Participation of peroxisomes gland of guinea pig lipid biosynthesis in the harderian

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					Biochem. J. (1989) 262, 677-680 (Printed in Great Britain)                                                                                   677
Participation of peroxisomes                                    in    lipid biosynthesis in the harderian
gland of guinea pig
Shuichi HORIE* and Tetsuya SUGA
Department of Clinical Biochemistry, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan

       Peroxisomal enzyme activities in the guinea-pig harderian gland, which has a unique lipid composition, were
       studied. Activities of catalase, acyl-CoA oxidase and the cyanide-insensitive acyl-CoA fl-oxidation system
       in this tissue were comparable with those in rat liver. The activities of dihydroxyacetone phosphate
       acyltransferase (DHAPAT, EC and alkyl-DHAP synthase (EC were appreciable, and the
       distributions of both activities were consistent with that of sedimentable catalase activity. Glycerol-3-
       phosphate acyltransferase (GPAT, EC, which is localized in both microsomes (microsomal
       fractions) and mitochondria in the rat liver, was a peroxisomal enzyme in the harderian gland, though the
       activity was only about one-tenth of the DHAPAT activity. These enzymes had different pH profiles and
       substrate specificity. The existence of high activities of enzymes of the acyl-DHAP pathway in peroxisomes
       suggests the physiological significance of peroxisomes in the biosynthesis of glycerol ether phospholipid and
       1 -alkyl-2,3-diacylglycerol in the guinea-pig harderian gland.

INTRODUCTION                                                               and the harderian glands were removed immediately
   Generally glycerol phospholipid and triacylglycerol                     after respiration ceased. Tissues were minced, then
are biosynthesized via phosphatidic acid, which is
                                                                           homogenized in 0.25 M-Tris/HCl, pH 7.4. The
synthesized by direct acylation of glycerol 3-phosphate                    homogenates were centrifuged by the method of De
                                                                           Duve et al. [14] with a slight modification. The nuclear
(GP) and subsequent acylation of the 1-acyl-GP [1-3].                      fraction was rehomogenized and spun at 600 g for
On the other hand, dihydroxyacetonephosphate                               10 min and the, resulting supernatant was combined
acyltransferase (DHAPAT, EC and alkyl-                           with the previous supernatant. Sucrose-density-gradient
DHAP synthase (EC participate in the pro-                        centrifugation was done using the light mitochondrial
duction of glycerol ether lipids [4-8]. Both enzymes are                   (LM) fraction (from 33000 to 21 000 g) of the harderian
localized mainly in peroxisomes of guinea-pig liver,                       gland. A linear gradient of 28-54 % (w/w) sucrose
though the ultimate glycerol lipids are biosynthesized in                  solution containing 10 mM-Tris/HCl (pH 7.4), 1 mm-
the endoplasmic reticulum [9-11]. Rock et al. [12]                         EDTA and 0.1 % ethanol was used. The LM fraction
reported that the pink portion of rabbit harderian gland                   was layered on the sucrose gradient and centrifuged at
contained a preponderance of alkyl glycerolipids. It is                    40000 rev./min. for 3 h in a Hitachi RPV-50T rotor.
known that these ether lipids are actively secreted from
this organ [13]. Yamazaki et al. [13] showed that guinea-                  Enzyme assays
pig harderian gland contains a unique lipid which consists
of 1-alkyl-2,3-diacylglycerol as the main lipid instead of                    Activities of catalase (EC, D-amino-acid oxi-
triacylglycerol.                                                           dase (EC, urate oxidase (EC, carnitine
   There have been many investigations on peroxisomal                      acyltransferases (EC 2.3.1.-), cyanide-insensitive acyl-
lipid metabolism, but few data are available concerning                    CoA fl-oxidation system, acyl-CoA oxidase, cytochrome
the peroxisomal enzymes in the harderian gland [12]. In                    c oxidase (EC, NADPH cytochrome c reductase
the present study we have compared the. peroxisomal                        (EC and acid phosphatase (EC were
enzyme activities of harderian gland with those of rat                     measured as described in [15,16]. DHAPAT, GPAT,
liver and investigated whether or not peroxisomes par-                     alkyl-DHAP synthase and acyl/alkyl-DHAP reductase
ticipate in the biosynthesis of the alkyl-type glycerol                    (EC were determined by the methods described
lipids in this tissue.                                                     in [17-19]. Protein contents were determined by the
                                                                           method of Lowry et al. [20].

EXPERIMENTAL                                                               Materials
                                                                              [32P]DHAP and [32P]GP were prepared as described
Animals and preparation of samples                                         [17] and [14C]hexadecanol was synthesized from
  Male Hartley guinea pigs, weighing about 300-400 g                       [14C]palmitic acid [18]. Palmitoyl-DHAP and 1-0-
were used. Animals were anaesthetized with diethyl ether,                  hexadecyl-DHAP were prepared by the method of Hajra

  Abbreviations used: DHAPAT, dihydroxyacetonephosphate acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; CAT, carnitine
acetyltransferase; COT, carnitine octanoyltransferase; CPT, carnitine palmitoyltransferase; M, mitochondrial; LM, light mitochondrial; P,
  * Present address and address for correspondence and reprint requests: Department of Clinical Biochemistry, Faculty of Pharmaceutical Sciences,
Teikyo University, Sagamiko, Kanagawa 199-01, Japan.
Vol. 262
678                                                                                                           S. Horie and T. Suga

et al. [21]. Radioactive compounds were purchased from                oxidase, NADPH: cytochrome c reductase and acid
Amersham Japan. CoA derivatives and nicotinamide                      phosphatase, which are representative of mitochondrial,
nucleotides were obtained from Sigma, and L-carnitine                 microsomal and lysosomal enzymes respectively, were
was donated by the Otsuka Pharmaceutical Factory.                     within the range of 0.3-0.4-fold of those of ihat liver. The
                                                                      activities of enzymes relating to the acyl-DHAP pathway
                                                                      were relatively high. The activities in harderian gland
RESULTS AND DISCUSSION                                                and rat liver were 568 and 68 nmol/min per g of tissue
                                                                      respectively. On the other hand, the activity of GPAT,
   The lipid composition of the harderian gland of guinea             which is localized in both microsomes and mitochondria
pig is very different from those of other tissues, inasmuch           of all the tissues reported [22-24], was only 0.23-fold of
as it contains alkyl- and branched-chain-fatty-acid                   that in rat liver.
moieties and small contents of unsaturated fatty acids                   Since high activities of CAT, COT and DHAPAT
[13]. Yamazaki et al. [13] reported that more than 90 %               were recognized in the harderian gland, the subcellular
of total lipid in the harderian gland of guinea pig was 1-            distributions of these enzymes were investigated (Fig. 1).
alkyl-2,3-diacylglycerol. On the other hand, it is known              Catalase activity was localized in both the light
that peroxisomes contain DHAPAT and alkyl-DHAP                       mitochondrial (LM) and the cytosolic fractions, though
synthase [9-11]. We therefore examined the distribution              the activity in the latter fraction might have been released
of enzymes participating in the formation of ether lipids            from peroxisomes during the homogenization procedure.
in this tissue.                                                      The activities of cytochrome c oxidase and NADPH:
   Table 1 shows the enzyme activities in the harderian              cytochrome c reductase were mainly distributed in the
gland of guinea pig. The catalase activity was approx.               mitochrondrial (M) and microsomal (P) fractions. The
-0.5- and 0.8-fold of that of rat liver when the activity was        acyl-CoA oxidase and CPT activities were localized
expressed per g of tissue and per mg of protein re-                  mainly in the LM and M fractions respectively, suggesting
spectively. The activities of D-amino-acid oxidase and               the presence of separate fatty acid ,-oxidation systems in
urate oxidase were not detected under the same assay                 peroxisomes and mitochondria of the harderian gland.
conditions employed for the rat liver enzymes. The                   The activities of CAT and COT were recognized in both
activities of fatty acyl-CoA oxidase, the first enzyme of            M and LM fractions. Since COT and CPT activities
the peroxisomal f-oxidation system, and the system                   showed different distribution patterns, it was considered
measured in terms of the reduction of NAD+, were also                that, in the harderian gland of guinea pig, the low
recognized in this organ. The activities of carnitine                activity of CPT was not due to the contribution of COT,
acetyltransferase (CAT) and carnitine octanoyl-                      which has some activity towards palmitoyl-CoA as a
transferase (COT) measured by using acetyl-CoA and                   substrate. However, at present we do not know whether
octanoyl-CoA as substrates respectively, were very high              CAT and COT activities are attributable to the same
compared with those in rat liver, whereas carnitine                  enzyme protein or not. The activities of DHAPAT,
palmitoyltransferase (CPT) activity was not high. On a               GPAT and alkyl-DHAP synthase were exclusively local-
per-gram-of-tissue basis, the activities of cytochrome c             ized in the LM fraction, and the activity of alkyl-DHAP

Table 1. Enzyme activities and protein contents of guinea-pig harderian gland and rat liver.
  Results are expressed as means + S.D. for five samples. One unit of activity of catalase, urate oxidase and cytochrome c oxidase
  was defined as the amount giving a k value of 1, where k is the decrease in the absorbance/min. One unit of other enzyme
  activities was expressed as nmol/min. Abbreviation: N.D., not detected.

                                                               Activity (units/g of tissue)
         Enzyme                                  Guinea-pig harderian gland (a)               Rat liver (b)    Ratio (a/b)

         Catalase                                           1086+ 192                         2214+186            0.49
         D-Amino-acid oxidase                                  N.D.                            1850+409
         Urate oxidase                                         N.D.                            2.87 + 0.40
         Acyl-CoA ,-oxidation system                         296+34                             765 + 181         0.39
         Acyl-CoA oxidase                                    318+29                             779 +1 27         0.41
         Carnitine acetyltransferase                       15010+ 1206                         1531 +305          9.80
         Carnitine octanoyltransferase                     16913 + 3501                        1840+224           9.19
         Carnitine palmitoyltransferase                     1254+68                           2450 + 234           0.51
         Cytochrome c oxidase                                588 + 138                         1336+ 102           0.44
         Glutamate dehydrogenase                               N.D.                           9141 + 1108
         NADPH :cytochrome c reductase                       964+114                          2424+ 190            0.40
         Acid phosphatase                                    635+11                            1799 + 69           0.35
         DHAPAT                                              568 + 79                          67.8+ 8.8           8.38
         GPAT                                                35.0+ 6.4                        157.2+ 11.9          0.22
         Alkyl-DHAP synthase                                122.8+ 19.4                        69.9+ 17.7          1.76
         Acyl/alkyl-DHAP reductase                          8563 + 1425                        8190+ 1015          1.05
         Protein (mg/g of tissue)...                        137.9+24.4                        224.7 18.0          D.61
Lipid biosynthesis in harderian-gland peroxisomes                                                                                                                                                       679

                                                                                               c     1.0 (a)                                  20[ (b)                                                   I


                                                                                               0 o~~~~~~~~~~~~~~~~~~~                                                                          I

                                                                                                    a0.8                                       15 -O,I

                                                                                               ED 0.6
                                                                                               0)                             I

                                                                                                                                              10                          ,,'
                                                                                        - (/
                                                                                          Q 4

                                                                                               E                    P1                            5             d
                                                                                               -E0.2 /                                                    ,cf
 0                                                                                             E
 0                                                                                                        0      100 200 300                          0             25     50     75
 cn                                                                                                            1/[DHAP] (mM-')                                       1/[GP] (mM-1)


                                    rO                                                  Fig.                             substrate   concentration          on      the    DHAPAT                      and
              r         400
 .)_                                                                                                 GPAT activities of harderian-gland peroxisomes
.)_                                                        4
                        )0200                                                             Assay conditions are given in the text. The palmitoyl-CoA
                                                           2                              concentration              was    60 /LM. GPAT activity was measured                                         at

         O    .
                          0     O            I             0 vL
                                                                                          pH 8.0. The assay mixture contained 3.1
                                                                                          protein. (a) DHAPAT activity with (0) and without (0)
                                                                                                                                                                ,ug of peroxisomal
nL                                  _
                                                                                          2.5 mM-GP. The
                                                                                          per       mg of
                                                                                                              for DHAPAT was 29.1 nmol/min
                                                                                                                             the   Km   for DHAPAT                  was         67   /M,       and
                         1.0                                                              the K1 for GP is 80 4tM. (b) GPAT activity with (0) and
         5                                              50                                without             (0)    2.5 mM-DHAP.           The
                                                                                                                                                      Vmax.         for GPAT                   was

                         0.5_                                                             4.1 nmol/min per mg of protein, the Vm... inhibite was
                                                                                          1.2 nmol/min per mg of protein, and the Km for GPAT was
         0        50   100 0            50           100 0               50       100     95,uM.
                              Protein (% of total)
Fig. 1. Cell fractionation of homogenate of guinea-pig harderian
   The homogenate was centriifuged as described in the                                  reductase showed bimodal distributions in the LM and P
   Experimental section. The collumns represent, from left to                           fractions. When palmitoyl-DHAP instead of hexadecyl-
   right, the nuclear, mitochondirial (M), light mitochondrial                          DHAP   was used as a substrate in the reaction mixture
   (LM), microsomal (P) and so luble fractions. (a) Catalase                            for the reductase, the same distribution pattern was
   (recovery, 103 %); (b) cytocbirome c oxidase (87%); (c)                              obtained. Furthermore, the intracellular distributions of
   NADPH:cytochrome c redu(ctase (106%); (d) acyl-CoA                                   these       enzyme          activities       were   confirmed by the result of
   oxidase (95%); (e) CPT (8S)%); (f) acid phosphatase                                  sucrose-density-gradient                        centrifugation                of             the       post-

   (97 %); (g) CAT (95 %); (h) C 10T (86 %); (i) alkyl DHAP                             nuclear supernatant from the harderian gland (S. Horie &
   synthase (112%); (j) DHW ucPAT (91 %) (k) GPAT                                       T. Suga, unpublished work). These results indicate that
   (103%); (1) alkyl-DHAP redi                                                          peroxisomes of guinea-pig harderian gland have a high
                                                                                        potency in the biosynthesis of alkyl glycerolipids.
                                                                                          It is known that GPAT is the first enzyme required for
                                                                                        the production of glycerolipid by direct acylation of GP,
       100                          100(b)                                              and         the       activity       contributes          to        the       formation                             of

  E                                                                                     phosphatidic acid in mammalian cells [1-3]. In                                                                 our
        75                           75                                                 present study, however, it was confirmed that the activity
  x                                                                 A.                  of GPAT was extremely low compared with that of
                                                                                        DHAPAT, and the distribution of the former enzyme
        50                           50                        /   50
                                                                                        activity       was      consistent with that of the latter                                     enzyme
                                                                                        activity. Then we examined some properties of both
 .5 25                               25                                                 enzymes. Fig. 2 shows the pH profiles of DHAPAT and
                                                                                        GPAT. The pH optimum for DHAPAT activity was
                                                      .,                                situated in the range pH 6.6-7.5, and no significant
                                        0        5     6       7         8    9         activity was recognized at pH 5.5-5.7, which is the
                                    pH                                                  environmental pH for the peroxisomal DHAPAT in the
                                                                                        livers of guinea pig and rat [24,25]. When microsomal
Fig. 2. Effect of pH on the activii ties of DHAPAT and GPAT in                          samples from                rat    brain and rabbit lung                     were        examined,
        the peroxisomal fraction                                                        low pH optima for DHAPAT activities                   also                               were
   Peroxisomal fraction was olbtained by sucrose-density-                               reported [17,26]. The GPAT activity tended to be active
   gradient centrifugation. Prep aration of peroxisomes and                             over a higher range of pH (about pH 8.5). Thus the two
   the assay conditions are descri bed in the text. (a) DHAPAT                          activities have very different pH optima. Rock et al. [12]
   (maximum activity 27.2 units,/ng of protein); (b) GPAT                               reported that the activity of DHAPAT in the pink
   (maximum activity 3.8 units)/mg of protein): *, Mes;                                 portion of rabbit harderian gland was stimulated by
   A, Tris/HCI.                                                                         treatment with cholate and deoxycholate. When we
Vol     262
680                                                                                                      S. Horie and T. Suga
determined the activities of DHAPAT and GPAT in the           REFERENCES
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harderian gland.                                               4.   Hajra, A. K. (1968) J. Biol. Chem. 243, 3458-3465
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slightly inhibited by 2.5 mM-GP in the reaction mixture.            1037-1044
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inhibition was observed (Fig. 3a). On the other hand,          8.   Wykle, R. L. & Snyder, F. (1976) in The Enzymes of
GPAT activity was strongly inhibited by the addition of             Biological Membranes (Martonosi, A., ed.), vol. 2, pp.
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DHAPAT. The inhibition of GPAT by DHAP did not                11.   Hajra, A. K. & Bishop, J. E. (1982) Ann. N.Y. Acad. Sci.
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                                                              12.   Rock, C. O., Fitzgerald, V. & Snyder, F. (1977) J. Biol.
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possible that DHAP associates with the active centre or       13.   Yamazaki, T., Seyama, Y., Otsuka, H., Ogawa, H. &
an adjacent site of GPAT. These results indicate that               Yamakawa, T. (1981) J. Biochem. (Tokyo) 89, 683-391
DHAPAT and GPAT in the harderian gland are different          14.   De Duve, C., Pressman, B. C., Gianetto, R., Wattiaux, R.
from those observed in other tissues such as liver [27],            & Appelmans, F. (1955) Biochem. J. 60, 605-617
extrahepatic tissues [28] and fibroblasts [29]. However,      15.   Suga, T., Watanabe, T., Matsumoto, Y. & Horie, S. (1984)
we cannot exclude the possibility that DHAPAT and                   Biochim. Biophys. Acta 794, 218-224
GAPT activities are manifested by a single protein having     16.   Yamada, J., Itoh, S., Horie, S., Watanabe, T. & Suga, T.
dual catalytic sites, i.e. a bifunctional enzyme.                   (1986) Biochem. Pharmacol. 35, 4363-4368
   Generally speaking, GAPT is found in microsomes            17.   Hajra, A. K. & Burke, C. (1978) J. Neurochem. 31,125-134
and mitochondria, whereas DHAPAT is mainly localized          18.   Davis, P. A. & Hajra, A. K. (1981) Arch. Biochem.
in peroxisomes and microsomes. In studies on rat liver,             Biophys. 211, 20-29
Hajra et al. [10,11] demonstrated that the peroxisomal        19.   Ghosh, M. K. & Hajra, A. K. (1986) Arch. Biochem.
DHAPAT was responsible for most of the hepatic DHAP                 Biophys. 245, 523-640
acylation, but that the DHAP pathway contributed to           20.   Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall,
only a minor extent to the overall hepatic glycerolipid             R. J. (1951) J. Biol. Chem. 193, 265-275
synthesis. On the other hand, the present data indicated      21.   Hajra, A. K., Saraswathi, T. V., Das, A. K. (1983) Chem.
that peroxisomes play a major role in overall glycerolipid          Phys. Lipids 33, 179-193
biosynthesis in the harderian gland. We conclude that         22.   Coleman, R. & Bell, R. M. (1978) J. Cell Biol. 76, 245-253
peroxisomes of harderian gland have a very important          23.   Nimmo, H. G. (1979) FEBS Lett. 101, 262-264
role in glycerolipid metabolism. Yamazaki et al. [13]         24.   Declercq, P. E., Haagsman, H. P., Veldhoven, P. V.,
showed that the aliphatic chains of I-alkyl-2,3-                    Debeer, L. J., Van Golde, L. M. G. & Mannaerts, G. P.
diacylglycerol in guinea-pig harderian gland were ex-               (1984) J. Biol. Chem. 259, 9064-9075
                                                              25    Jones, C. L. & Hajra, A. K. (1980) J. Biol. Chem. 255,
clusively saturated, and so it appears that the fatty acids         8289-8295
of glycerolipid in this tissue were produced endogenously     26.   Fisher, A. B., Huber, G. A., Furia, L., Bassett, D. &
rather than by uptake from the surroundings. Further                Rabinowitz, J. L. (1976) J. Lab. Clin. Med. 87, 1033-1040
studies are necessary on the properties of I-alkyl-2-         27.   Datta, N. S. & Hajra, A. K. (1984) FEBS Lett. 176, 264-268
lysophosphatidic acid acyltransferase and on further          28.   Veldhoven, P. V. & Mannaerts, G. P. (1985) Biochem. J.
metabolism in order to establish the physiological                  227, 737-741
significance of the high activities of CAT and COT in the     29.   Webber, K. O., Datta, N. S. & Hajra, A. K. (1987) Arch.
guinea-pig harderian gland.                                         Biochem. Biophys. 254, 611-620

Received 28 April 1989/20 June 1989; accepted 10 July 1989


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