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Glycerol phosphate dehydrogenase in mammalian peroxisomes
.\RCHIVES
OF
Glycerol
BIOCHEMISTRY
Phosphate
AND
BIOPHYSICS
161, 187-193 (1974)
Dehydrogenase
in Mammalian
ROBERT GEE, ESTELLE MCGROARTY, BERLIN AND N. E. TOLBERT Department
of Biochemistry,
Michigan
State University,
Received
August
Peroxisomes’
HSIEH, DIANA East La?&lLg,
Michigan
nI. WIED, 48824
31, 1973
Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD+:a-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 7Ooj,, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviviug intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min-’ mg-I protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD+:a-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver pcroxisomal and cytoplasmic NAD+:a-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands. Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1-2y0 of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K, values and migrated similarly to the anode at pH G.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K, value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD+:ol-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in auimal peroxisomes.
Peroxisomes (microbodies) are subcellular respiratory organelles containing flavin oxidase and catalase (1, 2). Essentiall\i nothing is knorvn concerning transport into and out, of the particle (3). Because of a very active isoenzyme of malate dehydrogenase in leaf peroxisomes (4) and plant glyoxysomes (5, S), as me11as an aspartate aminotransferase (5,7), a malate-oxaloacetate-aspartate shuttle betmeen plant microbodies and other 1 This work was supported in part by NIH grant HD 004441.02 and published as journal article No. 6514 of the Michigan agricultural Experiment Station.
2 A preliminary peared (8). 187
Copyright All rights
@ 1971 by Academic Press, of reproduction in my form
Inc. reserved.
subcellular compart’ments has been envisaged (3, 7). By analogy \vith mit’ochondrial shuttles, both the malate shuttle and the SAD+: a-glycerol phosphate shuttle may also be involved in transport into the animal peroxisomes. Homicver, as prchsentedin this report, only NAD+: c+glycerol phosphate dehydrogenasc was found in pcroxisomes from liver and kidney, but malate dehydrogenasc, \vas absent from the peroxisomcs of the animals studied.2 PeroxisomtLs also contain carnitinc acyl transferascs for acetyl report
of this
work
has ap-
188
GEE
and octanyl derivatives (9), which by analogy with their function in mitochondria may also be involved in a shuttle for fatty acyl compounds. METHODS Particles were separated by isopyncnic sucrose density gradient, and the organelles located by marker enzymes as described in the accompanying paper (10). Enzyme assays. NAD+:glycerol phosphate dehydrogenase was routinely assayed in a recording spectrophotometer as the NADH:dihydroxyacetone phosphate reductase. In a total volume of 1.0 ml, the reaction mixture contained 0.25 M glycylglycine at pH 7.5, 3 mM KCN, 0.1 PM rotenone, 0.162 mM NADH, 0.025% Triton X-100, and 0.465 mM dihydroxyacetone phosphate. The reaction rate was measured as the difference between the oxidation rates of NADH before and after the addition of dihydroxyacetone phosphate. The reaction, NAD+:or-glycerol phosphate reverse dehydrogenase, was run in a reaction mixture which in 1.0 ml contained 0.1 M Tricine at pH 8.3, 1.0 mM KCN, 3.3 mM NAD+, and 33 mM DL-OIglycerol phosphate. KCN was used in both assay directions to inhibit NADH oxidation in the mitochondrial fractions in order to run a complete gradient scan for the dehydrogenase activity. Rotenone, inhibiting mitochondrial complex I, was also used for the same purpose in the assay of NADH:dihydroxyacetone phosphate reductase in order to obtain nearly zero endogenous rates. The detergent Triton X-106 is routinely used to overcome latency in peroxisomal enzyme assays and was so used in the NADH:dihydroxyacetone phosphate reductase reaction. However, Triton X-100, deoxycholate, and other detergents tested all inhibited the NAD+:cY-glycerol phosphate dehydrogenase reaction in the peroxisomal fractions for unknown reasons. Because the amount of sucrose in the gradient fractions required for assay severely inhibited glycerol phosphate dehydrogenase, the sucrose was removed by dialysis to establish correction factors. From each peak of organelles, 2-ml fractions from the 3 peak tubes were combined and dialyzed against 2 liters of 100 mM Tris, 40 mM citrate, 16 rnM MgCls, and 1 mM NAD+. After dialysis for 3 hr fresh solution was added, and the dialysis was continued overnight. Malate dehydrogenase was assayed in a recording spectrophotometer by the rate of oxidation of NADH after the addition of oxaloacetate at pH 7.8. In a 1.0 ml total volume the reaction mixture contained 60 mM Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) at pH 7.8, 0.15%
ET AL. Triton X-100, 0.1 mM NADH, and enzyme. After a 1-min measurement of the endogenous rate, the reaction was initiated by the addition of neutralized oxaloacetate to a final concentration of
0.4 mM. isoenzymic pattern of Electrophoresis. The NAD+:glycerol phosphate dehydrogenase was examined by electrophoresis on 6% polyacrylamide gels as described in the accompanying paper (10). Samples from the peak fractions of the sucrose gradients were incubated in 0.2% Triton X-100 and 20 mM Tris-glycine buffer at pH 9.0 for at least 1 hr before electrophoresis, and the dehydrogenase was located by the procedure of Dewey and Conklin (11). Starch gel electrophoresis for malate dehydrogenase followed the method of Thorne et al. (12). The starch mixture, consisting of 36 g of hydrolyzed starch (Connaught Medical Research Lab., Toronto, Canada) and 309 ml of 8.6 mM sodium phosphate and 1.4 mM sodium citrate, pH 6.2, was stirred continuously and heated slowly to 70-8O”C, or until the solution cleared and became thicker. Then the hot mixture was quickly poured into a a-liter suction flask which was sitting in hot water. A vacuum was applied with an aspirator to boil the solution until only few bubbles remained. The gel was then poured into a Plexiglas form, and a mold for sample wells was put in place. The electrode buffer contained 32 mM phosphate and 4 mnn sodium citrate, pH 6.2. Clear separation of the isoenzymes was obtained by electrophoresis at 30 mA overnight at 4°C. Enzymatic activity on the gel was detected by incubation for 30 min at 37°C in 87 mu Bicine, pH 8.5,0.457 mM nitro blue tetazolium, 0.95 mM NAD+, 0.673 mM phenazine methosulfate, and 0.11 M L-malate. RESULTS
Gradient Procedures In order to separate the peroxisomes and mitochondria, sucrose gradients were made with minimal density changes in the fractions between 1.15 and 1.25 g/cc over a wide part of the gradient. This resulted in a steeper gradient at lower densities where organelles not being investigated were entrapped. In these gradients, about 40% of the catalase in liver
fraction,
homogenates
was in the peroxisomal
with the rest (60%) in the soluble
fraction. For kidney, which required more vigorous homogenation, only 15 % of the catalase survived in the peroxisomal fraction. Using the assumptions that most of the catalase is in the prroxisomes, that solu-
PEROXISOME
GLYCEROL
PHOSPHATE
bilization represented peroxisomal breakage, and that the solubilization factor for all the peroxisomal enzymes is similar, a correction factor of 2.5 was used for calculating the percentage distribution of a peroxisomal enzyme from rat liver homogenate and 6.6 for rat kidney. These values are estimates, since there could be differential loss of catalase and glycerol phosphate dehydrogcnase from the peroxisomcs, as well as varying amounts of both cytoplasmic and peroxisomal reservoirs of these enzymes. Since these assumptions are tenuous, the uncorrected data arc’ presented in the figures and tables and the calculated values corrected for particle breakage are placed in separate column in Table I. NAD+: a-glycerol phosphate dehydrogenase was inhibited by the sucrose in aliquots of the gradient fraction taken for analyses. Since the amount of sucrose increased nonlinearly from the top to bottom of the gradient, aliquot’s from each peak fraction were assayed before and after removal of the sucrose by dialysis. After dialysis overnight,, NAD+: glycerol phosphate dehydrogenase was 3.26-fold more active in the peroxisomal arra and 2-fold more active in the area near the mitochondria, but the activiby in the supcrnatant fraction remained the same. These corrections have been applied in the table for calculation of the percent, distribut,ion and specific activity. TABLE NAD+:
GLYCEROL
PHOSPHATE
Phosphate
Dehydroyenase
From six gradients of rat liver homogenates (Fig. 1 and Table I), an average of 2.8% (ranging from 1.9 to 5.5%) of the NAD+ : cu-glycerol phosphate dehydrogenasc was in the surviving pcroxisomal fract’ion, and most of the rest was in the cytoplasmic fraction. If the particles had first been concentrated by differential cent’rifugation, glycerol phosphate drhydrogenasc activity was also found in the pcroxisomal band, but the cytoplasmic fraction was mostly absent. From rat liver the average specific activity of the pcroxisomal fraction was about 326 nmoles min- mg-’ protein compared to a value of 147 for the cytoplasm. The dehydrogenase should be considered as a peroxisomal enzyme because of the higher specific act,ivit’y in this organ& than in the cytoplasm. A nearly similar distribution was obtained whether the assay was run as NAD : CYglycerol phosphate dehydrogenase or as the rcphosphate NAD : dihydroxyacctone ductase (data not shown). No NAD+:cuglycerol phosphate dehydrogenase was found in the mitochondrial fraction, where t)he FAD : a-glycerol phosphat,e dehydrogenase was located. l’rroxisomes from rat kidneys also had a distinct’ peak of KAD+:wglycerol phosphate dehydrogenase along with t,he other peroxisomal mzymes, and none of this dchydrogenasc activit,y was found in the mitoI ON Sumosr:
GRADIICNTS
FROM
Cytosol
Peroxisomes Measured with sucrose
Rat liver Rat kidney Chicken liver Dog liver
NAD: a-Glycerol
DEHYDROGENASIS IN FRACTIONS LIVER AND KIDNIEY
source
189
DEHYDROGENASE
Corrected for sucrose inhibition
Corrected for particle breakag@
y0 Total
7OTotal
nmoles mind1 mg-’ protein
co Total
2.Sn 2.9 5.5 1.1
8.4 9.5 17.9 3.6
326 147 460 11
21 63 45 9
nmoles min-’ mg-l protein 147 100 163 38
a An average of six similar experiments in which the percentage ranged from 1.9 to 5.5% of the total activity in the sucrose gradient fractions as presented in Figs. 1 and 2. Values as high as 9% have been observed for peroxisomes from rat liver. * See text for derivation of 2.5 for liver preparations and G.F for kidney tissue.
GEE
190
ET AL. I 28 I24 I20
II6 I.1 2
I 08 1.04 I 00
T :: x 9 E u, ?
o( -GLYCEROL PHOSPHATE DEHYDROGENASE
GRADIENT
VOLUME
(ML)
1. Distribution of NAD:a-glycerol phosphate dehydrogenase on isopycnic sucrose density gradients of a total homogenate from rat liver. In the upper half of the figure catalase is the marker enzyme for peroxisomes and cytochrome c oxidase for mitochondria. The dehydrogenase activity was measured in the presence of the sucrose from the gradient fraction and higher values shown in Table I for the peroxisomal fraction are obtained after removal of the sucrose by dialysis. In this gradient 2.2% of the total dehydrogenase activity as measured in the sucrose was in the peroxisomal fraction, while in six gradients an average of 2.8y0 was measured in the peroxisomal fraction. FIG.
PEROXISOME
GLYCEROL
PHOSPHATE
chondria (Fig. 2 and Table I). However, the recovery of peroxisomes from kidney tissue was poor (about 15 %), and the specific activity of this dehydrogenase in the kidney peroxisomal fraction was somewhat lower too. NAD+ : a-glycerol phosphate dehydrogenase activity was also present in the peroxisomal band from dog and chicken livers and coincided in distribution with peroxisomal catalase, urate oxidase, and Lu-hydroxy acid oxidase act’ivities (data not shown). The dehydrogenase activity in the dog liver preparations was less than that in the rat, T 1
CATALASE
2 2 06u s
04c
I
NAD- d-GLYCEROL PHOSPHATE DEHYDROGENASE
x
T z f
60
GRADIENT
VOLUME
x
(ML)
FIG. 2. Distribution of NAD:ol-glycerol phosphate dehydrogenase on isopycnic sucrose density gradients of a total homogenate from rat kidney. Data in the upper half of the figure characterize the gradient. In the lower half of the figure the cited dehydrogenase activity is that after correction for the inhibition by sucrose in the gradient.
DEHYDROGENASE
191
but the enzyme in chicken liver peroxisome was very active. No NAD+: a-glycerol phosphate dehydrogenase was found in the peroxisomal fraction from spinach leaves (data not shown). During this investigation, about 20 different liver preparations from rats were examined for peroxisomal glycerol phosphate dehydrogenase, and often two peaks of peroxisomal catalase activity were observed with both containing glycerol phosphate dehydrogenase. Figures 1 and 2 are selected because only one peak of catalase is shown at’ a maximum density of 1.246 (52% sucrose) with little mitochondrial contamination, but if the second peak occurred it was at a density of about 1.229 (49 % sucrose) and mitochondrial activit’y in it was significant. Total peroxisomal activity was calculated as the sum of both peaks, but specific activity was taken from the purest fraction in 51 to 52 % sucrose. Two other variables that need furthcr investigation are the age and the sex of the rats. For this investigation female Sprague Dawley rats of 130-200 g were used. Except for starving overnight no prctreatment was used, and even starving the rats overnight before sacrifice was not essential. The peroxisomal fraction from livers of male rats had nearly the same level of glycerol phosphate dehydrogenase activity as that from female rats. In one experiment with female Long Evans rats about t)he same amount of glycerol phosphate dehydrogcnase was observed in t,hc peroxisomrs as in Sprague Dawley rats. There was also some NAD+:a-glycerol phosphate dehydrogcnasc activity in the low density region of the gradient where membranes would be expected. In general, from rat livers only a small amount of the total dehydrogenase activity was in this membrane arca of the gradient (Fig. I), while from dog livers thcrc was more dehydrogenase activity in the membrane area than in the peroxisomal peak. For the dog liver gradient the distribution of NADPHcytochromc c rcductase, a marker for cndoplasmic reticulum, overlapped but did not coincide with the glycerol phosphat’r dehydrogcnase activity in this membrane area. Further identification of this parbiculate material might be achieved by flatter sucrose
192
GEE
gradients in the range containing this second peak of dehydrogenase activity. The peroxisomal fract’ion from rat livers was run on 6 % polyacrylamide gels by electrophoresis and stained for NAD+ : glycerol phosphate dehydrogenase. The enzyme from the isolated peroxisomes of rat liver and the soluble fraction both showed 2 closely moving bands with an Rf of about 0.55. Electrophoretic patterns of mitochondria preparations showed no enzymatic activity for NAD+: glycerol phosphate dehydrogenase. A slower moving band in all three cell fractions also developed on the gels with NAD+ but was independent of cY-glycerol phosphate. Malate Dehydrogenase Activity of this dehydrogenase was found to coincide with the mitochondrial marker as well as in the supernatant on the gradients of rat and pig liver and kidney homogenates. No activity could be attributed to the peroxisomes. For dogs a small amount of it was found in the peroxisomal fraction. There was about 2 % of the total malic dehydrogenase activity in the dog liver peroxisomal fraction and 0.5 % in the dog kidney peroxisomal fraction. Because of the great amount of malate dehydrogenase in plant peroxisomes, this activity in the peroxisomal fraction of dog liver and kidney was repeatedly examined. In some cases the profile of distribution of malate dehydrogenase and peroxisomal catalase did not coincide, but rather the malate dehydrogenase lay as an overlapping shoulder toward less dense sucrose. Whenever a significant amount of malate dehydrogenase was present in the peroxisomal area of the gradient, there was also a small shoulder of cytochrome c oxidase extending into this area. Such results suggested that some mitochondria or mitochondrial fractions were accounting for the malate dehydrogenase activity in the dog peroxisomal fractions. The specific activity of the malate dehydrogenase was about equal in the peroxisomal and mitochondrial fraction and varied between 1 and 2 pmoles min-l mg-’ protein. There was about 4 pmoles min-l mg-l protein in the supernatant fraction of dog liver. The pH optima for malate dehydrogenase was the same in dog liver peroxisomal and
ET AL.
mitochondrial fractions. In glycylglycine the pH optimum was at 8.8, and in Tris-phosphate buffer it was about 8.0. Malate dehydrogenase activity in the soluble fraction had a distinctly different pH activity profile with a maximum 0.9 of a pH unit lower. The K, (oxaloacetate) for the dog liver peroxisomal fraction was measured at 8 PM, for the mitochondrial fraction 11 PM, and for the supernatant fraction 52 PM. Starch gel electrophoresis of malate dehydrogenase from the different gradient fractions from the dog liver was run at different pH and with different buffers. At pH 7-7.5 with Tris-citrate or phosphatecitrate, as used by others, the peroxisomal and mitochondrial forms of malate dehydrogenase migrated little from the origin. Upon lowering the pH to 6.2 malate dehydrogenase in both the peroxisomes and mitochondria migrated toward the cathode in a major and minor band of activity (Fig. 3). The enzyme in the supernatant fraction was resolved into four bands that moved rapidly toward the anode, and one small band that moved to the cathode faster than the peroxisomal or mitochondrial isoenzymes. When starch gel electrophoresis was run at higher pH of around 8.6, all forms of malate dehydrogenase moved with poor resolution toward the anode, except for the one isoenzyme MITOCYTOSOL CHONDRIA (+I ANODE
PEROXISOMES la lb IC Id
0
-
2a 2b
3
t-j
CATHODE
FIG. 3. Starch gel electrophoresis of malate dehydrogenase from sucrose gradient fractions of dog liver. The electrophoreticgram was run at pH 6.3 from a citrate-phosphate buffer.
PEROXISOME
GLYCEROL
PHOSPHATE
in the supernatant that migrated to the cathode. Similar electrophoretic mobilities were obtained after passage of the fractions through Sephadex G-25 to remove sucrose. Subcellular fractions from pig liver were also used, and similar results were obtained, except that t,here was no peroxisomal malate dehydrogenase. DISCUSSION
Our data suggest that there are three pools of NAD:a-glycerol phosphate dehydrogenase: cytoplasmic, peroxisomal, and a membrane-like material which has not been investigated. The surviving peroxisoma1 fractions from liver and kidney contained by direct measurement of the gradient a significant 8-10 % of the total NAD:cP glycerol phosphate dehydrogenase. If this activity were corrected for peroxisomal breakage during homogenation, the percentage in the peroxisomal fraction could be as high as 2&60% of the total in the tissue. The specific activity of the enzyme in the isolated rat liver peroxisomes was over twice the activity in the cytosol. The enzyme in the cytoplasm and peroxisomes was electrophoretically similar, and there was no NAD : glycerol phosphate dehydrogenase in the mitochondria where the FAD-linked dehydrogenase is located. A wide range in the percentage of the total NAD:glycerol phosphate dehydrogenase was experimentally found in the peroxisomal fraction surviving on the gradients. Lesser amounts correlated with poorer peroxisomal recovery, but exploratory experiments on age, drugs, and hormones suggest that variable results may be related to physiological parameters. No evidence for a malate dehydrogenasc in mammalian peroxisomes was found in contrast to very high activity levels of this enzyme in plant microbodies. The leaf peroxisomal malate dehydrogenase was an isoenzyme
of that
in the mitochondria,
and
both were about equal in total activity (4). In liver and kidney of the pig and rat no malate dehydrogenase was found in the peroxisomal
fractions,
and
from
the
dog
liver and kidney a small amount of malate dehydrogenase in the peroxisomal fraction appeared to be due to mitochondrial contamination. The activity in both the mito-
DEHYDROGENASE
193
chondrial and peroxisomal fractions of the dog had the same electrophoretic mobility, same pH optimum, and same K, (oxaloacetate). The function of NAD:a-glycerol phosphage dehydrogenase in peroxisomes is not known. Under the general hypothesis that peroxisomes are involved in oxidative degradative processes, the peroxisomal glycerol phosphate dehydrogenase should be one of the last steps of lipid degradation, namely the conversion of glycerol phosphate to dihydroxyacetone phosphate. It is not thought at this time that this dehydrogenase in the peroxisome should be involved in lipid synthesis. This observation on the oxidation of glycerol phosphate by mammalian peroxisomes is also the first report of a phosphate ester substrate in microbodies. Other substrates of microbody metabolism have been organic acids or amino acids. Because phosphate buffer severely inhibits the aminotransferase in leaf peroxisomes (7), we have even avoided the use of phosphate buffers in peroxisomal assays. REFERENCES 1. DE DUVE, C. (1969) Physiol. Rev. 46,323. 2. TOLRERT, N. E. (1971) Ann. Rev. Plant Physiol. 22,45. 3. TOLBERT, N. E. (1973) Symposium 27, Sot.
Exp. Biol., Press.
p. 215. Cambridge
Univrsity
4. YAMAZAKI, R., AND TOLBERT, N. E. (1969) Biochim. Biophys. Acta 178, 11. 5. BREIDENBACH, R. W., AND BI~XVERS, H. (1967) Biochem. Biophys. Res. Commun. 27,462. 6. SCHNARRENBERGER, C., OE:SER, A., AND TOLBERT, N. E. (1971) Plant Physiol. 48,566. 7. REHFELD, D. W., AND TOLDERT, N. E. (1972) J. Biol. Chem. 247,4803. 8. MCGROARTY, E. J., HSIEH, B., GE:E, It., WED, D., AND TOLBERT, N. E. (1973) Fed. Proc. Fed. Amer. Sot. Exp. Biol. abs. 642. 9. MARKWELL, M. A. K., MCGROARTY, E. J., BIEBER, L. L., AND TOLHERT, N. E. (1973) J. Biol. Chem. 248,3426. 10. MCGROARTY, E., HSIEH, B., WIED, D. M., GEE, R., AND TOLBERT, N. E. (1974) Arch. Biothem. Biophys. 161, 194-210. 11. DI.:~EY, M. M., AND CONKLIN, J. L. (1960) Proc. Sot. Exp. Biol. Med. 106,492. 12. THORNIC, C. J. It., GROSSMAN, L. I., AND KAPLAN, N. 0. (1963) Biochim. Biophys. Acta 73, 193.
OF
Glycerol
BIOCHEMISTRY
Phosphate
AND
BIOPHYSICS
161, 187-193 (1974)
Dehydrogenase
in Mammalian
ROBERT GEE, ESTELLE MCGROARTY, BERLIN AND N. E. TOLBERT Department
of Biochemistry,
Michigan
State University,
Received
August
Peroxisomes’
HSIEH, DIANA East La?&lLg,
Michigan
nI. WIED, 48824
31, 1973
Peroxisomes isolated on sucrose density gradients from homogenates of rat, chicken, or dog livers and rat kidney contained NAD+:a-glycerol phosphate dehydrogenase. Since the amount of sucrose in the peroxisomal fraction inhibited the enzyme activity about 7Ooj,, it was necessary to remove the sucrose by dialysis. About 8.4% of the total dehydrogenase of rat livers was in the surviviug intact peroxisomes after homogenation. If corrected for particle breakage, this represented approximately 21% of the total activity. About 9.5% of the total enzyme was isolated in rat kidney peroxisomes, and because of severe particle rupture may represent over half of the total activity. No glycerol phosphate dehydrogenase was found in spinach leaf peroxisomes. A specific activity of 326 nmoles min-’ mg-I protein in the rat liver peroxisomal fraction was at least twice that in the cytoplasm. NAD+:a-glycerol phosphate dehydrogenase was also present in a membrane fraction which was not identified, but none was in the mitochondria. The liver pcroxisomal and cytoplasmic NAD+:a-glycerol phosphate dehydrogenase moved similarly on polyacrylamide gels and each resolved into two adjacent bands. Malate dehydrogenase was not found in peroxisomes from liver and kidney of rats and pigs, but 1-2y0 of the total particulate malate dehydrogenase was present in the peroxisomal area of the gradient from dog livers. However, this malate dehydrogenase in dog peroxisomal fractions did not exactly coincide with the peroxisomal marker, catalase. Malate dehydrogenase in dog liver mitochondria and in the peroxisomal fraction had similar pH optima and K, values and migrated similarly to the anode at pH G.5 on starch gels as a major and a minor band. The cytoplasmic malate dehydrogenase had a different pH optimum and K, value and resolved into five different isoenzymes by electrophoresis. It is concluded that NAD+:ol-glycerol phosphate dehydrogenase is in peroxisomes of liver and kidney, whereas malate dehydrogenase, present in peroxisomes of plants, is apparently absent in auimal peroxisomes.
Peroxisomes (microbodies) are subcellular respiratory organelles containing flavin oxidase and catalase (1, 2). Essentiall\i nothing is knorvn concerning transport into and out, of the particle (3). Because of a very active isoenzyme of malate dehydrogenase in leaf peroxisomes (4) and plant glyoxysomes (5, S), as me11as an aspartate aminotransferase (5,7), a malate-oxaloacetate-aspartate shuttle betmeen plant microbodies and other 1 This work was supported in part by NIH grant HD 004441.02 and published as journal article No. 6514 of the Michigan agricultural Experiment Station.
2 A preliminary peared (8). 187
Copyright All rights
@ 1971 by Academic Press, of reproduction in my form
Inc. reserved.
subcellular compart’ments has been envisaged (3, 7). By analogy \vith mit’ochondrial shuttles, both the malate shuttle and the SAD+: a-glycerol phosphate shuttle may also be involved in transport into the animal peroxisomes. Homicver, as prchsentedin this report, only NAD+: c+glycerol phosphate dehydrogenasc was found in pcroxisomes from liver and kidney, but malate dehydrogenasc, \vas absent from the peroxisomcs of the animals studied.2 PeroxisomtLs also contain carnitinc acyl transferascs for acetyl report
of this
work
has ap-
188
GEE
and octanyl derivatives (9), which by analogy with their function in mitochondria may also be involved in a shuttle for fatty acyl compounds. METHODS Particles were separated by isopyncnic sucrose density gradient, and the organelles located by marker enzymes as described in the accompanying paper (10). Enzyme assays. NAD+:glycerol phosphate dehydrogenase was routinely assayed in a recording spectrophotometer as the NADH:dihydroxyacetone phosphate reductase. In a total volume of 1.0 ml, the reaction mixture contained 0.25 M glycylglycine at pH 7.5, 3 mM KCN, 0.1 PM rotenone, 0.162 mM NADH, 0.025% Triton X-100, and 0.465 mM dihydroxyacetone phosphate. The reaction rate was measured as the difference between the oxidation rates of NADH before and after the addition of dihydroxyacetone phosphate. The reaction, NAD+:or-glycerol phosphate reverse dehydrogenase, was run in a reaction mixture which in 1.0 ml contained 0.1 M Tricine at pH 8.3, 1.0 mM KCN, 3.3 mM NAD+, and 33 mM DL-OIglycerol phosphate. KCN was used in both assay directions to inhibit NADH oxidation in the mitochondrial fractions in order to run a complete gradient scan for the dehydrogenase activity. Rotenone, inhibiting mitochondrial complex I, was also used for the same purpose in the assay of NADH:dihydroxyacetone phosphate reductase in order to obtain nearly zero endogenous rates. The detergent Triton X-106 is routinely used to overcome latency in peroxisomal enzyme assays and was so used in the NADH:dihydroxyacetone phosphate reductase reaction. However, Triton X-100, deoxycholate, and other detergents tested all inhibited the NAD+:cY-glycerol phosphate dehydrogenase reaction in the peroxisomal fractions for unknown reasons. Because the amount of sucrose in the gradient fractions required for assay severely inhibited glycerol phosphate dehydrogenase, the sucrose was removed by dialysis to establish correction factors. From each peak of organelles, 2-ml fractions from the 3 peak tubes were combined and dialyzed against 2 liters of 100 mM Tris, 40 mM citrate, 16 rnM MgCls, and 1 mM NAD+. After dialysis for 3 hr fresh solution was added, and the dialysis was continued overnight. Malate dehydrogenase was assayed in a recording spectrophotometer by the rate of oxidation of NADH after the addition of oxaloacetate at pH 7.8. In a 1.0 ml total volume the reaction mixture contained 60 mM Hepes (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) at pH 7.8, 0.15%
ET AL. Triton X-100, 0.1 mM NADH, and enzyme. After a 1-min measurement of the endogenous rate, the reaction was initiated by the addition of neutralized oxaloacetate to a final concentration of
0.4 mM. isoenzymic pattern of Electrophoresis. The NAD+:glycerol phosphate dehydrogenase was examined by electrophoresis on 6% polyacrylamide gels as described in the accompanying paper (10). Samples from the peak fractions of the sucrose gradients were incubated in 0.2% Triton X-100 and 20 mM Tris-glycine buffer at pH 9.0 for at least 1 hr before electrophoresis, and the dehydrogenase was located by the procedure of Dewey and Conklin (11). Starch gel electrophoresis for malate dehydrogenase followed the method of Thorne et al. (12). The starch mixture, consisting of 36 g of hydrolyzed starch (Connaught Medical Research Lab., Toronto, Canada) and 309 ml of 8.6 mM sodium phosphate and 1.4 mM sodium citrate, pH 6.2, was stirred continuously and heated slowly to 70-8O”C, or until the solution cleared and became thicker. Then the hot mixture was quickly poured into a a-liter suction flask which was sitting in hot water. A vacuum was applied with an aspirator to boil the solution until only few bubbles remained. The gel was then poured into a Plexiglas form, and a mold for sample wells was put in place. The electrode buffer contained 32 mM phosphate and 4 mnn sodium citrate, pH 6.2. Clear separation of the isoenzymes was obtained by electrophoresis at 30 mA overnight at 4°C. Enzymatic activity on the gel was detected by incubation for 30 min at 37°C in 87 mu Bicine, pH 8.5,0.457 mM nitro blue tetazolium, 0.95 mM NAD+, 0.673 mM phenazine methosulfate, and 0.11 M L-malate. RESULTS
Gradient Procedures In order to separate the peroxisomes and mitochondria, sucrose gradients were made with minimal density changes in the fractions between 1.15 and 1.25 g/cc over a wide part of the gradient. This resulted in a steeper gradient at lower densities where organelles not being investigated were entrapped. In these gradients, about 40% of the catalase in liver
fraction,
homogenates
was in the peroxisomal
with the rest (60%) in the soluble
fraction. For kidney, which required more vigorous homogenation, only 15 % of the catalase survived in the peroxisomal fraction. Using the assumptions that most of the catalase is in the prroxisomes, that solu-
PEROXISOME
GLYCEROL
PHOSPHATE
bilization represented peroxisomal breakage, and that the solubilization factor for all the peroxisomal enzymes is similar, a correction factor of 2.5 was used for calculating the percentage distribution of a peroxisomal enzyme from rat liver homogenate and 6.6 for rat kidney. These values are estimates, since there could be differential loss of catalase and glycerol phosphate dehydrogcnase from the peroxisomcs, as well as varying amounts of both cytoplasmic and peroxisomal reservoirs of these enzymes. Since these assumptions are tenuous, the uncorrected data arc’ presented in the figures and tables and the calculated values corrected for particle breakage are placed in separate column in Table I. NAD+: a-glycerol phosphate dehydrogenase was inhibited by the sucrose in aliquots of the gradient fraction taken for analyses. Since the amount of sucrose increased nonlinearly from the top to bottom of the gradient, aliquot’s from each peak fraction were assayed before and after removal of the sucrose by dialysis. After dialysis overnight,, NAD+: glycerol phosphate dehydrogenase was 3.26-fold more active in the peroxisomal arra and 2-fold more active in the area near the mitochondria, but the activiby in the supcrnatant fraction remained the same. These corrections have been applied in the table for calculation of the percent, distribut,ion and specific activity. TABLE NAD+:
GLYCEROL
PHOSPHATE
Phosphate
Dehydroyenase
From six gradients of rat liver homogenates (Fig. 1 and Table I), an average of 2.8% (ranging from 1.9 to 5.5%) of the NAD+ : cu-glycerol phosphate dehydrogenasc was in the surviving pcroxisomal fract’ion, and most of the rest was in the cytoplasmic fraction. If the particles had first been concentrated by differential cent’rifugation, glycerol phosphate drhydrogenasc activity was also found in the pcroxisomal band, but the cytoplasmic fraction was mostly absent. From rat liver the average specific activity of the pcroxisomal fraction was about 326 nmoles min- mg-’ protein compared to a value of 147 for the cytoplasm. The dehydrogenase should be considered as a peroxisomal enzyme because of the higher specific act,ivit’y in this organ& than in the cytoplasm. A nearly similar distribution was obtained whether the assay was run as NAD : CYglycerol phosphate dehydrogenase or as the rcphosphate NAD : dihydroxyacctone ductase (data not shown). No NAD+:cuglycerol phosphate dehydrogenase was found in the mitochondrial fraction, where t)he FAD : a-glycerol phosphat,e dehydrogenase was located. l’rroxisomes from rat kidneys also had a distinct’ peak of KAD+:wglycerol phosphate dehydrogenase along with t,he other peroxisomal mzymes, and none of this dchydrogenasc activit,y was found in the mitoI ON Sumosr:
GRADIICNTS
FROM
Cytosol
Peroxisomes Measured with sucrose
Rat liver Rat kidney Chicken liver Dog liver
NAD: a-Glycerol
DEHYDROGENASIS IN FRACTIONS LIVER AND KIDNIEY
source
189
DEHYDROGENASE
Corrected for sucrose inhibition
Corrected for particle breakag@
y0 Total
7OTotal
nmoles mind1 mg-’ protein
co Total
2.Sn 2.9 5.5 1.1
8.4 9.5 17.9 3.6
326 147 460 11
21 63 45 9
nmoles min-’ mg-l protein 147 100 163 38
a An average of six similar experiments in which the percentage ranged from 1.9 to 5.5% of the total activity in the sucrose gradient fractions as presented in Figs. 1 and 2. Values as high as 9% have been observed for peroxisomes from rat liver. * See text for derivation of 2.5 for liver preparations and G.F for kidney tissue.
GEE
190
ET AL. I 28 I24 I20
II6 I.1 2
I 08 1.04 I 00
T :: x 9 E u, ?
o( -GLYCEROL PHOSPHATE DEHYDROGENASE
GRADIENT
VOLUME
(ML)
1. Distribution of NAD:a-glycerol phosphate dehydrogenase on isopycnic sucrose density gradients of a total homogenate from rat liver. In the upper half of the figure catalase is the marker enzyme for peroxisomes and cytochrome c oxidase for mitochondria. The dehydrogenase activity was measured in the presence of the sucrose from the gradient fraction and higher values shown in Table I for the peroxisomal fraction are obtained after removal of the sucrose by dialysis. In this gradient 2.2% of the total dehydrogenase activity as measured in the sucrose was in the peroxisomal fraction, while in six gradients an average of 2.8y0 was measured in the peroxisomal fraction. FIG.
PEROXISOME
GLYCEROL
PHOSPHATE
chondria (Fig. 2 and Table I). However, the recovery of peroxisomes from kidney tissue was poor (about 15 %), and the specific activity of this dehydrogenase in the kidney peroxisomal fraction was somewhat lower too. NAD+ : a-glycerol phosphate dehydrogenase activity was also present in the peroxisomal band from dog and chicken livers and coincided in distribution with peroxisomal catalase, urate oxidase, and Lu-hydroxy acid oxidase act’ivities (data not shown). The dehydrogenase activity in the dog liver preparations was less than that in the rat, T 1
CATALASE
2 2 06u s
04c
I
NAD- d-GLYCEROL PHOSPHATE DEHYDROGENASE
x
T z f
60
GRADIENT
VOLUME
x
(ML)
FIG. 2. Distribution of NAD:ol-glycerol phosphate dehydrogenase on isopycnic sucrose density gradients of a total homogenate from rat kidney. Data in the upper half of the figure characterize the gradient. In the lower half of the figure the cited dehydrogenase activity is that after correction for the inhibition by sucrose in the gradient.
DEHYDROGENASE
191
but the enzyme in chicken liver peroxisome was very active. No NAD+: a-glycerol phosphate dehydrogenase was found in the peroxisomal fraction from spinach leaves (data not shown). During this investigation, about 20 different liver preparations from rats were examined for peroxisomal glycerol phosphate dehydrogenase, and often two peaks of peroxisomal catalase activity were observed with both containing glycerol phosphate dehydrogenase. Figures 1 and 2 are selected because only one peak of catalase is shown at’ a maximum density of 1.246 (52% sucrose) with little mitochondrial contamination, but if the second peak occurred it was at a density of about 1.229 (49 % sucrose) and mitochondrial activit’y in it was significant. Total peroxisomal activity was calculated as the sum of both peaks, but specific activity was taken from the purest fraction in 51 to 52 % sucrose. Two other variables that need furthcr investigation are the age and the sex of the rats. For this investigation female Sprague Dawley rats of 130-200 g were used. Except for starving overnight no prctreatment was used, and even starving the rats overnight before sacrifice was not essential. The peroxisomal fraction from livers of male rats had nearly the same level of glycerol phosphate dehydrogenase activity as that from female rats. In one experiment with female Long Evans rats about t)he same amount of glycerol phosphate dehydrogcnase was observed in t,hc peroxisomrs as in Sprague Dawley rats. There was also some NAD+:a-glycerol phosphate dehydrogcnasc activity in the low density region of the gradient where membranes would be expected. In general, from rat livers only a small amount of the total dehydrogenase activity was in this membrane arca of the gradient (Fig. I), while from dog livers thcrc was more dehydrogenase activity in the membrane area than in the peroxisomal peak. For the dog liver gradient the distribution of NADPHcytochromc c rcductase, a marker for cndoplasmic reticulum, overlapped but did not coincide with the glycerol phosphat’r dehydrogcnase activity in this membrane area. Further identification of this parbiculate material might be achieved by flatter sucrose
192
GEE
gradients in the range containing this second peak of dehydrogenase activity. The peroxisomal fract’ion from rat livers was run on 6 % polyacrylamide gels by electrophoresis and stained for NAD+ : glycerol phosphate dehydrogenase. The enzyme from the isolated peroxisomes of rat liver and the soluble fraction both showed 2 closely moving bands with an Rf of about 0.55. Electrophoretic patterns of mitochondria preparations showed no enzymatic activity for NAD+: glycerol phosphate dehydrogenase. A slower moving band in all three cell fractions also developed on the gels with NAD+ but was independent of cY-glycerol phosphate. Malate Dehydrogenase Activity of this dehydrogenase was found to coincide with the mitochondrial marker as well as in the supernatant on the gradients of rat and pig liver and kidney homogenates. No activity could be attributed to the peroxisomes. For dogs a small amount of it was found in the peroxisomal fraction. There was about 2 % of the total malic dehydrogenase activity in the dog liver peroxisomal fraction and 0.5 % in the dog kidney peroxisomal fraction. Because of the great amount of malate dehydrogenase in plant peroxisomes, this activity in the peroxisomal fraction of dog liver and kidney was repeatedly examined. In some cases the profile of distribution of malate dehydrogenase and peroxisomal catalase did not coincide, but rather the malate dehydrogenase lay as an overlapping shoulder toward less dense sucrose. Whenever a significant amount of malate dehydrogenase was present in the peroxisomal area of the gradient, there was also a small shoulder of cytochrome c oxidase extending into this area. Such results suggested that some mitochondria or mitochondrial fractions were accounting for the malate dehydrogenase activity in the dog peroxisomal fractions. The specific activity of the malate dehydrogenase was about equal in the peroxisomal and mitochondrial fraction and varied between 1 and 2 pmoles min-l mg-’ protein. There was about 4 pmoles min-l mg-l protein in the supernatant fraction of dog liver. The pH optima for malate dehydrogenase was the same in dog liver peroxisomal and
ET AL.
mitochondrial fractions. In glycylglycine the pH optimum was at 8.8, and in Tris-phosphate buffer it was about 8.0. Malate dehydrogenase activity in the soluble fraction had a distinctly different pH activity profile with a maximum 0.9 of a pH unit lower. The K, (oxaloacetate) for the dog liver peroxisomal fraction was measured at 8 PM, for the mitochondrial fraction 11 PM, and for the supernatant fraction 52 PM. Starch gel electrophoresis of malate dehydrogenase from the different gradient fractions from the dog liver was run at different pH and with different buffers. At pH 7-7.5 with Tris-citrate or phosphatecitrate, as used by others, the peroxisomal and mitochondrial forms of malate dehydrogenase migrated little from the origin. Upon lowering the pH to 6.2 malate dehydrogenase in both the peroxisomes and mitochondria migrated toward the cathode in a major and minor band of activity (Fig. 3). The enzyme in the supernatant fraction was resolved into four bands that moved rapidly toward the anode, and one small band that moved to the cathode faster than the peroxisomal or mitochondrial isoenzymes. When starch gel electrophoresis was run at higher pH of around 8.6, all forms of malate dehydrogenase moved with poor resolution toward the anode, except for the one isoenzyme MITOCYTOSOL CHONDRIA (+I ANODE
PEROXISOMES la lb IC Id
0
-
2a 2b
3
t-j
CATHODE
FIG. 3. Starch gel electrophoresis of malate dehydrogenase from sucrose gradient fractions of dog liver. The electrophoreticgram was run at pH 6.3 from a citrate-phosphate buffer.
PEROXISOME
GLYCEROL
PHOSPHATE
in the supernatant that migrated to the cathode. Similar electrophoretic mobilities were obtained after passage of the fractions through Sephadex G-25 to remove sucrose. Subcellular fractions from pig liver were also used, and similar results were obtained, except that t,here was no peroxisomal malate dehydrogenase. DISCUSSION
Our data suggest that there are three pools of NAD:a-glycerol phosphate dehydrogenase: cytoplasmic, peroxisomal, and a membrane-like material which has not been investigated. The surviving peroxisoma1 fractions from liver and kidney contained by direct measurement of the gradient a significant 8-10 % of the total NAD:cP glycerol phosphate dehydrogenase. If this activity were corrected for peroxisomal breakage during homogenation, the percentage in the peroxisomal fraction could be as high as 2&60% of the total in the tissue. The specific activity of the enzyme in the isolated rat liver peroxisomes was over twice the activity in the cytosol. The enzyme in the cytoplasm and peroxisomes was electrophoretically similar, and there was no NAD : glycerol phosphate dehydrogenase in the mitochondria where the FAD-linked dehydrogenase is located. A wide range in the percentage of the total NAD:glycerol phosphate dehydrogenase was experimentally found in the peroxisomal fraction surviving on the gradients. Lesser amounts correlated with poorer peroxisomal recovery, but exploratory experiments on age, drugs, and hormones suggest that variable results may be related to physiological parameters. No evidence for a malate dehydrogenasc in mammalian peroxisomes was found in contrast to very high activity levels of this enzyme in plant microbodies. The leaf peroxisomal malate dehydrogenase was an isoenzyme
of that
in the mitochondria,
and
both were about equal in total activity (4). In liver and kidney of the pig and rat no malate dehydrogenase was found in the peroxisomal
fractions,
and
from
the
dog
liver and kidney a small amount of malate dehydrogenase in the peroxisomal fraction appeared to be due to mitochondrial contamination. The activity in both the mito-
DEHYDROGENASE
193
chondrial and peroxisomal fractions of the dog had the same electrophoretic mobility, same pH optimum, and same K, (oxaloacetate). The function of NAD:a-glycerol phosphage dehydrogenase in peroxisomes is not known. Under the general hypothesis that peroxisomes are involved in oxidative degradative processes, the peroxisomal glycerol phosphate dehydrogenase should be one of the last steps of lipid degradation, namely the conversion of glycerol phosphate to dihydroxyacetone phosphate. It is not thought at this time that this dehydrogenase in the peroxisome should be involved in lipid synthesis. This observation on the oxidation of glycerol phosphate by mammalian peroxisomes is also the first report of a phosphate ester substrate in microbodies. Other substrates of microbody metabolism have been organic acids or amino acids. Because phosphate buffer severely inhibits the aminotransferase in leaf peroxisomes (7), we have even avoided the use of phosphate buffers in peroxisomal assays. REFERENCES 1. DE DUVE, C. (1969) Physiol. Rev. 46,323. 2. TOLRERT, N. E. (1971) Ann. Rev. Plant Physiol. 22,45. 3. TOLBERT, N. E. (1973) Symposium 27, Sot.
Exp. Biol., Press.
p. 215. Cambridge
Univrsity
4. YAMAZAKI, R., AND TOLBERT, N. E. (1969) Biochim. Biophys. Acta 178, 11. 5. BREIDENBACH, R. W., AND BI~XVERS, H. (1967) Biochem. Biophys. Res. Commun. 27,462. 6. SCHNARRENBERGER, C., OE:SER, A., AND TOLBERT, N. E. (1971) Plant Physiol. 48,566. 7. REHFELD, D. W., AND TOLDERT, N. E. (1972) J. Biol. Chem. 247,4803. 8. MCGROARTY, E. J., HSIEH, B., GE:E, It., WED, D., AND TOLBERT, N. E. (1973) Fed. Proc. Fed. Amer. Sot. Exp. Biol. abs. 642. 9. MARKWELL, M. A. K., MCGROARTY, E. J., BIEBER, L. L., AND TOLHERT, N. E. (1973) J. Biol. Chem. 248,3426. 10. MCGROARTY, E., HSIEH, B., WIED, D. M., GEE, R., AND TOLBERT, N. E. (1974) Arch. Biothem. Biophys. 161, 194-210. 11. DI.:~EY, M. M., AND CONKLIN, J. L. (1960) Proc. Sot. Exp. Biol. Med. 106,492. 12. THORNIC, C. J. It., GROSSMAN, L. I., AND KAPLAN, N. 0. (1963) Biochim. Biophys. Acta 73, 193.