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Spontaneous incorporation of biogenic amines into purified proteolipid
608 6 7 8 9
PRELIMINARY NOTES
G. G. I{. I~.
GORIN, Biochemistry, I (1962) 911. FASMAN AND C. NIEMAN, dr. Am. Chem. Soc., 73 (1951) 1646I'qATH AND T. PRADHAM, Bull. Calcutta School Trop. Med., 9 (1961) 63F. KOUBA, personal communication.
Received July 2Ist, 1965 Biochim. Biophys. Acta, lO 7 (1965) 605-608
P~,*
21 lO8
Spontaneous incorporation of biogenic amines into purified proteolipid Experiments demonstrating the ability of purified proteolipids to incorporate amino acids spontaneously z and irreversibly led to similar experiments on the binding of amines to proteolipids. Alkaloids and other amines are known to be bound reversibly to serum proteins2, 3 and nerve proteins 4. Work leading to the experiments reported here revealed that, while amines could be bound reversibly to proteolipids as to other proteins, there was a fraction of the bound amine which could not be dissociated except by destruction of the proteolipid. Bovine white matter proteolipid, prepared according to FOLCH, LEES AND SLOANE STANLEY5 and LEES, CARR AND FOLCHs, was precipitated from CHCls-CH30H solution by 4 vol, of diethyl ether at o °. The precipitate was washed once with ethanol (o °) and the centrifugal residue was drained of free ethanol. The residue was then emulsified in distilled water (o °) to a conch, of io mg/ml. In a typical incubation, 0.25 ml of this emulsion was added to 0.25 ml of a solution containing 0.02 M Tris-HC1 (pH 7.5) and 0. 4 mM Iz4Clamine at o °. The incorporation reaction was begun by immersing the tubes in a 39 ° bath. The reaction was stopped after 30 rain by the addition of i ml of a IO mM solution of the nonisotopic amine and 5 ml of CHC13 and CFI30H (i : I, v/v). The lower phase from this mixture was then washed at least five times according to FOLCHv with an upper phase mixture 5 mM in the appropriate non-isotopic amine. These proteolipid solutions were chilled to o °, and precipitated with 4 vol. of cold ether. After centrifugation the proteolipids were resuspended in cold ethanol and centrifuged. Finally the proteolipids were emulsified in cold distilled water, or dissolved in CHC13-CHsOH mixtures or dissolved in 88 % HCO2H, depending upon the subsequent treatment intended. The [z4Clamines were purified within a week of their use by elution from Amberlite IRC-5o by a formic acid gradient, and the purity of the eluted product was checked by paper chromatography in two or more solvent systems. When spots appeared having as much as 1 % of the radioactivity of the main amine spot, the amine was repurified. The biphasic washing procedure and the subsequent solvent precipitations of the proteolipid freed it of all the reversibly bound isotope. Starch column filtration s revealed no trace of unreacted or dissociable [z4C]amines although CHC13-CH3OH solutions of proteolipid and of the [14C]amines used are completely separable by this technique in any of the variations of eluants previously tried 1. More vigorous methods were next tested to separate the [14C]amine from re-isolated proteolipid. Table I illustrates the effect of some mild and some drastic reagent treatments Biochim. Biophys. Acta, lO 7 (1965) 6o8-61o
609
PRELIMINARY NOTES
on proteolipid labeled with [14Cltryptamine. Proteolipid was labeled, washed and precipitated as described above. In replicate tubes, 2 mg of the ethanol-washed residue was resuspended in I.O ml of the reagents listed under the heading "treatment". TABLE I ACTION OF REAGENTS ON ~14CZTRYPTAMINE-PROTEOLIPID
Treatment
Water Io mM t r y p t a m i n e o.i IV[ trichloroacetic acid o.i M N a 2 C 0 3 - o . I M p o t a s s i u m citrate 0.2 M NaCI E t h a n o l + H 2 0 (7:3, v/v) 88 % HCO2H
Recovered proteolipid Specific activity (counts/rain per rag)
Total (counts/rain)
435 403 322 43 ° 429 425 378
244 242 171 250 249 271 229
Counts/min released from proteolipid 6-5 o 87 4.5 o 2. 5 61
Each suspension was agitated gently for 50 min at 39 °. The proteolipid suspended in HCO2H went into solution and was recovered by precipitation at o ° with 5 ml diethyl ether. Each suspension was centrifuged at the end of the heating period and the supernatant solutions were checked for radioactivity. The centrifuged residues of proteolipid were dissolved in HCO2H, and the aliquots assayed for radioactivity. o.i M trichloroacetic acid and HCOzH degrade proteolipid with loss of nitrogen and phosphorus. Na,CO3-potassium citrate denatures proteolipid with loss of some of its constituent phospholipids. Only the two acid treatments, however, significantly reduced the specific activity of the reisolated proteolipid and released radioactivity into solution in the reagents of the treatment. Similar results were seen with proteolipid labeled with [14Clhistamine, and with [14Clmescaline. ]'he released radioactivity in each treatment migrated on paper chromatography to positions identical with those of the authentic amines. This indicates that the species released is probably the same as the amine used, but the species remaining bound is not identified by this technique. Enzymatic degradation of the [14C~amine labeled proteolipids was next attempted, i.o mg of the [14Cltryptamine-proteolipid was emulsified in 0.02 M Tris-HC1 (pH 7-5) containing the various combinations of I mg protease (Streptomyces griseus) 9 and o.41 mg pancreatic lipase (EC 3.1.1.3) purified according to MARCHIS-MOUREN, SARDA AND DESNUELLETM. After incubation for 5 h at 39 °, the remaining proteolipids were recovered by centrifugation and washing with cold distilled water, were dissolved in HCOeH and were assayed for radioactivity (Table II). The supernates from the incubation treatments were assayed for radioactivity and were subjected to paper chromatography. The only radioactive spot from the protease treatment corresponded in RF to authentic tryptamine. The lipase contained a contaminant enzyme which degraded tryptamine to some other substance. The product from the [14C!tryptamineproteolipid released by the lipase treatment corresponded to the product of the action of the lipase on authentic tryptamine in its chromatographic behavior. The purpose of the lipase was to make the peptide chain more accessible to the protease. However, another of the contaminant enzymes of the lipase is an amidase, which mav be responsible for the release of the amine. Biochim. Biophys. dcta, lO 7 (i965) 608 61o
61o
PRELIMINARY NOTES
T h e p r o p e r t i e s of t h e p r o t e o l i p i d - b o u n d a m i n e s in t h e s e e x p e r i m e n t s are cons i s t e n t w i t h t h e h y p o t h e s i s t h a t c o v a l e n t b o n d s are f o r m e d b e t w e e n t h e a m i n e a n d t h e p r o t e o l i p i d d u r i n g t h e i n c o r p o r a t i o n i n c u b a t i o n . E l e c t r o s t a t i c or V a n d e r W a a l s L o n d o n forces do n o t s e e m to c o n t r i b u t e s i g n i f i c a n t l y to t h e b i n d i n g . TABLE II ACTION OF ENZYMES ON [14C]TRYPTAMINE-PROTEOLIPID
Treatment
Buffer only Lipase Protease Lipase and protease
Recovered proteolipid Specific activity (counts~rain per mg)
Total (counts/min)
382 292 24o 268
386 248 255 251
Counts/min released from proteolipid
6 184 158 187
This work was supported by grants from the Ford Foundation and from the U.S. Public Health Service (Grant B-I30).
Research Laboratory, M c L e a n Hospital, Belmont, Mass. (U.S.A.) i 2 3 4 5 6 7 8 9 io
LEWIS C. MOKRASCH
L. C. IViOKRASCHAND P. MANNER, Biochim. Biophys. Acts, 6o (1962) 415 . R. H. MCMENAMY,J. Biol. Chem., 239 (1964) 2835. E. O. AKINRIMISI AND P. O. TS'O, Biochemistry, 3 (1964) 619. I~. KIYOTA, J. Neurochem., 9 (1962) 555J. FOLCH, M. LEES AND G. H. SLOANE STANLEY,J. Biol. Chem., 226 (1957) 497M. LEES, S. CARR AND J. FOLCH, Bioehim. Biophys. Acts, 84 (1964) 464 . J. FOLCH-PI, Exposes Ann. Biochim. M~d., 21 (1958) 81. B. LINDQVlST AND T. STORG.~RDS, Nature, 75 (1955) 511. M. NOMOTO, Y. N&RAHASHIAND M. ~¢[URAKAMI,J. Biochem., 48 (196o) 593. G. MARCHIS-MOUREN,L. SARDA AND P. DESNUELLE, Arch. Biochem. Biophys., 83 (1959) 309.
R e c e i v e d J u l y 5th, 1965
Biochim. Biophys. Acts, lO 7 (1965) 6o8-61o
PRELIMINARY NOTES
G. G. I{. I~.
GORIN, Biochemistry, I (1962) 911. FASMAN AND C. NIEMAN, dr. Am. Chem. Soc., 73 (1951) 1646I'qATH AND T. PRADHAM, Bull. Calcutta School Trop. Med., 9 (1961) 63F. KOUBA, personal communication.
Received July 2Ist, 1965 Biochim. Biophys. Acta, lO 7 (1965) 605-608
P~,*
21 lO8
Spontaneous incorporation of biogenic amines into purified proteolipid Experiments demonstrating the ability of purified proteolipids to incorporate amino acids spontaneously z and irreversibly led to similar experiments on the binding of amines to proteolipids. Alkaloids and other amines are known to be bound reversibly to serum proteins2, 3 and nerve proteins 4. Work leading to the experiments reported here revealed that, while amines could be bound reversibly to proteolipids as to other proteins, there was a fraction of the bound amine which could not be dissociated except by destruction of the proteolipid. Bovine white matter proteolipid, prepared according to FOLCH, LEES AND SLOANE STANLEY5 and LEES, CARR AND FOLCHs, was precipitated from CHCls-CH30H solution by 4 vol, of diethyl ether at o °. The precipitate was washed once with ethanol (o °) and the centrifugal residue was drained of free ethanol. The residue was then emulsified in distilled water (o °) to a conch, of io mg/ml. In a typical incubation, 0.25 ml of this emulsion was added to 0.25 ml of a solution containing 0.02 M Tris-HC1 (pH 7.5) and 0. 4 mM Iz4Clamine at o °. The incorporation reaction was begun by immersing the tubes in a 39 ° bath. The reaction was stopped after 30 rain by the addition of i ml of a IO mM solution of the nonisotopic amine and 5 ml of CHC13 and CFI30H (i : I, v/v). The lower phase from this mixture was then washed at least five times according to FOLCHv with an upper phase mixture 5 mM in the appropriate non-isotopic amine. These proteolipid solutions were chilled to o °, and precipitated with 4 vol. of cold ether. After centrifugation the proteolipids were resuspended in cold ethanol and centrifuged. Finally the proteolipids were emulsified in cold distilled water, or dissolved in CHC13-CHsOH mixtures or dissolved in 88 % HCO2H, depending upon the subsequent treatment intended. The [z4Clamines were purified within a week of their use by elution from Amberlite IRC-5o by a formic acid gradient, and the purity of the eluted product was checked by paper chromatography in two or more solvent systems. When spots appeared having as much as 1 % of the radioactivity of the main amine spot, the amine was repurified. The biphasic washing procedure and the subsequent solvent precipitations of the proteolipid freed it of all the reversibly bound isotope. Starch column filtration s revealed no trace of unreacted or dissociable [z4C]amines although CHC13-CH3OH solutions of proteolipid and of the [14C]amines used are completely separable by this technique in any of the variations of eluants previously tried 1. More vigorous methods were next tested to separate the [14C]amine from re-isolated proteolipid. Table I illustrates the effect of some mild and some drastic reagent treatments Biochim. Biophys. Acta, lO 7 (1965) 6o8-61o
609
PRELIMINARY NOTES
on proteolipid labeled with [14Cltryptamine. Proteolipid was labeled, washed and precipitated as described above. In replicate tubes, 2 mg of the ethanol-washed residue was resuspended in I.O ml of the reagents listed under the heading "treatment". TABLE I ACTION OF REAGENTS ON ~14CZTRYPTAMINE-PROTEOLIPID
Treatment
Water Io mM t r y p t a m i n e o.i IV[ trichloroacetic acid o.i M N a 2 C 0 3 - o . I M p o t a s s i u m citrate 0.2 M NaCI E t h a n o l + H 2 0 (7:3, v/v) 88 % HCO2H
Recovered proteolipid Specific activity (counts/rain per rag)
Total (counts/rain)
435 403 322 43 ° 429 425 378
244 242 171 250 249 271 229
Counts/min released from proteolipid 6-5 o 87 4.5 o 2. 5 61
Each suspension was agitated gently for 50 min at 39 °. The proteolipid suspended in HCO2H went into solution and was recovered by precipitation at o ° with 5 ml diethyl ether. Each suspension was centrifuged at the end of the heating period and the supernatant solutions were checked for radioactivity. The centrifuged residues of proteolipid were dissolved in HCO2H, and the aliquots assayed for radioactivity. o.i M trichloroacetic acid and HCOzH degrade proteolipid with loss of nitrogen and phosphorus. Na,CO3-potassium citrate denatures proteolipid with loss of some of its constituent phospholipids. Only the two acid treatments, however, significantly reduced the specific activity of the reisolated proteolipid and released radioactivity into solution in the reagents of the treatment. Similar results were seen with proteolipid labeled with [14Clhistamine, and with [14Clmescaline. ]'he released radioactivity in each treatment migrated on paper chromatography to positions identical with those of the authentic amines. This indicates that the species released is probably the same as the amine used, but the species remaining bound is not identified by this technique. Enzymatic degradation of the [14C~amine labeled proteolipids was next attempted, i.o mg of the [14Cltryptamine-proteolipid was emulsified in 0.02 M Tris-HC1 (pH 7-5) containing the various combinations of I mg protease (Streptomyces griseus) 9 and o.41 mg pancreatic lipase (EC 3.1.1.3) purified according to MARCHIS-MOUREN, SARDA AND DESNUELLETM. After incubation for 5 h at 39 °, the remaining proteolipids were recovered by centrifugation and washing with cold distilled water, were dissolved in HCOeH and were assayed for radioactivity (Table II). The supernates from the incubation treatments were assayed for radioactivity and were subjected to paper chromatography. The only radioactive spot from the protease treatment corresponded in RF to authentic tryptamine. The lipase contained a contaminant enzyme which degraded tryptamine to some other substance. The product from the [14C!tryptamineproteolipid released by the lipase treatment corresponded to the product of the action of the lipase on authentic tryptamine in its chromatographic behavior. The purpose of the lipase was to make the peptide chain more accessible to the protease. However, another of the contaminant enzymes of the lipase is an amidase, which mav be responsible for the release of the amine. Biochim. Biophys. dcta, lO 7 (i965) 608 61o
61o
PRELIMINARY NOTES
T h e p r o p e r t i e s of t h e p r o t e o l i p i d - b o u n d a m i n e s in t h e s e e x p e r i m e n t s are cons i s t e n t w i t h t h e h y p o t h e s i s t h a t c o v a l e n t b o n d s are f o r m e d b e t w e e n t h e a m i n e a n d t h e p r o t e o l i p i d d u r i n g t h e i n c o r p o r a t i o n i n c u b a t i o n . E l e c t r o s t a t i c or V a n d e r W a a l s L o n d o n forces do n o t s e e m to c o n t r i b u t e s i g n i f i c a n t l y to t h e b i n d i n g . TABLE II ACTION OF ENZYMES ON [14C]TRYPTAMINE-PROTEOLIPID
Treatment
Buffer only Lipase Protease Lipase and protease
Recovered proteolipid Specific activity (counts~rain per mg)
Total (counts/min)
382 292 24o 268
386 248 255 251
Counts/min released from proteolipid
6 184 158 187
This work was supported by grants from the Ford Foundation and from the U.S. Public Health Service (Grant B-I30).
Research Laboratory, M c L e a n Hospital, Belmont, Mass. (U.S.A.) i 2 3 4 5 6 7 8 9 io
LEWIS C. MOKRASCH
L. C. IViOKRASCHAND P. MANNER, Biochim. Biophys. Acts, 6o (1962) 415 . R. H. MCMENAMY,J. Biol. Chem., 239 (1964) 2835. E. O. AKINRIMISI AND P. O. TS'O, Biochemistry, 3 (1964) 619. I~. KIYOTA, J. Neurochem., 9 (1962) 555J. FOLCH, M. LEES AND G. H. SLOANE STANLEY,J. Biol. Chem., 226 (1957) 497M. LEES, S. CARR AND J. FOLCH, Bioehim. Biophys. Acts, 84 (1964) 464 . J. FOLCH-PI, Exposes Ann. Biochim. M~d., 21 (1958) 81. B. LINDQVlST AND T. STORG.~RDS, Nature, 75 (1955) 511. M. NOMOTO, Y. N&RAHASHIAND M. ~¢[URAKAMI,J. Biochem., 48 (196o) 593. G. MARCHIS-MOUREN,L. SARDA AND P. DESNUELLE, Arch. Biochem. Biophys., 83 (1959) 309.
R e c e i v e d J u l y 5th, 1965
Biochim. Biophys. Acts, lO 7 (1965) 6o8-61o