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The in vitro degradation of dihydrosphingosine
348
BIOCHIMICA
ET BIOPHYSICA
ACTA
BBA55536
THE
IN VITRO
R. W.
KEENAN***
DEGRADATION
AND
ALAN
OF DIHYDROSPHINGOSINE”
MAXAM**
The Department ofBiochemistry and the New England School of Medicine, Boston, Mass. (U.S.A.) (Received
October
4th,
Medical
Center Hospitals,
Tufts
University
1968)
SUMMARY
A cell-free system has been obtained in rat liver which catalyzes the formation of palmitic acid from dihydrosphingosine. The enzymatic activity appears to be localized mainly in the mitochondrial fraction and is quite labile. The breakdown of dihydrosphingosine has been found to require ATP and Mg2+. Dialyzed or washed enzyme
preparations
are stimulated
by the addition
of pyridoxal
phosphate
and
enzymatic activity is lost when incubation takes place in the presence of pyridoxalenzyme inhibitors such as semicarbazide or bisulfite. These facts are consistent with the proposal
that the cleavage
of dihydrosphingosine
is carried
out by a pyridoxal-
enzyme. Other data indicate that the reaction may proceed in a manner analogous to the cleavage of threonine by threonine aldolase and that palmitaldehyde may be an intermediate
in the reaction.
INTRODUCTION
Recent studies from several laboratories have shown that the in vivo degradation of dihydrosphingosine results in the formation of palmitic acid1-3. The data also indicate that the probable site of this degradation is the liver, since the largest quantities of radioactive palmitic acid were found in this tissue following the injection of labeled dihydrosphingosine. For this reason, we sought to demonstrate down of dihydrosphingosine with cell-free preparations of liver.
the break-
In this communication we report the presence of an enzyme system in the mitochondrial fraction of rat liver which is capable of degrading dihydrosphingosine to yield palmitic acid. Evidence is also presented which indicates that this process requires ATP and pyridoxal phosphate. A possible reaction sequence which explains these requirements and agrees with other observations is proposed. * A preliminary account of this work was presented at the 156th National Meeting of the American Chemical Society, in Atlantic City, N. J., U.S.A. ** Present address, Department of Biology, Brandeis University, Waltham, Mass., U.S.A. *** Present address, Department of Biochemistry, University of Texas. Medical School at San Antonio, San Antonio, Texas, 78229, U.S.A. Biochim.
Biophys.
Acta,
176 (1969) 348-356
350
R.W.KEENAN,A.MAXAM
were extracted by mixing with 0.20 ml of 0.1 M HCl and x.0 ml of chloroformmethanol (I :I, by vol.). Aliquots of the chloroform phase were dried under nitrogen and reacted with 2,4_dinitrophenylhydrazine. The 2,4_dinitrophenylhydrazones were chromatographed together with authentic samples of palmitaldehyde z,4-dinitrophenylhydrazone on Vaseline-impregnated filter paper. The details of this procedure are given in an earlier publication6. OtJzerassay methods. The quantitative determination of dibydrosphingosine was carried out using the ninhydrin method of SCHMIDT et aL7. Protein was estimated by the biuret methods using bovine serum albumin as a standard. Preparation of enzyme. Washed mitochondrial fractions were obtained by homogenizing whole livers from 125 to 150 g female wistar rats in g vol. of 0.25 M sucrose containing 10-3 &I EDTA (pH 7.0) and 0.075% dithiothreitol. The homogenate was centrifuged at 700 x g for IO min to remove whole cells and nuclei, and a crude mitochondrial fraction was prepared by centrifuging the supernatant at 7000 xg for xo min. The sediment was suspended in IO ml of the homogenizing medium and centrifuged at 24000 xg for IO min. The pellet was suspended in 0.25 M sucrose containing EDTA and dithiothreitol and used in the experiments described below. The mitochondrial and microsomal supernatant fractions were also found to catalyze the conversion of dihydrosphingosine to palmitic acid, but the mitochondrial fraction had at least twice the specific activity of these fractions. The presence of EDTA and dithiothreitol retarded the loss of enzymatic activity from the mitochondrial fraction, but in spite of these additions up to 90% loss of the initial activity took place in a single day at 4’. Several purification procedures were attempted, however none were found to be successful owing to the marked instability of this enzyme system. Freshly prepared mitochondria were used for all the experiments described below, except when the enzyme fraction was subjected to further washing or dialysis. Irzcubation conditiom. Unless otherwise indicated, the incubation mixture consisted of an ATP-generating system composed of 1.5 i&moles, ATP; 2.5 pmoles phosphoenolpyruvate; 2.5 pmoles MgCl,; 25 ,umoles KCl; and 2.5 pg pyruvate kinase. In addition, each tube also contained 2.5 pmoles NaF; IO pmoles Tris-HCl buffer (pH 7.6) ; 2.78 mpmoles [3H]dihydrosphingosine (1.2 -IO B disint. Imin) suspended with Cutscum; and 50 to IOO ,~dof enzyme solution in a total volume of 0.25 ml. Incubation was carried out in I2-ml conical centrifuge tubes in a 38” water bath under air. The control samples were incubated in the absence of enzyme and the enzyme was added immediately before stopping the reaction. The values reported in this paper are all corrected for the small and consistent amount (usually less than 59; of the incubated samples) of radioactivity present in the fatty acid spot taken from control samples. RESULTS
Preliminary experiments showed that both ATP and magnesium were required for the formation of fatty acid from dihydrosphingosine, unless fluoride was also present only a small proportion of the substrate was degraded. The reaction was markedly inhibited when the ATP concentration was 0.01 M or greater, possibly due to the fact that ATP forms an insoluble salt with the free amino group of the sphingolipids. For this reason lower concentrations of ATP were used and an ATP-generating system was Riochim.
Biophys.
Acta,
176 (1969) 348-356
DIHYDROSPHINGOSINE
P”
OF SVFER
lDWD
35=
BREAKDOWN
ml
moTEIN
Fig. I. The effectof the pH of the Tris-HCl buffer on the rate of [Wldihydrosphingosine breakdown. The incubation was carried out for 15 min in the presence of the Tris-KC1 buffer indicated. Each tube contained 1.4mg of protein and all other conditions are as described in the text. Fig. 2. Therate of dihydrosphingosine breakdown as a function of protein concentration. The protein concentration of each tube was varied as shown, and the sampies were incubated for r5 min as described in the text,
employed. Sodium fluoride could be present in concentrations as high as 0.05 M withinhibiting the reaction. E$ect of $H. The effect of the pH of the Tri-IICl buffer added to the reaction mixture is shown in Fig. I. Optimal reaction rates were found in the presence of a buffer of pH 7.7. The complete reaction mixture had a pH of 7.4 in the presence of TrisHCI buffer of pH 7.7, but since several reactions are involved, it was not possible to determine the pH optimum of the cleavage reaction. E$ect of protein concerstration and time. Fig. 2 shows that dihydrosphingosine breakdown is directly proportional to the protein concentration. The rate of the reaction was found to be linear with time up to 30 min. When the reaction was carried out for longer periods of time or with more enzyme, some loss of fatty acid was noted, possibly due to its oxidation. The effect of time on the extent of dihydrosphingosine breakdown is shown in Fig. 3. E#ect of avid&z. The requirement for ATP and Mga+ suggested the possibility that the lipid base might be activated prior to cleavage. One possible activation mechanism which had been suggested by STOFFEL AND STICHT~ was that the sphingolipid might be carboxylated and then cleaved by a reaction sequence which is essentially a reversal of the enzymatic synthesis of dihydrosphingosine from serine and palmityl-CoA (refs. g, IO). Since the most likely mechanism of such a carboxylation reaction would involve a biotin-containing enzyme, the effect of preincubating the enzyme with avidin was tested. The data obtained are given in Table I. The results of this experiment were that avidin had little or no effect on the conversion of dihydrosphingosine to fatty acid. Therefore it isunlikely that a biotin-dependent carboxylation could be involved. Cofactor requirements. The structural similarity between the /?-hydroxyamino acids and dihydrosphingosine raised the possibility that the lipid base might be cleaved by reactions similar to those which have been demonstrated for serine and out
&o&m. Biop/EyS. A&, 176 (1969) 348-356
352
R. W. KEENAN, A. MAXAM
c
UlSTANCE
FROM
ORIGIN
Icml
-
DISTANCE
FROM
ORlGlN
(cm)
-
Fig. 3. The effect of the incubation time on the extent of d~hydrosphin~~sine breakdown. The incubation was carried out as described in the text, but the incubation volume was increased IOfold. Samples (0.25 ml) were withdrawn at the intervals shown and analyzed as usual. Each o.zsml sample contained 0.82 mg of protein. Fig. 4, The distribution of radioactivity in chromatograms of samples which were reacted with 2, 4-dinitrophenylhydrazine (DNPH). A, Sample incubated as usual; B, zero time control; C, [3H] dihydrosphingosine. Free palmitic acid and unreacted dihydrosphingosine both run near the solvent front in this chromatographic system. See text for details.
pyridoxal phosphate functions as a coenzyme in these reactions, the effect of this compound on the cleavage of dihydros~hingosi~e was tested. No increase in the rate of fatty acid formation was produced when pyridoxal phosphate was added to the mitochnndrial fraction. It was only after dialysis or washing that an effect could be shown. The results of two such experiments are given in Table II. From these data it is evident that the reaction rate is increased in the presence of pyridoxal phosphate. The mitochondrial fraction was also treated with cysteine according to the procedure described by BRAUN AND %EI.L 1% in connection with their studies on sphingosine biosynthesis. Under these conditions pyridoxal is converted to the inactive thiazoiidme derivative. We were able to produce a several-fold increase in activity by adding ~II~WIIGY.Z Since
Bdochim.
Biophys.
Acta,
176 (IgBg) 348-356
353 TABLE
I
THE EFFECT
OF AVIDIN
Enzyme samples were incubated for 5 min with T&+-NC1 buffer in the presence and absence of avidin as shown below. The usua1 amounts of substrate, NaF, and the ATP-generating system were then added, and the incubation was continued for an additional 15 min. Each tube contained 2.5 mg of protein, and the final volumes were 0.25 ml. I_.__.__I._.. ~~~~~~~~~~~~iR fttqy acadS P%%~G&afi;on ~~~d~~~~s jdisint. jmin) xo [dmoles IW pmoles xo pmoles ro pmoles IO pmoles
TABLE THE
Tris Tris Tris Tris Tris
buffer buffer buffer buffer buffer
(pH (pH (pII (pW (pH
7.6) 7.6) 7.6) + 4.3 units Aviclin 7.6) + 2.2 units Avidin 7.6) f 1.x units Avidin
18705 20483 r79ro rgro8 20231
II
EFFECTS
DEGRADATION
OF
PYRIDOXAL
PHOSPHATE
AND
NECGTINAMIDE
ADENINE
IlINUCLEOTIDE
ON
THE
OF DIIiYDRDSFNINGOSINE
%he enzyme used in Expt. s was prepared as usual, but washed by suspending the mitochondrial fract.ion in fresh homogenizing medium and resedimenting two additional times. Each tube contained 2.35 mg of protein. The enzyme used in Expt. II was prepared as usual, but dialyzed for 24 h against isotonic sucrose containing IO-~ M EDTA and 0.075% dithiothreitol. Each tube contained x.8 mg protein. All other conditions were the same as described in the text except that the complete system also contained 0,125 pmole of nicotinamide adenine dinucleotide (NAD) and 0.125 #mole of pyridoxal phosphate (PLP). .~__l_-.l____ k?$t. ~~~~~~C~~~~~~ in fncubat~un Sptt-8% Per cent fatty acids time (WEin) No. complete (disilzt./min) systsm ._i Complete 100 ‘5 57 7oo 60500 Minus NAD 15 105 lMilaus PLP 15 44 9oo 78
IT
15
15 15 15
30 30 30 30
Complete &~i%.&sNAD IMiWS PLP Minzls NAD and PLP Complete &‘inus NAD MiWcs PLP iCTinus NAD and PLP
8 540 9000 6 66o 5960 22 500 I.5600 8000 7490
IO0
IO2
75 67 X00 125 64 60
pyridoxal phosphate to samples which had been treated with cysteine. Pyridoxal phosphate also significantly stimulated the rate of degradation of dihydrosp~gosine by mitochondrial fractious which had been washed by sedimeating them and resuspending the enzyme fraction in fresh buffer several times. In all of the above experiments large losses in the total enzymatic activity occurred. Other evidence which implicates pyridoxal phosphate is that the reaction was almost completely inhibited in the presence of semicarbazide (4 mM) which is known to react with the aldehyde function of the coenzyme. The effect of adding NAD to the dialyzed or washed enzyme preparations is also given in Table IT. In several experiments, some increase in the reaction rate was noted when NAD was added without adding pyridoxal phosphate, but in the presence of pyridoxal phosphate no effect or a slight inhibition was noted. NADP, CoA, CTP, Biochim.
Biophys.
Acta, 176 (1969) 348-356
R. W. KEENAN,A. MAXAM
354
UTP, and nicotinamide also failed to stimulate the reaction when added to dialyzed enzyme preparations. Identi$catiou of palmitaldehyde. The fact that neither NAD or NADP significantly stimulated the breakdown of the long-chain lipid base led us to suspect that the cleavage of ~llydrosphingosine does not proceed by the oxidation of the 3-hydroxyl group to yield a 3-keto-dihydrosphingosine derivative. If oxidation did not occur, one might expect that a C,, aldehyde would be an intermediate in the conversion of dihydrosphingosine to palmitic acid. This possibility was examined by carrying out the incubation as usual, and extracting the lipids without saponification. Aliquots of the lipid extracts were then reacted with 2, 4-dinitrophenylhydrazine and the hydrazones separated by chromatography on Vaseline-impregnated paper. The details of this procedure are given under EXPERIMENTAL.The chromatograms were cut into small strips and counted in the scintillation counter. Fig. 4 clearly shows the presence of a radioactive peak corresponding to the location of a sample of the dinitrophenylhydrazone of palmitaldehyde. This peak is not found in the control samples, but a small peak between the palmitaldehyde derivative and the solvent front is apparent. This peak was found to be an artifact produced in the reaction between the labeled dihydrosphingosine and 2,4_dinitrophenylhydrazine. Since there is considerably less dihydrosphingosine in the samples which have been subjected to the action of the enzyme, the artifact is less conspicious in the incubated samples than in the controls. This procedure has been carried out on about 6 different samples, incubated under varying conditions, and in every case where the long-chain base was degraded, a radioactive peak corresponding to the dinitrophenylhydrazone of palmitaldehyde was found. DISCUSSION The enzymatic breakdown of ~hydrosphingosine which had been previously reported to occur ifs vi~ol-~ has now been demonstrated using washed mitochondrial fractions from rat liver. BARENHOLZANDGATES have recently reported the degradation of phytosphingosine by a cell-free system from rat liver, but few details are given. The instability and low levels of enzymatic activity which were found in the present study required the preparation of fresh enzyme for each experiment. An investigation has been undertaken to find a better source of the enzyme system as we11 as discovering means by which the rapid loss of activity may be prevented. At the present time, it is not certain that the reaction rates which were obtained in these studies are a true measure of the enzymatic activity. It is possible that the physical state of the substrate or the presence of the nonionic detergent may have had an inhibitory effect, although the high specific activity of the tritiated dihydrosphingosine made it possible to use very low levels of both the substrate and Cutscum in the reaction mixtures. In one experiment not described in this paper, a z-fold increase in the concentration of substrate and Cutscum did not alter the rate of reactior The unexpected finding that both ATP and ma~esium (other divalent cations were not tested) were required to demonstrate dihydrosphingosine breakdown is very interesting. It is logical to assume that the ATP requirement represents an energy source for the activation of the lipid base. The likelihood that activation could occur Biochim.
Biophys.
Acta,
x76 (1969)
348-356
DIHYDROSPHINGOSINE BREAKDOWN
355
through the mediation of a biotin-requiring carboxylation step seems remote, since no inhibition was noted following the preincubation of the enzyme with avidin. A simpler explanation is that ATP might serve as a phosphate donor for the phosphorylation of the primary hydroxyl group. Carbons I and 2 might be split off to yield phosphoryl ethanolamine. Some support for this speculation is provided by the fact that STOFFEL AND STICHT~ reported is z&o experiments in which the radioactivity from hydroxyterminal labeled dihydrosphingosine was recovered in the ethanolamine moiety of phosphatidyl ethanolamine. There is however no evidence which provides direct confirmation that the role of the ATP is to activate the lipid base, but we have begun a search for a phosphorylated intermediate. The fact that the degradation of dihydrosphingosine is inhibited by both semicarbazide and bisulfite and the demonstration that the addition of pyridoxal phosphate to dialyzed, washed or cysteine-treated enzyme fractions results in a marked stimulation in the reaction rate is good evidence that pyridoxal phosphate or a related compound participates in the breakdown of the lipid base. NAD and NADP failed to produce any significant stimulation in the rate of dihydrosphingosine breakdown by dialyzed enzyme preparations. This fact, together with the demonstration that radioactive palmitaldehyde is produced during the reaction makes it logical to speculate that dihydrosphingosine or an activated form of this compound may undergo a B,-catalyzed reaction which is analogous to the cleavage of threonine by threonine aldolase. Threonine aldolase has been shown to cleave threonine to yield glycine and acetaldehyde 13. It is not surprising that only small amounts of radioactivity can be isolated from incubation mixtures in the form of the dinitrophenylhydrazone derivative of palmitaldehyde, considering the known instability of the long-chain aldehydes and the occurrence of a number of aldehyde-oxidizing systems in liver. Attempts which have been made to cause the accumulation of the aldehyde have not been successful. The compounds usually employed to trap aldehydes such as semicarbazide and bisulfite have been found to inhibit dihydrosphingosine breakdown, probably because they also react with pyridoxal. Another possibility is that the aldehyde is an artifact which arises from palmitic acid and that the reaction actually proceeds by the conversion of the lipid base to the 3-keto derivative. STOFFEL, LEKIM AND STICHT’* have shown that 3-keto-dihydrosphingosine is rapidly converted to palmitic acid in vivo. It is also possible that there is more than one pathway for the degradation of dihydrosphingosine, The final resolution of this problem will have to await further studies. Acknowledgments The authors wish to express their gratitude to Professor GERHARD SCHMIDT for his support and the many valuable criticisms given during the course of these studies. Vote are also indebted to Dr. JONATHAN NISHIMURA for a number of stimulating discussions. This investigation was supported by Grants HE-o3-457 and NB-00356 from the National Institutes of Health, Grant 271 from the National MultipIe Sclerosis Society, and a grant from the Godfrey Hyams Trust, Boston, Mass.
Biochim. Biophys. Acta, 176 (1969) 348-356
R. W. KEENAiS, A. MAXAM
356 REFERENCES
I W. STOFFEL AND G. STICHT, 2. Physiol. Chum., 348 (1967) 1345. 2 Y. BARENHOLZ AND S.GATT, Biochemistry, 7 (1968) 2603. 3 R. W. XEENAN AND K. OKABE, Biochemistry, 7 (1968) 2696. J D. C. MALINS AND H. K. MANGOLD, 1.Am. Oil Chemists Sm., 17 (1960) 576. _' j 1:.SB~DER AZ-XI N. STEPHENS,A~~a~.“3~*~hem., 4 (1962) 128. _' 6 R. W. KEENAN AXD B. I-I. MARKS, 3~ach~~~. Biophys. Acta, 39 (1960) 533. 7 G. SCH~~IDT, E. L. HOGAN, A. KJETA-FYDA, T. TANAKA, J. JOSEPH,N. I.FELDMAN, R. A. COLLINS,AND R. W. KEENAN in S.M. ARONSON AND B. W. VOLK, Inborn Disorders ojSphingolipid Metabolism, Pergamon Press, 1966, p. 325. 8 E. LAYNE, in S. P. GOLOWICK AXD N. 0. KAPLAN, Methods in Enzymology, Vol III, Academic Press, 1957. P. 450. g W. STOFFEL,D. LEKIM API'D G. STICHT, Z. Physiol. Chem., 349 (1968) 664. 10 P. E. BRAUN ANn E. E. SNELL,J.Biol. Chem., 243 (1968) 3775. II P. E. BRAUN ANn E. E. SNELL.Pvoc. Nat& Acad. Sci. U.S., ;8 (1967)I_ 298. 12 Y;.BARENHOLZ AND S.GATT,&&hem. Biopizys. Res. Comm&z., 32 (1968) 588. 13 M. KARASEK AND D. M. GREENBERG,J.B~OE.Chem., 227 (1957) Igr. 14 W. STOFFEL,D. LEX~IMAND G. STICHT, Z. PhysioZ.Chem., 34X(1967) 1570. Biochim.
Biophys.
Acta,
176 (1969)
348-356
BIOCHIMICA
ET BIOPHYSICA
ACTA
BBA55536
THE
IN VITRO
R. W.
KEENAN***
DEGRADATION
AND
ALAN
OF DIHYDROSPHINGOSINE”
MAXAM**
The Department ofBiochemistry and the New England School of Medicine, Boston, Mass. (U.S.A.) (Received
October
4th,
Medical
Center Hospitals,
Tufts
University
1968)
SUMMARY
A cell-free system has been obtained in rat liver which catalyzes the formation of palmitic acid from dihydrosphingosine. The enzymatic activity appears to be localized mainly in the mitochondrial fraction and is quite labile. The breakdown of dihydrosphingosine has been found to require ATP and Mg2+. Dialyzed or washed enzyme
preparations
are stimulated
by the addition
of pyridoxal
phosphate
and
enzymatic activity is lost when incubation takes place in the presence of pyridoxalenzyme inhibitors such as semicarbazide or bisulfite. These facts are consistent with the proposal
that the cleavage
of dihydrosphingosine
is carried
out by a pyridoxal-
enzyme. Other data indicate that the reaction may proceed in a manner analogous to the cleavage of threonine by threonine aldolase and that palmitaldehyde may be an intermediate
in the reaction.
INTRODUCTION
Recent studies from several laboratories have shown that the in vivo degradation of dihydrosphingosine results in the formation of palmitic acid1-3. The data also indicate that the probable site of this degradation is the liver, since the largest quantities of radioactive palmitic acid were found in this tissue following the injection of labeled dihydrosphingosine. For this reason, we sought to demonstrate down of dihydrosphingosine with cell-free preparations of liver.
the break-
In this communication we report the presence of an enzyme system in the mitochondrial fraction of rat liver which is capable of degrading dihydrosphingosine to yield palmitic acid. Evidence is also presented which indicates that this process requires ATP and pyridoxal phosphate. A possible reaction sequence which explains these requirements and agrees with other observations is proposed. * A preliminary account of this work was presented at the 156th National Meeting of the American Chemical Society, in Atlantic City, N. J., U.S.A. ** Present address, Department of Biology, Brandeis University, Waltham, Mass., U.S.A. *** Present address, Department of Biochemistry, University of Texas. Medical School at San Antonio, San Antonio, Texas, 78229, U.S.A. Biochim.
Biophys.
Acta,
176 (1969) 348-356
350
R.W.KEENAN,A.MAXAM
were extracted by mixing with 0.20 ml of 0.1 M HCl and x.0 ml of chloroformmethanol (I :I, by vol.). Aliquots of the chloroform phase were dried under nitrogen and reacted with 2,4_dinitrophenylhydrazine. The 2,4_dinitrophenylhydrazones were chromatographed together with authentic samples of palmitaldehyde z,4-dinitrophenylhydrazone on Vaseline-impregnated filter paper. The details of this procedure are given in an earlier publication6. OtJzerassay methods. The quantitative determination of dibydrosphingosine was carried out using the ninhydrin method of SCHMIDT et aL7. Protein was estimated by the biuret methods using bovine serum albumin as a standard. Preparation of enzyme. Washed mitochondrial fractions were obtained by homogenizing whole livers from 125 to 150 g female wistar rats in g vol. of 0.25 M sucrose containing 10-3 &I EDTA (pH 7.0) and 0.075% dithiothreitol. The homogenate was centrifuged at 700 x g for IO min to remove whole cells and nuclei, and a crude mitochondrial fraction was prepared by centrifuging the supernatant at 7000 xg for xo min. The sediment was suspended in IO ml of the homogenizing medium and centrifuged at 24000 xg for IO min. The pellet was suspended in 0.25 M sucrose containing EDTA and dithiothreitol and used in the experiments described below. The mitochondrial and microsomal supernatant fractions were also found to catalyze the conversion of dihydrosphingosine to palmitic acid, but the mitochondrial fraction had at least twice the specific activity of these fractions. The presence of EDTA and dithiothreitol retarded the loss of enzymatic activity from the mitochondrial fraction, but in spite of these additions up to 90% loss of the initial activity took place in a single day at 4’. Several purification procedures were attempted, however none were found to be successful owing to the marked instability of this enzyme system. Freshly prepared mitochondria were used for all the experiments described below, except when the enzyme fraction was subjected to further washing or dialysis. Irzcubation conditiom. Unless otherwise indicated, the incubation mixture consisted of an ATP-generating system composed of 1.5 i&moles, ATP; 2.5 pmoles phosphoenolpyruvate; 2.5 pmoles MgCl,; 25 ,umoles KCl; and 2.5 pg pyruvate kinase. In addition, each tube also contained 2.5 pmoles NaF; IO pmoles Tris-HCl buffer (pH 7.6) ; 2.78 mpmoles [3H]dihydrosphingosine (1.2 -IO B disint. Imin) suspended with Cutscum; and 50 to IOO ,~dof enzyme solution in a total volume of 0.25 ml. Incubation was carried out in I2-ml conical centrifuge tubes in a 38” water bath under air. The control samples were incubated in the absence of enzyme and the enzyme was added immediately before stopping the reaction. The values reported in this paper are all corrected for the small and consistent amount (usually less than 59; of the incubated samples) of radioactivity present in the fatty acid spot taken from control samples. RESULTS
Preliminary experiments showed that both ATP and magnesium were required for the formation of fatty acid from dihydrosphingosine, unless fluoride was also present only a small proportion of the substrate was degraded. The reaction was markedly inhibited when the ATP concentration was 0.01 M or greater, possibly due to the fact that ATP forms an insoluble salt with the free amino group of the sphingolipids. For this reason lower concentrations of ATP were used and an ATP-generating system was Riochim.
Biophys.
Acta,
176 (1969) 348-356
DIHYDROSPHINGOSINE
P”
OF SVFER
lDWD
35=
BREAKDOWN
ml
moTEIN
Fig. I. The effectof the pH of the Tris-HCl buffer on the rate of [Wldihydrosphingosine breakdown. The incubation was carried out for 15 min in the presence of the Tris-KC1 buffer indicated. Each tube contained 1.4mg of protein and all other conditions are as described in the text. Fig. 2. Therate of dihydrosphingosine breakdown as a function of protein concentration. The protein concentration of each tube was varied as shown, and the sampies were incubated for r5 min as described in the text,
employed. Sodium fluoride could be present in concentrations as high as 0.05 M withinhibiting the reaction. E$ect of $H. The effect of the pH of the Tri-IICl buffer added to the reaction mixture is shown in Fig. I. Optimal reaction rates were found in the presence of a buffer of pH 7.7. The complete reaction mixture had a pH of 7.4 in the presence of TrisHCI buffer of pH 7.7, but since several reactions are involved, it was not possible to determine the pH optimum of the cleavage reaction. E$ect of protein concerstration and time. Fig. 2 shows that dihydrosphingosine breakdown is directly proportional to the protein concentration. The rate of the reaction was found to be linear with time up to 30 min. When the reaction was carried out for longer periods of time or with more enzyme, some loss of fatty acid was noted, possibly due to its oxidation. The effect of time on the extent of dihydrosphingosine breakdown is shown in Fig. 3. E#ect of avid&z. The requirement for ATP and Mga+ suggested the possibility that the lipid base might be activated prior to cleavage. One possible activation mechanism which had been suggested by STOFFEL AND STICHT~ was that the sphingolipid might be carboxylated and then cleaved by a reaction sequence which is essentially a reversal of the enzymatic synthesis of dihydrosphingosine from serine and palmityl-CoA (refs. g, IO). Since the most likely mechanism of such a carboxylation reaction would involve a biotin-containing enzyme, the effect of preincubating the enzyme with avidin was tested. The data obtained are given in Table I. The results of this experiment were that avidin had little or no effect on the conversion of dihydrosphingosine to fatty acid. Therefore it isunlikely that a biotin-dependent carboxylation could be involved. Cofactor requirements. The structural similarity between the /?-hydroxyamino acids and dihydrosphingosine raised the possibility that the lipid base might be cleaved by reactions similar to those which have been demonstrated for serine and out
&o&m. Biop/EyS. A&, 176 (1969) 348-356
352
R. W. KEENAN, A. MAXAM
c
UlSTANCE
FROM
ORIGIN
Icml
-
DISTANCE
FROM
ORlGlN
(cm)
-
Fig. 3. The effect of the incubation time on the extent of d~hydrosphin~~sine breakdown. The incubation was carried out as described in the text, but the incubation volume was increased IOfold. Samples (0.25 ml) were withdrawn at the intervals shown and analyzed as usual. Each o.zsml sample contained 0.82 mg of protein. Fig. 4, The distribution of radioactivity in chromatograms of samples which were reacted with 2, 4-dinitrophenylhydrazine (DNPH). A, Sample incubated as usual; B, zero time control; C, [3H] dihydrosphingosine. Free palmitic acid and unreacted dihydrosphingosine both run near the solvent front in this chromatographic system. See text for details.
pyridoxal phosphate functions as a coenzyme in these reactions, the effect of this compound on the cleavage of dihydros~hingosi~e was tested. No increase in the rate of fatty acid formation was produced when pyridoxal phosphate was added to the mitochnndrial fraction. It was only after dialysis or washing that an effect could be shown. The results of two such experiments are given in Table II. From these data it is evident that the reaction rate is increased in the presence of pyridoxal phosphate. The mitochondrial fraction was also treated with cysteine according to the procedure described by BRAUN AND %EI.L 1% in connection with their studies on sphingosine biosynthesis. Under these conditions pyridoxal is converted to the inactive thiazoiidme derivative. We were able to produce a several-fold increase in activity by adding ~II~WIIGY.Z Since
Bdochim.
Biophys.
Acta,
176 (IgBg) 348-356
353 TABLE
I
THE EFFECT
OF AVIDIN
Enzyme samples were incubated for 5 min with T&+-NC1 buffer in the presence and absence of avidin as shown below. The usua1 amounts of substrate, NaF, and the ATP-generating system were then added, and the incubation was continued for an additional 15 min. Each tube contained 2.5 mg of protein, and the final volumes were 0.25 ml. I_.__.__I._.. ~~~~~~~~~~~~iR fttqy acadS P%%~G&afi;on ~~~d~~~~s jdisint. jmin) xo [dmoles IW pmoles xo pmoles ro pmoles IO pmoles
TABLE THE
Tris Tris Tris Tris Tris
buffer buffer buffer buffer buffer
(pH (pH (pII (pW (pH
7.6) 7.6) 7.6) + 4.3 units Aviclin 7.6) + 2.2 units Avidin 7.6) f 1.x units Avidin
18705 20483 r79ro rgro8 20231
II
EFFECTS
DEGRADATION
OF
PYRIDOXAL
PHOSPHATE
AND
NECGTINAMIDE
ADENINE
IlINUCLEOTIDE
ON
THE
OF DIIiYDRDSFNINGOSINE
%he enzyme used in Expt. s was prepared as usual, but washed by suspending the mitochondrial fract.ion in fresh homogenizing medium and resedimenting two additional times. Each tube contained 2.35 mg of protein. The enzyme used in Expt. II was prepared as usual, but dialyzed for 24 h against isotonic sucrose containing IO-~ M EDTA and 0.075% dithiothreitol. Each tube contained x.8 mg protein. All other conditions were the same as described in the text except that the complete system also contained 0,125 pmole of nicotinamide adenine dinucleotide (NAD) and 0.125 #mole of pyridoxal phosphate (PLP). .~__l_-.l____ k?$t. ~~~~~~C~~~~~~ in fncubat~un Sptt-8% Per cent fatty acids time (WEin) No. complete (disilzt./min) systsm ._i Complete 100 ‘5 57 7oo 60500 Minus NAD 15 105 lMilaus PLP 15 44 9oo 78
IT
15
15 15 15
30 30 30 30
Complete &~i%.&sNAD IMiWS PLP Minzls NAD and PLP Complete &‘inus NAD MiWcs PLP iCTinus NAD and PLP
8 540 9000 6 66o 5960 22 500 I.5600 8000 7490
IO0
IO2
75 67 X00 125 64 60
pyridoxal phosphate to samples which had been treated with cysteine. Pyridoxal phosphate also significantly stimulated the rate of degradation of dihydrosp~gosine by mitochondrial fractious which had been washed by sedimeating them and resuspending the enzyme fraction in fresh buffer several times. In all of the above experiments large losses in the total enzymatic activity occurred. Other evidence which implicates pyridoxal phosphate is that the reaction was almost completely inhibited in the presence of semicarbazide (4 mM) which is known to react with the aldehyde function of the coenzyme. The effect of adding NAD to the dialyzed or washed enzyme preparations is also given in Table IT. In several experiments, some increase in the reaction rate was noted when NAD was added without adding pyridoxal phosphate, but in the presence of pyridoxal phosphate no effect or a slight inhibition was noted. NADP, CoA, CTP, Biochim.
Biophys.
Acta, 176 (1969) 348-356
R. W. KEENAN,A. MAXAM
354
UTP, and nicotinamide also failed to stimulate the reaction when added to dialyzed enzyme preparations. Identi$catiou of palmitaldehyde. The fact that neither NAD or NADP significantly stimulated the breakdown of the long-chain lipid base led us to suspect that the cleavage of ~llydrosphingosine does not proceed by the oxidation of the 3-hydroxyl group to yield a 3-keto-dihydrosphingosine derivative. If oxidation did not occur, one might expect that a C,, aldehyde would be an intermediate in the conversion of dihydrosphingosine to palmitic acid. This possibility was examined by carrying out the incubation as usual, and extracting the lipids without saponification. Aliquots of the lipid extracts were then reacted with 2, 4-dinitrophenylhydrazine and the hydrazones separated by chromatography on Vaseline-impregnated paper. The details of this procedure are given under EXPERIMENTAL.The chromatograms were cut into small strips and counted in the scintillation counter. Fig. 4 clearly shows the presence of a radioactive peak corresponding to the location of a sample of the dinitrophenylhydrazone of palmitaldehyde. This peak is not found in the control samples, but a small peak between the palmitaldehyde derivative and the solvent front is apparent. This peak was found to be an artifact produced in the reaction between the labeled dihydrosphingosine and 2,4_dinitrophenylhydrazine. Since there is considerably less dihydrosphingosine in the samples which have been subjected to the action of the enzyme, the artifact is less conspicious in the incubated samples than in the controls. This procedure has been carried out on about 6 different samples, incubated under varying conditions, and in every case where the long-chain base was degraded, a radioactive peak corresponding to the dinitrophenylhydrazone of palmitaldehyde was found. DISCUSSION The enzymatic breakdown of ~hydrosphingosine which had been previously reported to occur ifs vi~ol-~ has now been demonstrated using washed mitochondrial fractions from rat liver. BARENHOLZANDGATES have recently reported the degradation of phytosphingosine by a cell-free system from rat liver, but few details are given. The instability and low levels of enzymatic activity which were found in the present study required the preparation of fresh enzyme for each experiment. An investigation has been undertaken to find a better source of the enzyme system as we11 as discovering means by which the rapid loss of activity may be prevented. At the present time, it is not certain that the reaction rates which were obtained in these studies are a true measure of the enzymatic activity. It is possible that the physical state of the substrate or the presence of the nonionic detergent may have had an inhibitory effect, although the high specific activity of the tritiated dihydrosphingosine made it possible to use very low levels of both the substrate and Cutscum in the reaction mixtures. In one experiment not described in this paper, a z-fold increase in the concentration of substrate and Cutscum did not alter the rate of reactior The unexpected finding that both ATP and ma~esium (other divalent cations were not tested) were required to demonstrate dihydrosphingosine breakdown is very interesting. It is logical to assume that the ATP requirement represents an energy source for the activation of the lipid base. The likelihood that activation could occur Biochim.
Biophys.
Acta,
x76 (1969)
348-356
DIHYDROSPHINGOSINE BREAKDOWN
355
through the mediation of a biotin-requiring carboxylation step seems remote, since no inhibition was noted following the preincubation of the enzyme with avidin. A simpler explanation is that ATP might serve as a phosphate donor for the phosphorylation of the primary hydroxyl group. Carbons I and 2 might be split off to yield phosphoryl ethanolamine. Some support for this speculation is provided by the fact that STOFFEL AND STICHT~ reported is z&o experiments in which the radioactivity from hydroxyterminal labeled dihydrosphingosine was recovered in the ethanolamine moiety of phosphatidyl ethanolamine. There is however no evidence which provides direct confirmation that the role of the ATP is to activate the lipid base, but we have begun a search for a phosphorylated intermediate. The fact that the degradation of dihydrosphingosine is inhibited by both semicarbazide and bisulfite and the demonstration that the addition of pyridoxal phosphate to dialyzed, washed or cysteine-treated enzyme fractions results in a marked stimulation in the reaction rate is good evidence that pyridoxal phosphate or a related compound participates in the breakdown of the lipid base. NAD and NADP failed to produce any significant stimulation in the rate of dihydrosphingosine breakdown by dialyzed enzyme preparations. This fact, together with the demonstration that radioactive palmitaldehyde is produced during the reaction makes it logical to speculate that dihydrosphingosine or an activated form of this compound may undergo a B,-catalyzed reaction which is analogous to the cleavage of threonine by threonine aldolase. Threonine aldolase has been shown to cleave threonine to yield glycine and acetaldehyde 13. It is not surprising that only small amounts of radioactivity can be isolated from incubation mixtures in the form of the dinitrophenylhydrazone derivative of palmitaldehyde, considering the known instability of the long-chain aldehydes and the occurrence of a number of aldehyde-oxidizing systems in liver. Attempts which have been made to cause the accumulation of the aldehyde have not been successful. The compounds usually employed to trap aldehydes such as semicarbazide and bisulfite have been found to inhibit dihydrosphingosine breakdown, probably because they also react with pyridoxal. Another possibility is that the aldehyde is an artifact which arises from palmitic acid and that the reaction actually proceeds by the conversion of the lipid base to the 3-keto derivative. STOFFEL, LEKIM AND STICHT’* have shown that 3-keto-dihydrosphingosine is rapidly converted to palmitic acid in vivo. It is also possible that there is more than one pathway for the degradation of dihydrosphingosine, The final resolution of this problem will have to await further studies. Acknowledgments The authors wish to express their gratitude to Professor GERHARD SCHMIDT for his support and the many valuable criticisms given during the course of these studies. Vote are also indebted to Dr. JONATHAN NISHIMURA for a number of stimulating discussions. This investigation was supported by Grants HE-o3-457 and NB-00356 from the National Institutes of Health, Grant 271 from the National MultipIe Sclerosis Society, and a grant from the Godfrey Hyams Trust, Boston, Mass.
Biochim. Biophys. Acta, 176 (1969) 348-356
R. W. KEENAiS, A. MAXAM
356 REFERENCES
I W. STOFFEL AND G. STICHT, 2. Physiol. Chum., 348 (1967) 1345. 2 Y. BARENHOLZ AND S.GATT, Biochemistry, 7 (1968) 2603. 3 R. W. XEENAN AND K. OKABE, Biochemistry, 7 (1968) 2696. J D. C. MALINS AND H. K. MANGOLD, 1.Am. Oil Chemists Sm., 17 (1960) 576. _' j 1:.SB~DER AZ-XI N. STEPHENS,A~~a~.“3~*~hem., 4 (1962) 128. _' 6 R. W. KEENAN AXD B. I-I. MARKS, 3~ach~~~. Biophys. Acta, 39 (1960) 533. 7 G. SCH~~IDT, E. L. HOGAN, A. KJETA-FYDA, T. TANAKA, J. JOSEPH,N. I.FELDMAN, R. A. COLLINS,AND R. W. KEENAN in S.M. ARONSON AND B. W. VOLK, Inborn Disorders ojSphingolipid Metabolism, Pergamon Press, 1966, p. 325. 8 E. LAYNE, in S. P. GOLOWICK AXD N. 0. KAPLAN, Methods in Enzymology, Vol III, Academic Press, 1957. P. 450. g W. STOFFEL,D. LEKIM API'D G. STICHT, Z. Physiol. Chem., 349 (1968) 664. 10 P. E. BRAUN ANn E. E. SNELL,J.Biol. Chem., 243 (1968) 3775. II P. E. BRAUN ANn E. E. SNELL.Pvoc. Nat& Acad. Sci. U.S., ;8 (1967)I_ 298. 12 Y;.BARENHOLZ AND S.GATT,&&hem. Biopizys. Res. Comm&z., 32 (1968) 588. 13 M. KARASEK AND D. M. GREENBERG,J.B~OE.Chem., 227 (1957) Igr. 14 W. STOFFEL,D. LEX~IMAND G. STICHT, Z. PhysioZ.Chem., 34X(1967) 1570. Biochim.
Biophys.
Acta,
176 (1969)
348-356