ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 286, No. 1, April, pp. 277-283, 1991

Rhizomelic Chondrodysplasia Punctata: Studies of Peroxisomes Isolated from Cultured Skin Fibroblasts

Biochemical

I. Singh,*,’ 0. Lazo, M. Contreras, W. Stanley,? and T. Hashimoto$ *Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425; SDepartment of Biochemistry, Shinshu University School of Medicine, Japan; and TDepartment of Laboratory Medicine, Children’s National Medical Center and Department of Pediatrics, George Washington University, Washington, D.C. 20052

Received August 6, 1990, and in revised form November

26, 1990

Peroxisomes isolated from cultured skin fibroblasts of two patients with rhizomelic chondrodysplasia punctata (RCDP) and two controls were compared for biochemical studies. These experiments provided the following results: (1) peroxisomes isolated from RCDP-cultured skin fibroblasts had the same density (1.175 g/ml) as control (2) dihydroxyacetone phosphate acylperoxisomes; transferase activity, the first enzyme in the synthesis of plasmalogens, was deficient (0.5% of control) in RCDP peroxisomes and this activity was not observed in any other region of the gradient; (3) the rate of activation (lignoceroyl-CoA ligase) and oxidation of lignoceric acid was normal in RCDP peroxisomes; and (4) peroxisomes from RCDP contained 3-ketoacyl-CoA thiolase in the unprocessed form (44-kDa protein), whereas control peroxisomes had both processed (41-kDa protein) and unprocessed forms of 3-ketoacyl-CoA thiolase. The presence of both processed and unprocessed 3-ketoacylCoA thiolase in control peroxisomes and the unprocessed form in RCDP peroxisomes suggests that processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. Although the specific activity and percentage of activity of 3-ketoacyl-CoA thiolase in RCDP peroxisomes was only 22-26% of control, the normal oxidation of lignoceric acid in RCDP peroxisomes indicates that unprocessed 3ketoacyl-CoA thiolase is active. The remaining peroxisomal3-ketoacyl-CoA thiolase activity in RCDP was observed in a protein fraction (peroxisome ghosts) lighter than peroxisomes. The normal oxidation of fatty acids in peroxisomes and the absence of such activity in peroxisome ghosts (d = 1.12 g/ml) containing peroxisomal proteins in RCDP suggest that RCDP has only one population of functional peroxisomes (d = 1.175 g/ml). G 1991

Academic

Press,

Inc.

0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

The rhizomelic form of chondrodysplasia punctata (RCDP)’ is a fatal autosomal recessive disorder (1). Clinically, it is characterized by the abnormal calcification of extremities, dwarfism, cataracts, skin changes, wide open fontanels, and severe mental retardation. The abnormal biochemical findings are a reduced amount of plasmalogens and an increased amount of phytanic acid as a result of deficient activities of dihydroxyacetone phosphate acyltransferase (DHAP-AT), alkyl dihydroxyacetone phosphate synthetase (alkyl DHAP synthetase), and phytanic acid oxidation (2-5). Based on these clinical and biochemical findings RCDP is considered a member of the peroxisomal disorder group (2). There is a significant resemblance in the clinical and biochemical findings between RCDP and Zellweger’s cerebro-hepato-renal syndrome (CHRS) (2-8). However, RCDP is distinguished from CHRS by the presence of peroxisomes and by the normal levels of very long chain (VLC) fatty acids (>C,,) (2-5). The levels of VLC fatty acids and the rate of their oxidation in homogenates of RCDP cultured skin fibroblast is normal, whereas in CHRS, VLC fatty acids accumulate as a result of an abnormality in their peroxisomal oxidation. In addition to the abnormality in the metabolism of plasmalogens and phytanic acid in RCDP, there is also a defect in the processing of peroxisomal 3-ketoacyl-CoA thiolase, an enzyme in the peroxisomal P-oxidation pathway (4-8). The 3-ketoacyl-CoA thiolase in cultured skin fibroblasts (5, ’ To whom correspondence should be addressed at Department of Pediatrics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425. FAX: (803) 792-9223. ’ Abbreviations used: RCDP, rhizomelic chondrodysplasia punctata; DHAP-AT, dihydroxyacetone-acyltransferase; CHRS, cerebro-hepatorenal syndrome; VLC, very long chain; SDS, sodium dodecyl sulfate.

277 Inc. reserved.

278

SINGH

7) and in liver (5) from RCDP patients was present in the precursor form (44 kDa) instead of the mature form (41 kDa). In subcellular fractionation studies, Balfe et al. (7) observed the localization of the majority of 3-ketoacylCoA thiolase in fractions lighter than peroxisomes (peroxisome ghosts) whereas most of the acyl-CoA oxidase and bifunctional enzymes were present in catalase containing peroxisomes with normal density. Based on the presence of both peroxisomal membrane proteins (9) and the P-oxidation enzymes (7) in these lighter density organelles (peroxisome ghosts) it was suggested that these organelles are not simply immature peroxisomes (7). Thus, these observations raise the question as to whether RCDP has a mixed population of peroxisomes; one with normal density (peroxisomes) and others of lighter density (peroxisome ghosts) as observed in CHRS (7, 9). Additionally, another question is whether these peroxisome ghosts oxidize fatty acids to normalize the deficient activity observed in peroxisomes isolated from RCDP (8). In this study, we report that normal density peroxisomes in RCDP are functional whereas peroxisome ghosts are nonfunctional with respect to fatty acid oxidation. Residual activity of DHAP-acyltransferase in RCDP was also found in normal density peroxisomes but not in peroxisome ghosts. The 3-ketoacyl-CoA thiolase in RCDP peroxisomes had 24% of normal activity but this was enough to provide normal fatty acid oxidation. The processing of 3-ketoacyl-CoA thiolase takes place in peroxisomes. MATERIALS

AND

METHODS

Malate, FAD, NAD+, L-carnitine, and a-cyclodextrin were purchased from Sigma Chemical Co. (St. Louis, MO). ATP and CoASH were obtained from P-L Biochemicals. [1-i4C]Palmitic acid (58.7 mCi/mmol) and Ki4CN (52.0 mCi/mmol) were purchased from New England Nuclear (Boston, MA). Nycodenz was obtained from Accurate Chemical and Scientific (Westbury, NY). [1-i4C]Lignoceric acid was synthesized by treatment of n-tricosanoyl bromide with K”CN as described (10). [li4C]Phytanic acid (55 mCi/mmol) was purchased from Amersham International (Arlington Heights, IL). [U-‘*C]Glycerol 3-phosphate (27 mCi/mmol) was purchased from ICN Radiochemicals (Irvine, CA) and converted to [U-i4C]dihydroxyacetone phosphate as described previously (11). Fractionation of cultured skin fibroblasts by differential and density gradient eentrifugation. The subcellular fractionation of skin fibroblasts from RCDP and controls was performed according to our previously described procedure (13). Briefly, cells were lysed by using four to six hand strokes in a Teflon-glass homogenizer until approximately 90% of the cells were broken. The homogenate was centrifuged at 500g for 5 min, and the supernatant (postnuclear fraction) was further fractioned by isopycnic equilibrium centrifugation in continuous Nycodenz gradients. Tubes (39 ml) for a JV-20 Beckman rotor were layered with 4 ml of 35% Nycodenz and then 28 ml of a continuous gradient consisting of O-30% Nycodenz in homogenization medium as described previously (13). Samples (5 ml) of the postnuclear fraction were placed on top of the gradients. The tubes were sealed and then centrifuged at 33,700g for 60 min at 8°C in a 52-21 M Beckman centrifuge with low acceleration and deceleration. The gradient was collected from the bottom and each fraction was analyzed for marker enzyme activities. The densities of

ET AL. gradient fractions Type 500).

were determined

with a hand refractometer

(Atago

Marker enzyme assays. The gradient fractions were analyzed for the following subcellular enzyme markers: cytochrome c oxidase for mitoe reductasefor microsomes(rs), chondria (14), NADPH-cytochrome and catalase for peroxisomes (16). Enzyme assay for activation and oxidation of 1-Y!-labeled fatty acids to acetate (water-soluble products). Acyl-CoA ligase activity was measured as reported previously (17). Enzyme activity for the oxidation of 1-“C-labeled fatty acids to acetate was measured as described (18) except that the fatty acid substrate was solubilized with a-cyclodextrin and was added to the assay medium. The enzyme reaction was stopped with 1.25 ml of 1 M potassium hydroxide in methanol, and the denatured protein was removed by centrifugation. The supernatant was incubated at 60°C for 1 h, neutralized with acid, and partitioned with chloroform and methanol. The amount of radioactivity in the upper phase is an index of the amount of I-“C-labeled fatty acid oxidized to acetate. For solubilization of the fatty acid substrate with a-cyclodextrin, the fatty acid (20 X 10s dpm) was first dried in a tube under nitrogen and then resuspended in 3.5 ml (20 mg/ml) of a-cyclodextrin by sonication for 1 h at 4°C. For oxidation of [1-‘*C]phytanic acid, the release of CO2 was measured as previously described (18). Enzyme assay for dihydroxyacetone phosphate acyltransferase activity. The enzyme activity of DHAP-AT was measured according to the procedure described previously (11). Briefly, [U-14C]dihydroxyacetone phosphate was synthesized from glycerol g-phosphate. The reaction mixture contained 10 &i of [U-“C]glycerol3-phosphate (27 mCi/mmol) and 0.6 mM glycerol 3-phosphate, 5 mM pyruvate, 1 mM NAD+, lactate dehydrogenase (10 units) and a-glycerol-3-phosphate dehydrogenase (10 units), and 50 mM triethanolamine buffer, pH 7.6. After 60 min of incubation at 25”C, the reaction was stopped by the addition of an equal volume of chloroform and after mixing vigorously the upper phase containing [U-“Cldihydroxyacetone phosphate was transferred to another set of tubes. Under these conditions, the conversion of [U-“Clglycerol 3-phosphate to [U-i”C]dihydroxyacetone phosphate was quantitative (12). For assay of dihydroxyacetone phosphate acyltransferase, a reaction mixture containing 0.1 mM [U-“CIDHAP, 8 mM MgClr, 8 mM sodium fluoride, 0.4 mg albumin, 0.15 mM palmitoyl-CoA, and 75 mM acetate buffer, pH 5.4 in 0.12 ml was incubated at 37°C for 2 h. The reaction was stopped with 0.45 ml of chloroform: methanol (2:l) followed by additions of 150 ~1 chloroform and 150 ~1 2 M KCl/0.2 M HsPO,. The lower phase (100-200 al) was applied to filter papers (Whatman 3 MM), which were dried at room temperature and then washed with 20 ml lo%, 10 ml 5%, and 10 ml 1% trichloroacetic acid, respectively. Filter papers were dried overnight and the radioactivity was counted. Enzyme assay for thiolase activity. Thiolase activity was determined by cleavage of acetoacetyl-CoA following the decrease in absorbance at 303 nm at room temperature (19) in a double beam spectrophotometer. The sample was premixed with 2% Triton X-100 (1:l). The reaction mixture containing the following was then added for a final volume of pH 8.3,25 mM MgClz, 100 pM EDTA, and 52 1 ml: 100 mM Tris-HCl, pM acetoacetyl-CoA. The reaction was started with the addition of CoASH (250 pM final concentration) and followed for 3 min. The molar coefficient of 21,400 M-’ X cm-’ for acetoacetyl-CoA was used for the calculations. Western blot analysis. One hundred micrograms of protein was precipitated with trichloracetic acid (10% final concentration) and washed with ether. The protein sample was resuspended in sample buffer (60 mM Tris-HCl buffer, pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol, and 0.01 mg/ml bromphenol blue) heated for 5 min in a boiling water bath and then subjected to electrophoresis in 10% acrylamide according to the procedure of Laemmli (20). Protein transfer and immunoblot analysis was performed as described previously (21). The nitrocellulose paper was incubated with antibody to 3-ketoacyl-CoA thio-

THIOLASE

ACTIVITY

IN RHIZOMELIC

lase for 5-7 h at room temperature in saline buffer containing 0.05% Tween 20. The blot was incubated with [‘*‘I]protein-A (Amersham) and then washed and dried at room temperature and exposed to Kodak film at -70°C and developed.

RESULTS Table I shows that the marker enzyme activities for peroxisomes (catalase), mitochondria (cytochrome oxidase), and microsomes (cytochrome c reductase) and the enzyme activities for fatty acid activation and oxidation from two RCDP cells lines were similar to controls. However, the activities for oxidation of phytanic acid and DHAP-acyltransferase were only 4% of the controls. Although RCDP has often been clinically confused with Zellweger, the normal oxidation of lignoceric acid in association with deficient activities of DHAP-acyltransferase and oxidation of phytanic acid in cellular homogenates clearly identifies these cell lines as RCDP. Moreover, the similar distribution of catalase activity in the cytosol (free activity) and in peroxisomes (membrane bound activity) from RCDP and controls also supports this conclusion (Table I). To study the metabolic status of peroxisomes in RCDP, subcellular organelles from cultured skin fibroblasts were prepared according to the procedure described previously (13). The subcellular distribution of marker enzymes and the enzyme activities of fatty acid metabolism and DHAPacyltransferase are shown in Figs. 1A and lB, respectively. As judged by the distribution of marker enzyme activities, the peroxisomes, mitochondria, and microsomes were well resolved from each other in these gradients (Fig. 1A). In RCDP, the density of catalase containing peroxisomes in RCDP (1.174-1.176 g/ml) was similar to that of control peroxisomes (1.172-1.178 g/ml). The bimodal distribution of lignoceroyl-CoA ligase in peroxisomes and microsomes and the trimodal distribution of palmitoyl-CoA ligase in peroxisomes, mitochondria, and microsomes in RCDP were also similar to control. The oxidation of palmitic acid in mitochondria and peroxisomes and the oxidation of lignoceric acid in peroxisomes from RCDP were also similar to the control (Fig. 1B). Moreover, the specific activities for the oxidation of palmitic and lignoceric acids in peroxisomes isolated from RCDP were similar to peroxisomes from control (Table II). DHAP acyltransferase activity, a peroxisomal membrane protein, was observed only in peroxisomes (Fig. 1B) but its activity was 4-6% of control peroxisome activity (Table II). Peroxisomal3-ketoacyl-CoA thiolase, the last enzyme of the fatty acid P-oxidation system, has been described in RCDP cells as being in the immature form (44 kDa) (4-8) and more than 95% (as judged from western blots) of it has been found in organelle fractions other than peroxisomes (7). In another study, immunoreactive material to peroxisomal 3-ketoacyl-CoA was detected in peroxisomes from control cells whereas no such material was detected in any fraction of a gradient from RCDP-cultured

CHONDRODYSPLASIA

TABLE Specific

279

PUNCTATA I

Activities in Postnuclear Fraction from Cultured Skin Fibroblasts from Control and RCDP Control

RCDP Marker

enzyme activities

Cytochrome c oxidase NADPH cytochrome c reductase Total catalase activity in postnuclear fraction Percentage free catalase activity Percentage membrane-bound catalase activity Enzyme activities Lignoceric acid oxidation Palmitic acid oxidation Lignoceroyl-CoA ligase Palmitoyl-CoA ligase DHAP-acyltransferase Phytanic acid oxidation”

RCDP/Control

(mU/mg protein)

0.94

0.87

1.08

4.51

4.80

0.94

6.07

5.94

1.02

33

25

1.3

68

75

0.91

(nmol/h/mg 0.14 1.49 0.35 8.90 0.53 0.27

protein) 0.12 1.57 0.33 9.10 13.64 7.4

Note. These results are averages of duplicate cell lines each. ’ (pmol/h/mg protein).

1.16 0.95 1.06 0.98 0.039 0.036

tests on two different

skin fibroblasts (8). To understand and quantitate the subcellular localization of peroxisomal 3-ketoacyl-CoA thiolase in RCDP, we examined the levels of 3-ketoacylCoA thiolase activity and the levels of 3-ketoacyl-CoA thiolase protein in gradient fractions from control and RCDP fibroblasts (Table III and Fig. 2). Although the total 3-ketoacyl-CoA thiolase activity in RCDP-cultured skin fibroblasts was similar to controls (Table III), thiolase activity was bimodally distributed between peroxisomes and mitochondria in control cells while in RCDP it was trimodally distributed between peroxisomes, mitochondria, and membrane fractions (peroxisome ghosts) which band between mitochondria and microsomes (1.12 gm/ml) (Fig. 2A). The specific activity of 3-ketoacyl-CoA thiolase in the mitochondrial fraction from RCDP was similar to control whereas peroxisomes from RCDP had only 24% of the control activity (Table III). This decrease in 3-ketoacyl-CoA thiolase activity in peroxisomes coincided with an increase in 3-ketoacyl-CoA thiolase in peroxisome ghost fractions. Immunoblot analysis of different subcellular fractions from the gradient with antibodies to peroxisomal3-ketoacyl-CoA thiolase is shown in Fig. 2B. These antibodies in control peroxisomes detected both immature (44 kDa) and mature (41 kDa) forms of 3-ketoacyl-CoA thiolase whereas peroxisomes from RCDP had only the immature form (44 kDa) of 3-ketoacyl-CoA thiolase. The 3-ketoacyl-CoA thiolase in peroxisome ghosts in RCDP was also of the immature form of the protein

280

SINGH

ET AL. 0

CONTROL

RCDP

Lignoceric

Cylochrome

F oridars

LL

Palmilic

DHAP-AC~I

0

100

acid oildation

acid oxldalion

Iranhrass

0

100

FIG. 1. Fractionation of postnuclear fraction from cultured skin fibroblasts by Nycodenz isopycnic gradient. The postnuclear fractions from control and RCDP fibroblasts were further fractionated by an isopycnic continuous Nycodenz gradient as described previously (13). The distribution of marker enzymes (A) and the enzyme activities for the activation and oxidation of fatty acids and dihydroxyacetone phosphate acyltransferase (B) are presented. The relative specific activity (ordinate) plotted against the cumulative volume (abscissa) is an average of two experiments from two different cell lines. The density profile of the gradient is also shown. The recovery of the enzyme activities ranged from 83 to 112%.

(Fig. 2B). The antibody cross-reactivity and distribution of enzyme activity suggest that peroxisomal 3-ketoacylCoA thiolase in RCDP was present in peroxisomes and peroxisome ghosts. No cross-reactivity material was observed in the mitochondrial fraction, suggesting that this antibody was specific for peroxisomal 3-ketoacyl-CoA thiolase. DISCUSSION

Peroxisomal disorders are generally divided into two groups: Group I with multiple enzyme deficiencies and Group II with single enzyme deficiencies (22-24). Based on this classification, RCDP falls into Group I. However, it differs from all other members of this group (e.g., Zellweger syndrome, Infantile Refsum’s syndrome, and hyperpipecolic acidemia) in that RCDP has membrane bound catalase (Table II), peroxisomes which can be morphologically identified, and normal levels of fatty acids (2, 5-7). Although peroxisomes, catalase containing organelles, are absent in CHRS, some peroxisomal proteins were identified in membrane structures of lighter density

called peroxisome ghosts (9). The subcellular fractionation of cultured skin fibroblasts from RCDP revealed that acyl-CoA oxidase and bifunctional protein are present in peroxisomes whereas over 95%, as judged by the density of the thiolase band, was present in extraperoxisomal fractions including peroxisome ghosts (7). In another study, 3-ketoacyl-CoA thiolase was detected in gradient fractions from control fibroblasts but not in gradient fractions from RCDP fibroblasts (8). Moreover, the oxidation of palmitoyl-CoA in peroxisomes from RCDP has also been described as deficient (8). This observation is not consistent with previous studies that showed normal oxidation of lignoceric acid in homogenates of RCDP fibroblasts (4,5). Since VLC fatty acids are mainly oxidized in peroxisomes (13), there is a conceptual difficulty in understanding how VLC fatty acids are oxidized normally in homogenates from RCDP-cultured skin fibroblasts if the P-oxidation enzymes are localized in two different membranes or organelles, peroxisomes and peroxisome ghosts, in RCDP as compared with only one organelle, peroxisomes, in normal tissue (17) and why there is deficient oxidation of palmitoyl-CoA in peroxisomes isolated

THIOLASE TABLE

ACTIVITY

IN RHIZOMELIC

CHONDRODYSPLASIA

281

PUNCTATA

II

Enzyme Activities and Densities of Peroxisomes Isolated from Control and RCDP Cultured Skin Fibroblasts RCDP Density of peroxisomes’ Experiment I Experiment II

Control

1.178 1.172

1.176 1.174

Enzyme activities Lignoceric acid oxidation Experiment I Experiment II Palmitic acid oxidation Experiment I Experiment II DHAP-acyltransferase Experiment I Experiment II

(nmol/h/mg 0.17 0.19

RCDP/Control

1.00 1.00

protein) 0.18 0.20

13.8 15.6

13.9 12.7

1.3 0.7

222.3 187.6

0.94 0.95

100

0

‘/a

VOLUME

0.99 1.22 0.006 0.004

Note. These results are averages of duplicates. a Peak of catalase activity in the Nycodenz gradient, g/ml. Experiments I and II were performed on two different cell lines each from control and RCDP fibroblasts.

from RCDP-cultured skin fibroblasts (8). To answer these questions, we isolated peroxisomes from cultured skin fibroblasts from RCDP and controls using our newly established procedure (13) (Fig. 1). Consistent with our

TABLE

III

Specific Activity and Distribution of Thiolase in Different Fractions from Control and RCDP-Cultured Skin Fibroblasts RCDP Thiolase Postnuclear fraction Experiment I Experiment II Normal peroxisomes Experiment I Experiment II Mitochondrial peak fraction Experiment I Experiment II Peroxisome ghosts Experiment I Experiment II Thiolase activity as percentage of control in peroxisomes Experiment I Experiment II

activity

Control ( pmol/h/mg

23.4 22.3

RCDP/Control

protein)

25.1 23.2

0.93 0.96

41.0 (2.70)” 39.4 (2.53)

182.1 (2.49) 160.2 (2.75)

0.22 (1.08) 0.24 (0.92)

58.2 (19.6) 60.3 (21.8)

59.1 (21.3) 63.4 (22.9)

0.98 (0.92) 0.95 (0.95)

95.8 (9.2) 89.2 (8.5)

5.9 (8.0) 6.0 (9.6)

1

previous

3.7 2.9

14.1 13.2

16.2 (1.15) 14.8 (0.88)

0.26 0.22

‘Percentage protein recovery from the gradient in that particular fraction. Experiments I and II were performed on two different cell lines each from control and RCDP fibroblasts.

2

3

4

567

8

FIG. 2. (A) Distribution of thiolase activity in a gradient from cultured skin fibroblasts. The thiolase activity in gradients from control and RCDP was determined by the cleavage of acetoacetyl-CoA as described previously (19). (-) Activity in control gradient, (- * -) activity in the RCDP gradient. (B) Immunoblot analysis of peroxisomal 3-ketoacylCoA thiolase. Peroxisomal and lighter peroxisomal peaks were electrophoresed and immunoblotted as described under Materials and Methods. Lanes 1 and 2 are control and RCDP peroxisomal peaks (fractions 3, 4, and 5 from the bottom of the gradient). Lanes 3,4, and 5 are fractions 11, 12, and 13 (lighter peroxisomal peak) from control gradient and lanes 6, 7, and 8 are fractions 11, 12, and 13 (lighter peroxisomal peak) from the RCDP gradient. Lanes 1 and 2 were charged with 100 pg of protein from control and RCDP, respectively. Lanes 3-8 were charged with 90 pg protein from respective samples.

findings

(13, 18) lignoceric

acid was oxidized

in

peroxisomes whereas palmitic acid was oxidized in both mitochondria and peroxisomes (Fig. 1B) and contrary to the observations of Heikoop et al. (8) the rate of oxidation of fatty acids, lignoceric acid, was normal in peroxisomes isolated from RCDP (Table II). There was no activity for oxidation of lignoceric acid in the region of peroxisome ghosts (Fig. 1B). This normal peroxisomal fatty acid oxidation is consistent with the absence of excessive accumulation of VLC fatty acids in RCDP patients (2-6). For normal oxidation of lignoceric acid in RCDP, all the enzymes needed for peroxisomal P-oxidation, including 3ketoacyl-CoA thiolase must be present in peroxisomes (Fig. 1B and Tables II and III). The 3-ketoacyl-CoA thio-

282

SINGH

lase in the control gradient had a bimodal distribution in peroxisomal and mitochondrial fractions as compared to a trimodal distribution in peroxisomes, mitochondrial, and peroxisome ghosts (density of 1.12 gm/ml) in RCDP gradients (Fig. 2). The residual 3-ketoacyl-CoA thiolase activity (22-26% of control) in RCDP peroxisomes was enough for normal oxidation of lignoceric acid (Fig. 1B and Tables II and III). These studies support the conclusion that even though peroxisome ghosts in RCDP have a number of peroxisomal proteins (7,9, and Fig. 2B), they are nonfunctional with respect to oxidation of fatty acids. The enzyme activity for DHAP acyltransferase, a peroxisomal membrane component, was deficient both in homogenates and in peroxisomes isolated from RCDPcultured skin fibroblasts and this residual activity was only observed in the region of normal peroxisomal population (Fig. 1B and Table II). Moreover, the normal distribution of peroxisomal activities of lignoceroyl-CoA and palmitoyl-CoA ligases (Fig. 1B) suggest that these peroxisomal membrane proteins (25) have normal distribution in RCDP. All the peroxisomal proteins studied so far are synthesized on free polyribosomes and are then transported into peroxisomes and new peroxisomes are made by fission of existing peroxisomes (22, 26). The targeting of peroxisomal proteins, synthesized on free polysomes, into this organelle is an area of intensive research. The signal for directing transport of peroxisomal proteins into peroxisomes is localized on the C-terminus or interior of the proteins and it may be as small as a Ser-Lys-Leu tripeptide (27) or longer sequences (28). This signal on 3-ketoacyl-CoA thiolase is not at the C-terminus but rather at amino acids residues from 252-254 (29). All other peroxisomal proteins studied so far except 3-ketoacyl-CoA thiolase are synthesized in the final form (30). The 3ketoacyl-CoA thiolase in RCDP is of unprocessed form (44 kDa) and over 95%, as judged by the densities of the bands, was present in extraperoxisomal membrane fractions including peroxisomal ghosts (7). Heikoop et al. (8) observed the presence of 3-ketoacyl-CoA thiolase in peroxisomes from control fibroblasts but no such protein was detected in any fraction of the gradient from RCDP (8). Similar to Balfe et al. (7), we also observed the presence of 3-ketoacyl-CoA thiolase in RCDP but in our study it was localized in peroxisomes and peroxisome ghosts (Fig. 2B). Moreover, we also observed that normal peroxisomes contain both unprocessed (44 kDa) and processed forms (41 kDa) of 3-ketoacyl-CoA thiolase (Fig. 2B). These observations suggest that 3-ketoacyl-CoA thiolase is transported into peroxisomes in the unprocessed form and processing takes place inside the peroxisomes. These observations are consistent with the presence of only the unprocessed form of 3-ketoacyl-CoA thiolase in CHRS due to the lack of peroxisomes (8, 31). The fact that peroxisomes from RCDP had only unprocessed precursor

ET AL.

form of 3-ketoacyl-CoA thiolase (Fig. 2B) but had normal P-oxidation of fatty acids (Table III and Fig. 2B) demonstrates that the precursor form of 3-ketoacyl-CoA thiolase has normal activity (Table III). The presence of the unprocessed form of 3-ketoacyl-CoA thiolase in peroxisomes from RCDP also suggests that the targeting signal on 3-ketoacyl-CoA thiolase and the peroxisomal transport system may not be severely affected. The defect in the processing of 3-ketoacyl-CoA thiolase inside the peroxisomes may be either due to the lack of a proteolytic enzyme activity required for this reaction or a mutation in the 3-ketoacyl-CoA thiolase enzyme. ACKNOWLEDGMENTS This work was (NS-22576) and 1079). We thank Shuler for typing

supported by grants from National Institutes of Health from March of Dimes Birth Defects Foundation (lMs. Jan Ashcraft for technical support and Ms. Fran this manuscript.

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11,190-212. 2. Heymans, H. S. A., Oorthuys, J. W. E., Nelck, G., Wander, R. J. A., Dingemans, K. P., and Schutgens, R. B. H. (1986) J. In&r. Metab. Dis. 9, 329-331. 3. Schutgens, R. B. H., Heymans, H. S. A., Oorthuys, J. W. E., Nelck, G., and Wanders, R. J. A. (1985) N. Engl. J. Med. 313,187-188. 4. Schutgens, R. B. H., Heymans, H. S. A., Wanders, R. J. A., Oarthuys, J. W. E., Tager, J. M., Schrakamp, G., Van den Bosch, H., and Beemer, F. A. (1988) Adu. Clin. Enzymol. 6,57-65. 5. Hoefler, G., Hoefler, S., Watkins, P. A., Chen, W. W., Moser, A. E., Baldwin, Y., McGillivary, B., Charrow, J., Friedman, J. M., Rutledge, L., Hashimoto, T., and Moser, H. W. (1988) J. Pediatr. 112, 726-

733. 6. Poulos, A., Sheffield,

7. 8.

9. 10. 11.

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