig. 3). branes carry some alkaline phosphatase activity. This possibility was investigated in a further experiment DISCUSSION (Fig. 3). Rate dependent banding was carried out essentially The localization of xanthine oxidase and dehydro- as in Fig. 2 and a similar distribution of markers, genase in the guinea-pig small i'ntestinal mucosa indi- Fig. 3. A rate dependent banding experiment was carried out as described for Fig. 2. Some of the enzyme distributions are shown in column a. Aliquots of equal volume of fractions I to 13 (sample i indicated in column a) were combined and 10 ml was loaded on 10 m! of 20% w/w sucrose which rested on 13 m! ofa 25 to 50% (w/w) sucrose gradient on a cushion of 3 ml of 55% (w/w) sucrose. The tube was centrifuged at 25,000 rpm for 3 h in a 6 x 38 S.D. (M.S.E.) Totor; the resulring enzyme distributions are shown in column b. In addition aliquots of fractions 8. 9 and 10 (sample ii indicated in column a) were pooled. 20011 was loaded on a J3 ml gradient and centrifuged as for sample i; the resulting distributions are in column c. (A) Catalase (63%) and succinate dehydrogenase (dashed, 120%1.(D) K + p-nitrophenyl phosphatase (92%) and I-naphthyl acetate esterase (dashed, 88%). (C) Alkaline phosphatase (61%), xanthine oxidase (dashed, 89%) and xanthine dehydrogenase (dotted, 80%). (D) Density. (E) and (I) catalase (106 and 81%). (F) and (1) K + p-nitrophenyl phosphatase (67 and 94%). (0) and (K) alkaline phosphatase (67 and IOO%). (H) and (L) density. . cates that all conversion of hypoxanthine to uric acid L-x-hydroxyacid oxidase was undetectable; this was takes place in the soluble phase. In the goldfish intes- also the case in the goldfish intestine (Temple el al., tine xanthine dehydrogenase is entirely soluble phase 1979). This enzyme is widely found in reno-hepatic while the oxidase was undetectable (Temple el al., peroxisomes (Masters & Holmes, 1977). 1979).The oxidase of the mammalian liver is an ex- NADP linked isocitrate dehydrogenase is mainly clusively soluble activity (De Duve & Baudhuin, (about two thirds) soluble phase with the remainder 1966).The dehydrogenase, though, is partiaJly peroxi- in the mitochondria. This Jocalization resembles that somal in the liver and kidney of chicken (Scott et al.. in the goldfish intestine (Temple er al., 1979).An ad- 1969)but is wholly soluble in those organs of Tat and ditional contribution from the peroxisomes has been pig (Tolbert, 1973). detected in Mytilus digestive gland (our unpllblished Enzymes jn small intestine mucosa 331

Observations)and in the livers of rat (Leighton et al., REFERE1'\CES 1968) and carp (Goldenberg et al., 1978). BATES E. J. & SAGGERSOX E. D. (1979) A study of the Glyceride synthesis in the intestinal mucosa can glycerol phosphate acyltransferase and dihydroxyace- roceed via monoglyceride, sn-glycerol-3-phosphate tone phosphate acyltransferase activities in rat lh.er ~r dihydroxyacetone phosphate (Brindley, 1974). In mitochondrial and microsomal fractions. Biocllem. J. liver the latter pathway is initiated in peroxisomes 182,751-762. and that via sn-glycerol-3-phosphate is mitochondrial BAUDIIUI:'-OP.. BEAUFAYII, RAmIAs-LI Y., SELLlXGER 0.. WATTIAUX R., JACQUESP. & DE DUVE C. (1964) Tissue and microsomal (Bates & Saggerson, 1979; Jones & fractionation studies no. 17. Bioc/zelll. J.92, 179-184. Hajra, 1977). The data reported here indicates that BRI;\,DLEYD. N. (1974) Intracellular phase of fat absorp- the intestinal monoglyceride pathway is microsomal tion. In Biomembranes. Vol. 4B (Edited by S~lvrn D. H.) (probably ER) and has no microperoxisomal com- pp. 621-671. Plenum Press, London. ponent. This is in agreement with other fractionation BURTO;\,K. (1956) A study of the conditions and mechan- work on the mammalian intestine (Brindlcy, 1974; ism of the diphenylamine reaction for the colorimetric Hulsmann & Kurpershoek-Davidov, 1976).Similarly, estimation of deoxyribonucleic acid. Biocllem. J. 62, a histochemical study of the rat intestinal enterocyte 315-323. localizedit mainly in smooth ER (Higgins & Barrnett, CO;\,;\,OCKM. J., ELKIS A. & POVER W. F. R. (1972) The 1971). preparation of brush borders from the epithelial cells of K + p-nitrophenyl phosphatase had a complex dis- the guinea-pig small intestine by zonal centrifugation. JIisrocllem. J.3, 11-22. tribution suggesting at Icast two carriers. One is a COSSOCK M. J.. KIRK P. R. & STURDEE A. P. (1974) A slow moving membranc (density, l.I55 g/ml). The zonal rotor method for the preparation of microperoxi- second sediments with alkaline phosphatase suggest- somes from epithelial cells of guinea-pig small intestine. ing a brush border localization. However, Lewis et al. + J. Cell BioI. 61, 123-133. (1975) concluded that the Na+ -K stimulated DE DUVEC. (1967) General principles. In Enzyme Cytology ATPase in the brush border-rich fractions from (Edited by ROODYN D. B.). pp. 1-26. Academic Press, guinea-pig enterocytes is due to "tags" of basolateral London. plasma membrane attached to brush borders. This DE DUVE C. & BAUDIIUIS P. (1966) Peroxisomes (Micro- must also apply to K + p-nitrophenyl phosphatase bodies and related particles). Phpiol. Ret.. 46, 323-357. DOSALDSOX R. P.. TOLBERTN. E. & SCIISARRENBERGERC. sincc that enzyme carries out the second part of the (1972) A comparison of microbody membranes with ATPase mediated reaction (Garrahan et al., 1969). It + microsomes and mitochondria from plant and animal is possible thatlysosomes also carry a part of the K tissue. Arc/Is Biodlem. Bioph}'s. 152, 199~215. p-nitrophenyl phosphatase. If so, this would not be GARRAHAN P. J., POUCHAN M. I. & REGA A. F. (1969) altogether surprising as phagosomes derived from the Potassium activated phosphatase from human red blood plasma membrane fuse with primary lysosomes to cells. The mechanism of potassium activation J. Pllysiol. form secondary lysosomes. Furthermore, Wattiaux et 202, 305-327. al. (1978) observed that highly purified rat liver Iyso- GAUNT G. I.. & DE DUVE C. (1976) Subcellular distribution somes contain significant amounts of enzymes which of D-amino acid oxidase and catalase in rat brain. J. are otherwise confined to the plasma membrane. Nellrochem. 26. 749-759. GOLDE;\,BERGH.. HUTTISGER KA~IPFER P. & KRAMAR With respect to alkaline phosphatase the experi- M." R: (1978) Preparation of peroxisomes from carp liver by ments described here combined with our earlier zona! density gradient centrifugation. JIisrochem. J. 10, observations point to the following conclusions. After 103-113. isopycnic banding the main peak equilibrated at a GUILBAULT G. G., BRIGSAC P. & ZlmlER M. (1968) Homo- density of 1.24 glml but there was usually significant \'anillic acid as a nuoromelric substrate for oxidative activity at a density of U8 to 1.21. This was some- enzymes. Analytical applications of the peroxidase. glu- times resolvcd into a second, smaIler peak. In about cose oxidase and xanthine oxidase systems. Analyt. half of rate-dependent banding expcriments a sub. Cllem.40, 190-196. stantial minority of the alkaline phosphatase activity 1IIGGtss J. A. & BARR:-;ETT R. J. (1971) Fine structural (up to 40%) has a slow rate of migration overlapping localization of acyltransr~rases. The monoglyceride and :t-gl)'cerophosphate pathwa)'s in intestinal absorplive with that observed for the microperoxisomes. This ac- cells. J. CeIl BioI. 50, 102-120. tivity is largely independent of the basolateral plasma HRUBAS Z.. VIGIL E. 1.., SLESSERSA. & Hop KISS E. (1972) membrane as shown by the differences between its Microbodies. Constituent organel1es of animal cel1s. Lab. distribution and that of K + p-nitrophenyl phospha- lurest. 27, 184-191. tase after isopycnic banding (Fig. 3, columns b and c). HULSMA;\'S \Y. C. & KURPERSIIOEK-DAVIOOV R. (1976) This fact and the wide variation between experiments . Topographic distribution of enzymes involved in in both the relative size and rate of migration of the glycerolipid synthesis in rat small intestinal epithelium. slow moving alkaline phosphatase peak suggests that Biochim. biophys. Acta 450, 288-300. JOSES C. I.. & HAJRA A. K. (1977) The subcellular distribu- it probably represents fragments of brush border tion of acyl-CoA: dihydrox yacctone phosphate acyl which are heterogeneous in their size and their ratio transferase in guinea-pig liver. Bioche"m. biopllys. Res, of lipid to protein content. This is probably the origin C01ll11l1111.76, 1138-1143. of the low density peak of alkaline phosphatase ac- KRISASS S. K. & LAZAROW P. B. (1978) Presence of palmi- tivity observed in isopycnic banding experiments. In tate: CoA in rat liver peroxisomes. J. CeIl Bioi. 79, agreement with Lewis et al. (1975) and others we con- 210a. clude that the basolateral plasma membrane of the LA BELLE E. F. & HAJRA A. K. (1972) Biosynthesis of acyl guinea-pig enterocyte lacks alkaline phosphatase dihydroxyacetone phosphate in subcel1ular fractions of activity. . nil li,"er. J. bioI. Cllem. 247, 5835-5841. LAZAROW P. B. (1977) Role of.peroxisomes in the hepatic Acknowledgement-We gratefully acknowledge the finan- p-oxidation of long chain fatty acyl-CoA's, J. Cell BioI. cial support of the Wellcome Trust... 75,203a. . 332 P. A. MARTI~, N. J. TBIPLE and M. J. CONr-'OCK

LEIGHTO~ F., POOLE B., BEAUfAYH., BAUDHUIN P., COFFEY NOVIKOFF A. B., NOVIKOFf P. M., DAVIS C. & QUINT " J. W., FOWLER S. D. & DE DUVE C. (1968) The large N. (1973) Studies on microperoxisomes. V. Arc miq~ scale separation of peroxisomes, mitochondria and Iyso- peroxisomes ubiquitous in mammalian cells? J. Hisra. somes from livers of rats injected with Triton WR-1339. clrl!l1I.Cylochem. 21, 737-755. J. Cell Bioi. 37. 482-513. SANCHEZ M., NICHOLLS D. G. & BRINDLEY D, N. (197] LEWIS B. A., ELKIN A., MtCHELL R. M. & COLBIAN R. The relationship between palmitoyl-coenzyme A syOth I (1975) Basolateral plasma membranes of intestinal epith- tasc activity and esterification of sn-Glycerol 3'Phoe: elial cells. Biochem. J. 152, 71-84. phate in rat liver mitochondria. Bioclll!m. J. 13t LIPPEL K~ ROBn.;so:-l J. & TRA~IS E. G, (1970) Intracellular 697-706. SCOTT P. J., VISENTIN L. P. & ALLE:-I J. M. (1969) distribution of palmitoyl-CoA synthetase in rat Ih'er. TIlt Biochim. biop"ys. Acta 206, 173-177. enzymatic characteristics of peroxisomcs of amphibian LOWRY O. H., ROSEBROUGHN. 1., FARR A. 1. & RANDALL and avian liver and kidney. AlIn, N. Y. Acad. Sei. lIiS, R. J. (1951) Protein measurement with the Folin Phenol 244-264. reagent. J. bio/. Chern. 193,265-276. SHORT V. J., BRINDLEYD. N: & DILS R. (1974) A new assay MARKWELL M. A. K., BtEBERL. 1. & TOLBERTN. E. (1977) procedure Cor monoglyccndc acyltransfcrase. Biochem. J. Differential increase of hepatic peroxisomal mitochon- 141.407-411. drial and microsomal carnitine acyltransferases in TDIPLE N. J., MARTI,NP. A. & CONNOCKM., J. (1976) clofibrate-fed rats. Biochem. Pharmac. 26, 1697- Zonal rotor analYSIs of the subcellular localization of 1702. ::c-glyccrophosphate dehydrogenase, (X-naphthyl pa1!)ji. MARTIN P. A., TEMPLE N. J. & CONI\'OCK M. J. (1979) tate and fJ-naphthyllaurate in the mUCOsa of Zonal rotor study of the subcellular distribution of aeyl- the guinea-pig small intestine. Histochem. J. 8, 159-175. CoA synthetases, carnitine acyl transferascs and phos- THIPLE .N. J., M~TIN P. A. & CONNOCK M. J. (1979) phatidate phosphatase in the guinea-pig small intestine. Intestmal peroxlsomes of goldfish (Carassills auralUs~ Eur J. Cell BioI. 19, 3-10. Examination for hydrolasc, dehydrogenase and carnitine MASTERS C. & HOLMES R. (1977) Peroxisomes: New acetyltransferase activities. Compo Biochem. Physiol. 648 aspects of cell physiology and biochemistry. Physiol. ReI'. ~U ' TOLBERT N. E. (1973) Compartmentation and contr'o! in 57.816-882. ' MURER H., AMMANNE., BrBERJ. & HOFfER U. (1976) The microbodies. In Rare COlllrol oj Biological Processes. No. s'urface membrane of the small intestinal epithelial cell. I. 27 (Edited by DAVIES D. D.) pp. 215-239. Cambridge Localization of adenyl cyclase. Bioc"il1l. biophys. Acla University Press, 433.509-519. WATTIAUX R., WATTIAUX.DE CO:-';INCK S., RON\'EAUX- NORUM K. R. & BRE~IER1. (1967) The localization of acyl DUPAl M. & DUBOIS F. (f978). Isolation of rat tiler coenzyme A-carniline acyltransferases in rat liver ceIls. Iysosomes by isopycnic centrifugation in a metrizamide J. bioI. Chent. 242,407-411. gradient. J. Cell BiD/. 78, 349-368.