STUDIES ON THE BIOSYNTHESIS 0F SPHINGOUPID BASES Dissertatien for the Degree of Ph. D. MECHiGAN STATE UNWERSITY KAN?! K‘RiSNANGKURA £974 b.1133“? 4'1 “flu-mix This is to certify that the thesis entitled STUDIES ON THE BIOSYNTHESIS OF SPHINGOLIPID BASES presented by KANIT KRISNANGKURA has been accepted towards fulfillment of the requirements for Ph.D. Biochemistry degree in MM; Major proféo {~- 9/12/74 Date 0-7639 Lfilth’fifb... .} 5““. «omits 300x amnm mc LIBRARY SINGERS J’H!N§'fl.1 “In“!sll ll -... ....-.. . _ ill" _. . a i. Mann.“ _ ' "I I m (I m B‘IOSWfile'" 41' “wrung! up ”315 ‘h' [8711: l! ' n i-u-xtirf . fit “when” 2! syn... liu ‘- '>~ .L ”.1 I’M and *019-9'L .n yum We? 3.... W. .- z 3 V. 7:». M- O! army-7L? 81:.“ . I; "A Mi: “khaki! P1,? MMH‘A». A. v. R." h “(in of A prob... n_,.. n. H, ‘ V .4”. w hy‘rc-lya: «t ”w gun}, _ - I. “U man “no. :cvtauyw -. .‘ “WM luhtttatr 111‘". in“; .4695: mm“ "31M lino!“ .fi-Ofi‘. any, ‘7) , ‘ “ 0C [1, 3.3- Influmn by ucmrotw Mfg? "wot; new in» . ‘ Wan-upsu- my in an". «MW .wrlttigyr‘ifiwucx 7'91 1) gal-«m .1 3 brought-cunt. tom-n- are. My. ’ an a pug-pennant... (on... u. mm mm $2‘ :‘1 ‘ mom-“MM ‘ ”IQ-Wham. (kWh-mm. M " 3:. in '4'". fdfl‘li ' d". w STUD The bit microsomes a! isotOpes. Cc Vith the 1m formation 0 Maximo-34ll 1Wed by a. gimme aft °f Config as the g: boxyl 81'0 zation of 4-1 1) red“Q hy‘imm “ism of Oxygen on C‘Q mitate ABSTRACT STUDIES ON THE BIOSYNTHESIS 0F SPHINGOLIPID BASES BY Kanit Krisnangkura The biosynthesis of sphingolipid bases was studied in rat liver microsomes and whole cells of yeast, Hansenula ciferri, using stable isotopes. Condensation of serine and palmitoyl CoA was accomplished with the initial loss of a hydrogen atom on 0-2 of serine through the formation of serine-PLP Schiff's base. The proposed intermediate, 2-amino-3-ketoacid-PLP Schiff'base, underwent decarboxylation, fol- lowed by addition of a proton from the medium to yield 3-ketosphin- ganine after hydrolysis. The overall process occurred with retention of configuration since 3-ketosphinganine has the same configuration as the é-serine substrate with the palmitoyl group replacing the car- boxyl group of serine. A kinetic isotope effect was observed in utili— zation of [2,3,3-2H3Jserine by microsomal enzyme system from rat liver. 4-Hydroxy8phinganine could be derived from 3-ketosphinganine by: 1) reduction of 3-ketosphinganine, followed by hydroxylation; and 2) hydroxylation of 3-ketosphinganine, followed by reduction. The mecha- nism of the hydroxylation step is still unknown. Water and molecular oxygen were ruled out as the direct oxygen donors to the hydroxyl group on C-4 of 4-hydroxysphinganine (Thorpe and Sweeley, 1967). [1-180]Pal- mitate, [3-180]serine, [1-180]sphinganine, [3-180Jsphinganine, [Z-laoklucose, 31 except those pre: cxygen donors to The oxygen donor water in the medi 4.: m1: Krisnangkura Lichen present in the yeast extract were all ruled out as the I .‘ Widener: to the hydroxyl group on 0-4 of I. E,’ , _" .. ‘ 3:; gen defia- iwas heat-stble and did not exchange its oxygen with I. “‘0 1...: ”I: in the medium at autoclave temperature. .' .‘v {to} VT , n i . , STUDIES on THE BIOSYNTHESIS OF SPHINGOLIPID BASES .h g u . . ,‘5 I“. I we” ‘ .’ . ‘fhfi EIUI‘: . x ‘ ‘ Kanit Krisnangkura .;I ,31‘ A, I.) ‘7‘”1‘5" Y14M‘--‘ have rec v . ’. C', u l A DISSERTATIM Submitted to Michigan State University in partial fulfillment of the requirements for the degree of WSW PHILOSOPHY Department of Biochemistry - .. _ . | _ 3‘93 ‘ V ‘Z ‘ . ‘ " a7... . , -- ‘ 7 ,, :3 ~ .._'»\- n2:- . .1974 . . ~“33.73.; 3 t”! " “ ' I ‘2' -.P- x}; ' ‘A.’ “ “ ~ a .Lfifl". .-'-.’_ P ‘1. P f ‘ z ‘ ‘o ,. ii" 1 ‘ *- ‘ I .!~‘“;. 11-." ‘ w V Mould like Sweeley for his 8“ of this study. Sir :embers of my Ph-E L. Bieber, Dr. Roi Special ackv flatten for their studies, IESpeCti‘ I have recieved f Bieber and other mistry. W" ACKNOWLEDGEMENTS I would like to express my sincere gratitude to Dr. Charles C. Sweeley for his guidance and constant encouragement during the course or this study. Sincere appreciation is also expressed to the other members of my Ph.D. guidance committee: Dr. William W. Wells, Dr. Loran L. Bieber, Dr. Robert S. Bandurski and Dr. John C. Speck, Jr. Special acknowledgements are made to Mr Bernd Soltmann and Jack E. Harten for their expert in high and low resolution mass Spectrometry studies, respectively. Finally, I would like to thank for the helps I have recieved from Dr. Roger A. Laine, Dr. Ray K. Hammond, Dr. Mark A. Bieber and other associates from this lab and the Department of Bioche- mistry. ii , 1 ACKWIEDGEM LIST OF TAB LIST OF FIC- LIST 01-‘ A331 mm or L: A. Structure 3' Nomenclat 6' ISOlation D- Chemical 2' BiOsynthe F' Degradati TABLE OF CONTENTS ACKOWLEDGEMENT ................................................. LIST OF TABLES ................................................. LIST OF FIGURES .................... . ........................... LIST OF ABBREVIATIONS .......................................... REVIEW OF LITERATURE ON THE SPHINGOLIPID BASES ........... . ..... A. Structure and Stereochemistry ................ . .............. B. Nomenclature ................................................ C. Isolation and Separation .................................... D. Chemical Synthesis .......................................... E. Biosynthesis .................. . .............. . .............. F. Degradation.... ...................................... . ...... G. Biological Properties. ........ . ............................. INTRODUCTION ...................... . ............................ MATERIALS AND METHODS. ......................................... A. Materials. ............................... . .................. B. Methods. .............. . ..................................... 1. Gas-Liquid Chromatography ..... . ....... . .................. 2. Mass Spectrometry ...... ...... ........... . ....... ......... ‘ 5. Preparation of [2-1501Glucose ................... . ........ ' h. Synthesis of [5-180]Serine ...... . ........ ..... ........... | 5. Preparation of [1-180]Palmitate ............... ........... iii Page 12 19 21 25 30 55 35 57 37 37 38 39 ho 6 Synthe 7. Prepax 5.Isolat 9. Prepal 10. Grovti sphing 11. Hydro] RESUL'IS ..... L & Mass Spec stUdiES c h-Hydroxy . Incorpora . Studies c 1' Deter“ 2' h'HYdI GIUCOS . Studies c 1. Deter“ 2' u'HYdr PreSer . Studies c IncorPOra 61.0% 0n 1. Yeast 2. Yeas C V‘FE‘ TABLE OF CONTENTS (CONT'D) Synthesis of Deuterated Sphinganines ..................... . Preparation of Microsomal Enzymes ........................ . Isolation and Purification of Sphinganine ................ \Omsl 0‘ Preparation of Volatile Derivative of Sphingolipid Bases. 10. Growth of Yeast and Isolation of Tetraacetyl-h-hydroxy- sphinganine .............................................. 11. Hydrolysis of Sphingolipids .............................. RESULTS ........................................................ A. Mass Spectrum of Tris-O-TMSi-N-Acety1-h-hydroxysphinganine.. B. Studies on the Incorporation of [1,1,5-2H31Sphinganine into h-Hydroxysphinganine .......................... ‘ .............. C. Incorporation of fl,l,3-2H3]Sphinganine into h-Sphingenine.. D. Studies on Yeast Grown on [2-1SOJG1ucose .................... 1. Determination of 18O on C-2 of Glucose ................... Glucose .................................................. E. Studies on Yeast_Grown in the Presence of [5-1BOJSerine ..... 1. Determination of 18O on 0-3 of Serine ..... . ......... ..... 2. h-Hydroxysphinganine Produced by Yeast Grown in the Presence of [3-180]Serine .............. . ...... ........... F. Studies on Yeast Grown in the Presence of [1-1801Pa1mitate.. G. Incorporation of H530 into h-Hydroxysphinganine by Yeast Grown on Different Media ...... ..... ............... .......... 1. Yeast Grown on IMkl and LM—2 ....... . ................ ..... 2. Yeas Grown on Ethanol as the Principal Carbon Source ..... 2. h-Hydroxysphinganine Produced by Yeast Grown on [2-180]- iv Page hl 1+5 Ah hh us 45 47 1+7 1+9 51 55 55 56 59 59 62 6h 67 67 TO H. Mass Spectra I. incorporatim 1. Cnaracter metry. . .. 2. Incorpora Rat Liver J. Incorporatio 1" IncorPotatio sphinganine. ' IncorPol'atio bif'ie.'.-1st..,, DISCUSSION. . . ., R’ZI’E‘I‘ENCES ..... TABLE or CONTENTS (CONT'D) H. Mass Spectra of Bis-O-TMBi-N-Acetylsphinganines ............ I. Incorporation of [2,5,3-2H3]Serine into Sphinganine ........ 1. Characterization of [2,5,5-2H3]Serine by Mass Spectro- metry .......... ......................................... 2. Incorporation of [2,5,5-2H3]Serine into Sphinganine by Rat Liver Microsomes .................................... J. Incorporation of Deuterium from 2H20 into Sphinganine ...... K. Incorporation of Serine/ 2,5,5-Trideuteroserine into Sphinganine ...... .. .......... . ............................. L. Incorporation of [2,3,3-2H3]Serine into Sphingolipid bases by Yeast ......... . ........... . ............... ........ ...... DISCUSSION.... ........... . ................................ .... REFERENCES..... ..... . ................... . ..................... Page 76 88 88 91 96 98 108 129 hon \M \J‘ (D . Chromatogrz - AVA Analys: [1,1,5-2H3j Presence 0: ' AVA Analysj ' AVA AnalyS . AVA Analys; in “('5 in (691%)... . AVA Analys . AVA Analys in 111-1 in in I'M‘l ir LIST OF TABLES Page 1. Chromatographic Separation of Long-Chain Bases ......... '.... 8 2. AVA Analysis of [1,l-2H2]-h-Hydroxysphinganine and [1,1,5-2H3]-h-Hydroxysphinganine from Yeast Grown in the Presence of[1,1,3-2H3]Sphinganine .......................... 50 3. AVA Analysis of 18O on Carbon-2 of Synthetic Glucose ....... 57 h. AVA Analysis of 180 on Carbon-3 of Synthetic Serine ........ 61 5. AVA Analysis of 18O in h-Hydroxysphinganine from Yeast Grown in LM-j in the Presence of 11: Glucose and 0.5% [51-8018erine (6.911%) ................................... . ..... .. ......... 63 6. AVA Analysis of 1‘30 in Methyl Palmitate .................... 65 7. AVA Analysis of 180 in h-Hydroxysphinganine from Yeast Grown in LMel in the Presence of [1380]Palmitate (1 mg/ml) ....... 66 8. AVA Analysis of 18O in h-Hydroxysphinganine from Yeast Grown in LM-l in sgeo (50.15%). .................................. 68 9. AVA Analysis of 180 in h-Hydroxysphinganine from Yeast grown in 124-2 in 11550 (19.1%) .................................... 69 10. AVA Analysis of 180 in h-Hydroxysphinganine from Yeast Grown in 111-5 and 2% Ethanol ‘in 1122,80 (13.7%) ................ 71 11. AVA Analysis of 18O on C-2 of Glucose from Yeast Grown in 124-5 and 2% Ethanol in Hgao (13.7%). ...... . ........ . ....... 72 12. Composition of Reaction Mixture for the Incorporation of [2,5,3-2H518erine into Sphinganine by Rat Liver Microsomes. 92 vi \ 17. . Composition Deuterium f ' $011188 ...... . Composition [am-2a.] Hicrosomes . Deteminati the Mixture - Detenminatj SPhinganine "1‘“ [2,5,3 50mg EXamp by Retenti 15. 1h. 15. 16. 17. LIST or TABLES (CONT'D) Page Composition of Reaction Mixture for the Incorporation of Deuterium from 2H20 into sphinganine by Rat Liver Micro- somes ...................................................... 97 Composition of Reaction Mixture for the Incorporation of [2,3,3-2H3]Serine/Serine into Sphinganine by Rat Liver Microsomes ................................................ 99 Determination of the Amount of [2,3,5-2H3JSerine/Serine in the Mixture by AVA ......................................... 101 Determination of the Amount of Sphinganine and [1,1-2H2]- Sphinganine Formed in the Microsomal Reaction Incubated with [2,3,5-2H318erine/Serine Mixture .............. ........ 103 Some Example of PLP-Dependent Enzymes which Are Accompanied by Retention of Configuration .............................. 120 ._. n) \JT .A‘, (1‘) 15. 16' H353 . lntennedial . Degradatior zymes whici activities - T90 broad 1 sYnthesis.. 'HaSS Specti ' ProPosed m. 'M‘33 Specti 'Hass Spect: B} iSOIate Carbon SOu ' PESS spect °Hass ‘Mass 'lhss U. M. Spect sPect Spect Mass Mass Mass SPEct SPECt 8Pee: 8Pee: nine ...... 1 _- -4- LIST OF FIGURES Page 1. Intermediary metabolism of long-chain bases ................ 22 2. Degradation of complex sphingolipids, illustrating the en- zymes which are characteristic of human diseases if their activities are attenuated .................................. 29 5. Two broad hypothetical mechanisms of 5-ketosphinganine bio- synthesis .................................................. 3h h. Mass spectrum of tris-O-TMSi-N-acetyl-h-hydroxysphinganine. #8 5. Proposed mechanism of h-hydroxysphinganine biosythesis ..... 52 6. Mass spectrum of bis-O-TMSi-N-acetyl-h-sphinganine ......... 5h 7. Mass spectrum of bis-O-TMSi-N-acetylserine ................. 6O 8. Mass spectra of TMSi-glucose methoxime; A) reference and B) isolated from yeast grown on ethanol as the principal carbon source ........................... .. ................. 7h 9. Mass spectrum of bis-O-TMSi-N-acetylsphinganine ............ 8h 10. Mass spectrum of bis-O-[2H9]TMSi-N-acetylsphinganine ....... 8h 11. Mass spectrum of bis-O-TMSi-[5-2H]N-acetylsphinganine. ..... 85 12. Mass spectrum of bis-O-TMSi-[1,1-2H2]N-acetylsphinganine... 85 15. Mass spectrum of bis-O-TMSi-I},5-2H2]N-acetylsphinganine... 86 1%. Mass spectrum of bis-O-TMSi-N-E2H3]acetylsphinganine ....... 87 15. Mass spectrum of bis-O-TMSi-[1,1,5-2H3]N-acetylsphinganine. 87 16. Mass spectrum of bis-O-TMSi-[1,1,2,5,h,h-ZHéJN-acetylsphinga- nine... ..... ..... ...... .... ........ . ....... . ............... 87 viii 1'. Mass spectr >—‘ \D L) 26. - r. t. B} [5)5'2HE :. Mass spectr microsomal -Mass spectr CI'OSOmal re Mass spectr l‘i'l'U’Cll'Oiltysp Sence of [2’ ‘ ClY‘301ysis ‘ “Obable me . Possible n 17. 18. 19. 20. 21 25. 2h. 25. 26 LIST OF FIGURES (CONT'D) Page Mass spectra of bis-O-TMSi-N-benzylidineserine; A) serine B) [5,5-2H2]serine and C) [2,3,5-2H333erine ....... . ....... .. 89 Mass spectrum of TMSi-N-acetylsphinganine isolated from microsomal reaction incubated withl?,3,3-2H3]serine ......... 95 Mass spectrum of TMSi-N-acetylsphinganine isolated from mi- crosomal reaction carried out in 2H20 ....................... 95 Mass spectra of TMSi-N-acetylsphinganine and TMSi-N-acetyl- h-hydroxysphinganine isolated from yeast grown in the pre- sence of [2,5,5-2113] serine. . . .. .............................. 107 Glycolysis of [2-130Jg1ucose .............................. . . . 112 . Probable mechanisms of 5-ketosphinganine biosynthesis....... 118 Structural relationship of greetine and gyj-ketosphinganine. 121 Possible reaction sequences of 5-ketosphinganine biosynthe- sis ...... ........ ......... ... ................ . ........... .. 122 Probable conformations of PLP-serine Schiff's base in the transition state... ....... .. ....... . ........... ............ 126 Biosynthesis of S-aminolevulinic acid.... ......... ......... 128 ix Ac AVA Bu Cbz Cer DNP DIT Et Gal GalNAc Glc GLC 114-1 114-2 LIST or ABBREVIATIONS ._ acetyl 'f.\.-.. . SC?,:'_=;.‘ . 3 J 7:7 7 accelerating voltage alternator ' "butyl 1'86! . », 7 . . (7 37$: ' carbobenzoxycarbonyl 1- ] ' fro; sz- ‘ '7‘; ° ‘. £1“ ' ' ' ceramide ."%-".n12:; _ . t t' {‘Mm' ' dihitrophenyl \',§"I, ID”? "..1 ' .. .7 V‘. 511' dithiothreitol 3. . . 1* "73m“ " ethyl 81:13:; 1&ife “'0 galactose .? } AW ' 'N-acetylgalactosamine ' .' “g. [$39th on ' glucose "" ';".&in"' ' w gas-liquid chromatography I r k s . w m!" 3' liquid medium-1 ' 7 “L'L' ' liquid medium-2 ' w W" “ “ i'Iqu‘Id median-3 c-17 . ‘ )5?“ methyl flvryrs u =.--.x , - amiss spectrometry .st'. "‘U . ., 12"» 1' " ' ' ‘ '1‘ Mr N-acetylneuraminic acid ‘ 1.5‘};ch ti1c.11.hi.a..y1 .1 fed. m pyridoxal phosphate ‘ ' ' ' ' ' "' "' 'Li'b “PIG 11": 171.11.. 1 ‘. :e‘f‘. ' 7 ‘ 1, - . " . '4. 1 7 prépyl ' 7. {3" ifhinl 1'a'ye'i" chromatbgrapfiy " ' ' ‘ ” . 7-" “ "3 ' -" “isms « ~ . ' . ~ y‘I'iIIyl ~'.- -. .u- : ' ’ “is f 1 x .5 1' A. Structur In stu (15011) 1501 (from Greek formula (c1. close for t] Hornet and '. nine, indie.- (1919) char; comPound by sphinSOSine sine and d1} respectiVelj sphingtmine tween 0-1,. er and west: 1C I Widean the --< — vvvv' REVIEW OF LITERATURE ON THE SPHINGOLIPID BASES A. Structure and Stereochemistry. In studies on the chemical composition of human brain, Thudichum (188k) isolated an alkaloid-like compound which he called "sphingosin" (from Greek, sphingein, which means to bind tightly). The empirical formula (C17335N02) given by Thudichum was incorrect but remarkably close for that time, especially using the free base for analysis. Worner and Thierfelder (1900) reported that sphingosine absorbed bro- mine, indicating that it was an unsaturated compound. Levene and Jacobs (1919) characterized sphingosine as a monoaminodihydroxy unsaturated compound by hydrogenation and preparation of triacetyl derivative of sphingosine and the reduced product. Chromic acid oxidation of sphingo- sine and dihydrosphingosine yielded myristic acid and palmitic acid, respectively (Klenk, 1929; Klenk and Diebold, 1951), suggesting that sphingosine was a Ola-straight chain compound with a double bond be- tween C-h and C-5. Ozonolysis of sphingosine or its triacetyl deriva- tive yielded a nitrogen fragment containing four carbon atoms (Levene and West, 191h; Klenk and Diebold, 1951), providing another piece of evidence that the double bond was at C-h. Dihydrosphingosine was re- ported to consume 2 moles of periodate whereas its N-acetyl or N-benzoyl derivative were not attacked by periodate under a variety of conditions (Carter7gg 21., 19h2 and 19h7), suggesting that the two hydroxyl groups were not adjacent. Accordingly, l,5-dihydroxy-2-aminooctadec-h-ene was prooosed 1 Naturallj absorbed in t' vas character and Stotz, 19 sphingosine t gested that r Xy-Q-aminoom 2 was proposed for the structure of sphingosine. Naturally occurring sphingosine, cerebrosides and sphingomyelins absorbed in the infrared region around 975 cm.1 (or 10.5 mu) which was characteristic of the trans double bond (Mislow, 1952; Marinetti and Stotz, 195k). Kiss 33 g}. (l95h) correlated the structure of sphingosine to 2-erythro-2-amino-5,h-dihydroxybutyric acid and sug- gested that naturally occurring sphingosine was Q-erythro-1,5-dihydro- xy-2-aminooctadec-h-ene, as shown below. Sphingosine (1) During studies on the chemistry of sphingosine, Carter and Norris (19h2) usually detected a small amount of dihydrosphingosine. The structure and stereochemistry of this compound was subsequently shown to be72-eEzthro-l,5-dihydroxy-2-aminooctadecane (Carter and Shapiro, 1953)- DihydrOSphingosine (2) Sphing kingdom wer although th room by Zel cerebrin be (1951+) and hides of and pentade 0-1 to C-4 I my) deriva: the N-benzo: Same COnfigi tion of N- b. assigned the oxidant”,1 01 hydrOXYPalm established The eaSe of group 0n 0-: the hydrOxy: group 0n C‘Z group on Ca phymsphing octadecane ( HMHW-‘ 5 Sphingolipid bases which occurred predominantly in the plant kingdom were found to contain phytosphingosine (Carter 2; $1., 195k), although this compound, phytosphingosine, was first detected in mush- room by Zellner (1911). Several structures had been prOposed for yeast cerebrin before the correct structure was proposed by Carter _t _l. (l95h) and Carter and Hendrickson (1965). Phytosphingosine consumed 5 moles of periodate with a concomitant production of formaldehyde and pentadecanal, suggesting that h polar functional groups were on 6-1 to C-h of a 013' straight chain (Carter _5 _1., 195k). The N-ben- zoyl derivative, however, consumed only one mole of periodate, and the N-benzoylserinal that formed from periodate oxidation had the same configuration as N-benzoylserinal derived from periodate oxida- tion of N-benzoylglucosaminitol. The amino group on C-2 was, therefore, assigned the 2 configuration (Carter _£ _l., 195k). Partial periodate oxidation of phytosphingosine followed by Ag20 oxidation yielded 23- hydroxypalmitate (Carter and Hendrickson, 1965). This experiment established that the hydroxyl group on C-h had the 2 configuration. The ease of acyl migration from the amino group on C-2 to the hydroxyl group on 0-5 of anhydrophytosphingosine suggested that the amino and the hydroxyl groups were in the gig configuration. Since the amino group on C—2 had the 2 configuration, it followed that the hydroxyl group on C-5 should have the 3 configuration and naturally occurring phytosphingosine was then assigned as 2-ribo-1,5,h-trihydroxy-2-amino- octadecane (Carter and Hendrickson, 1965). ...N. ‘E"*4 '11: .~ . - Periodate 1959) reveal; dihydrosphini length homol. Pounds, To d. (Karisson, 1. 3' Nomenclat The Com recolilllended flamed dihYdr 2-amir100cta d The name substituents 1101101088 Shc systematic 1 atom as the Example. Cla~h0m010g The Con} Phytosphingosine (5) Periodate oxidation of Sphingolipid bases (Sweeley and Moscatelli, 1959) revealed, besides the three major long-chain bases (sphingosine, dihydrosphingosine and phytosphingosine), the occurrence of chain- 1ength homologs as well as the 332- and anteiso- branched chain com- pounds. To date about 6h sphingolipid bases have been detected (Karlsson, 1970; Weiss and Stiller, 1972 and 1975). B. Nomenclature. The Commission of Biochemical Nomenclature of IUPAC-IUB (1967) recmnmended the name sphinganine (2) for the compound previously named dihydrosphingosine (2g-aminooctadecane-1,5-diol or'B-erythro- 21aminooctadecane-1,5-diol or (2S,5R)-2-aminooctadecane-1,5-diol). The name, sphinganine, may be modified to indicate additional substituents or higher or lower homologs. The prefixes to designate homologs should be derived by deleting the terminal "ne" from the systematic name of hydrocarbons that have the same number of carbon atoms as the principal chains of the long-chain bases. Examples. Eicosasphinganine for Geo-homolog; sphinganine for Ole-homolog; hexadecasphinganine for Gig-homolog. The configuration of the additional substituents should be specified indicates tion at C- if they d: "c: 0r,E re: orleft, Fischer p the term Exam named phy The of the co " n* _ l1 ene", "a Orientati specifiec indicates EXar sphin808: Sine. 5 specified by the prefixes "3-" or tap" following the number that indicates the position of the substituted carbon atom. The configura- tion at C-2 and C-5 should be specified in the same manner, but only if they differ from those in sphinganine. In every case, the prefixes g orlL refer to the orientation of the functional group to the right or left, respectively, of the carbon chain written vertically in Fischer projection with C—l on top. If the configuration is unknown, the term f§-" should be used as prefix to the name. Example. EB-hydroxysphinganine for the compound previously named phytosphingosine. The names of unsaturated compounds are derived from the names of the corresponding saturated compounds by replacing the ending * "ane" with the appropriate ending denoting unsaturation such as ene , "adiene",, "yne". A double bond is presumed to have the trans orientation of the carbon chain unless cis or unknown geometry is specified by the term figi§-' or "3-' preceeding the number that indicates the position of the double bond. Examples. h-Sphingenine for the compound previously called sphingosine; gig-h-sphingenine for the geometric isomer of sphingo- sine. The trivial name "sphingosine" may be retained. If trivial names other than sphingosine are used, they should be defined in each paper in terms of this nomenclature, or of the general nomenclature of orga- nic chemistry. * "ane" is not the correct ending of sphinganine, it should be "anine" ........"enine", adienine", "ynine". The te logs and st tives of th ~sphir -g1ycc and one or -ceran «ceret -gang1 acid. Certai POinted Out SYnonyms of sPecific in example, 5F £0110w the in additioc will be use the stereoc repreSQnt 2 “38329-"7EE quently enc “ate the 814 811mm 3%4. ......A 6 The term long-chain base may be used for sphinganine, its homo- logs and stereoisomers, and for the hydroxy and unsaturated deriva- tives of these compounds; -sphingolipid, for any lipid containing a long-chain base; -glycosphingolipid, for any lipid containing a long-chain base and one or more sugars; -ceramide, for N-acyl long-chain base; -cerebroside, for a monoglycosylceramide; -ganglioside, for a glycosphingolipid containing a neuraminic acid. Certain disadvantages of the aforementioned nomenclature were pointed out by Karlsson (1970), who said "Several authors have used synonyms of the terms Sphingolipid long-chain base(s), not including specific information on chain length, stereochemistry, etc., for example, sphingosine bases, sphinganines, sphingosines. This does not follow the proposal given, but may indicate a need for a general term in addition to long chain bases". Accordingly, the term sphinganine will be used here to represent 2-am1nooctadecane-1,5-diol, ignoring the stereochemistry on C-2 and C-5. Similarly, h-sphingenine will represent 2-aminooctadec-h-ene-1,5-diol. The terms "erythro-", "threo-","ribo-", "arabino-", "1230-" and "xyl -" which were fre- quently encountered in the literature will also be used here to desig- nate the stereochemistry of the whole molecule of long-chain bases. Examples. B-Erythro-sphinganine for the recommended sphinganine; Q-erythr -h-sphingenine for sphingosine; 2-ribo-h-hydroxysphinganine groups, C. Isola Alt] (Kar18507 with the bound wit hydrolysi bases fro, Yields of noIiC acic Berline and genine (We h‘hydroxy37 siderable a (1965) Obs suppress th thEhYdroly the conditic for the comp0und previously called phytosphingosine. The term sphingolipid base will be used interchangeably with the term long-chain base; -dihydroxy long-chain base will be used for sphingolipid base which has two hydroxyl groups; -trihydroxy base, for sphingolipid base which has three hydroxyl groups, on C-1, C-5 and C-li. C. Isolation and Separation. Although free long—chain bases were recently found in nature (Karlsson, 1970), they represented a very small amount in comparison with the total long-chain bases in that tissue, most of which are bound with fatty acids and sugars or phosphorylcholine. Alkaline hydrolysis or acid hydrolysis can be used to liberate the long-chain bases from these complex molecules. Alkaline hydrolysis gave poor yields of sphingolipid bases (Robbin ggpgl., 1956). Anhydrous metha- nolic acid hydrolysis, however, tended to give 5-0-methyl-h-sphin- genine and 5-methoxy-5-deoxy-5-sphingenine by-products of h-sphin- genine (Weiss, 196%). If the long-chain base used in the study was h-hydroxysphinganine, anhydro-h-hydroxysphinganine was formed in con- siderable amounts (O'Connell and Tsein, 1959). Gaver and Sweeley (1965) observed that a trace amount of water in methanol could suppress the amount of ether by-products. Optimal conditions for the hydrolysis of complex sphingolipids were pursued and reported by Gaver and Sweeley (1965). Stoffel and Assmannn (1972) claimed that the conditions used by Gaver and Sweeley (1965) were not strong 11“ .momom cfifinolwcoa 5x0hokcuhh bum AXOMDAZwQ No COwuothow *lfltflfi CHISUINEOA N0 COuuflhUQDW UHSQSMQOUUEOHSU .H BHAEH .msauquunEOHSO momma-cgnu «DAB manaoquuoEouno ofisvfla-wom «Que “Lacunaouuficaouzam .mzn mfimuooo ao< «Hzawmaznuoaauuafimza ”muoauod>ounn< Aoemav Loaaaum can mess: uezeoeauuon -ouuazna no occuoo< oneness .onoHSLHouaauuo< Amwwmav some can Naoeemuum amuse swam neosvnno movmz zm can moor-mHo=o m as» season easaoo Amwmav assesses mzo mnmnm .mouz-nao=o-o=uxo= .Lommuz mm was o How uuaaam Aawmmav .wm mm amnaoum u<-z Hum .=Ouz-maumo Ammmav Aofioosm one uo>oo uamunasm woman «one Hues o: .mosaz zm-=ouz-maomo Asmmav Hfiucm new canon woman «one enmmnmm .om=-:ouz-naomo .o How uuaaam mam Anomav :OmmHqu Anomav moawmam can no>ou HmZHuo Aewmav subsoSm can masons amze-o new u<-z on-um mmm .mOmom cause-mac; mxouohsanh one axouohnwn mo eoHuouoaom *uouom cacao-moon we coauouomom canaoquuuaousm .H candy ACOT~V Eonuox0«3 DES U~ODOum Unwm oeu coaoxeom Anomav HOSHUOZ oau oouo>wmnnaom Abwmav Hamam ocu canon Ammmav conga was enough Awwmav canon one ocanzm 0/ “Hemfiv Amamosm one condom AHNmHV mofiwosm one OuHHom Anomav hoHo03m oco uo>ou AMNQHV zoflooam ocu ocofiflmm “meav hoHoosm one common “momfiv onooam our uo>uo Aowmgv Eusuoxoaz one «gooOum mzn cm we on .waoxo=-=oux-mao=o .Lomumoz am can a How suaaam u<-o was -z mumm .mooz-maomo o<-z mnsm .mouz-maomo .mozm< mom wen u Hum museum Lucauo: .mosmz zm-=oux-naumo : mmumm .om=-=oo=-mHoao a": .mooz-oaomu Ham «occuooo amza-o ecu u<-z om-mx am=H-o amza-o can o<-z on-um mam .mOmom cacao-ween ocquSuumcb one oouonnuom mo coauunoaom u<-o oer -z oaauuaaouoooo Apmmfiv no>mo one nausea AmmmHv moaoosm one uo>eu Ammmnv conununz Ammmav aoaooam one uo>so Amwmav hoaookm oao uo>mo 10 Aommnv nommnnaa Awmmav Hu>oM one hooum Anomav cadence: mehno Hmzhuo oco oum onuum mmm .noHuouomom :uwnon mango caumnmm .omn-zouu-noaz .naaouuoaunonnm ononm ouuo>u¢ canvases mummm «noes no snooH .nomz-naono .u unaasH< Ae.uaouv a «Haas Anomav anonm one cause moose sous aummnmo .Onr-=0o2-c~uru so How s0u~wm UQH ND.UC00» e twoflh 11 AmbmHv. luo newaHunM Ammmav nHAnN one oannh Awwmuv nunun one oannm w . Anmmdv oanuuz one uouu>Hnonaum H m m . Abmmuv anon» one nonnm an.“ a mouse scum nouns scum mouse scum _noann swam .“W. on 6H "on 3:83.851}? :5 1.5.. : .a..01qp‘.N-\.u . .:_._r..i... . 7 .325 a... 0 Ha. 83». I .H. N agomooflunflvlu H a 88058 HSH .3 .832 am. .8838 , 3. mm ....mm LU. ..M r» . e r . I.“ «V c i... filing-en £11.; I -‘ enough t1 that sph tive auno lins wit' prior to tive amo differen matograp solved 1.; Kisic it D- Chemi 1. Co Sanine, h- Senatior activit) C‘S and bases tc aqerus 1975). : hydroger dOuble 1 .-.—v“— ...-L 12 enough to cleave the phosphate diester linkage in sphingomyelin and that sphingenyl-l-phosphorylcholine was formed in almost quantita- tive amount. Michalec (1967) and Karlason (1968) treated sphingomye- lins with phospholipase C to hydrolyze the phosphate diester linkage prior to alkaline hydrolysis and these authors reported a quantita- tive amount of h-sphingenine. Separation of long-chain bases into different classes or individual compounds can be achieved by chro- matographic techniques, shown in Table 1. Optical isomers were re- solved with optically active organic acids (Ellner 35751., 1970; Kisic _e_t a_1., 1971; Sticht e_t a_l., 1972). D. Chemical Synthesis. 1. Conversion of h-Sphingenine to Sphinganine and h-Hydroxysphin- ganine. h-Sphingenine was converted to sphinganine by catalytic hydro- genation (Weiss and Stiller, 1967). When tritium gas was used, radio- activity was not evenly distributed. About h7$ was on C-h,57% was on 0-5 and 16% was on C-6 to 0-18. Reduction of unsaturated long-chain bases to saturated long-chain bases could be accomplished by using aqueous hydrazine (Renkonen and Hirvisalo, 1969; Hammond and Sweeley, 1975). The use of hydrazine had certain advantages over catalytic hydrogenation in that it did not cause isomerization or migration of double bond (Aylward and Sawisloka, 1962). Conversion of h—sphingenine to h-hydroxysphinganine was inde- pendently reported by Weiss and Stiller (1965) and Prostenik 25 l. (1965). Tribenzoyl-h-sphingenine was oxidised with perbenzoic acid and the epc derivative 2. S S 113111111 (19 the oxime ester (6) oxime inte H ,/pd "EN Grc rePOrted 13 and the epoxide intermediate was then reduced to h-hydroxysphinganine derivative. 1 2. Synthesis. Sphinganine was first chemically synthesized by Gregory and Malkin (1951). 5-Ketoacid ester was oximinated with butyl nitrite and the oxime intermediate (5) was catalytically hydrogenated to amino ester (6) which was further reduced to sphinganine with LiAlH4. The oxime intermediate (5) could be directly reduced to sphinganine with Luna (Fisher, 1952). Butyl nitrite ‘h 9 9. clsualb- 0112- coocrg clsnalc- -cooc11a -on o lie/L... clsnalfl- gm-coocua M5_>C15H31CH-§§- caaos 2 2 (6) (2) Grob and Jenny (1952) and Egerton gt a}. (1952) independently reported the condensation of 2-nitroethanol and palmitaldehyde, yielding 2-nitro-1,3-dihydroxyoctadecane (9). The nitroalcohol (9) was catalytically hydrogenated to sphinganine. - on I clssmcno + uo-cnacsanoa 3-. clsmflcn-SH-cngon 02 OH JL’. 015H31éfi- (.m- CH20H N32 (2) A sim (1957), Who synthesis 0 could not b (12) either C13H27' A dij ””9“ by : of Ethyl a: acid ester sence of a1 hydrazone . Save qUant; kem aCid . one 0:- two 1h A similar synthetic sequence was reported by Crab and Gadient (1957), who used 2-hexadecyna1 in place of palmitaldehyde for the synthesis of h-sphingenine. The nitro-diol intermediate (11), which could not be hydrogenated in this case, was reduced to an amino-diol (12) either by aluminium amalgum or zinc in hydrochloric acid. on 011 I | c13H27-(Ec-cso + H0-CH2-CH2-N02 -—°}—r—>— C13H27-CEC-CH-CH-GHZ N02 (10) (8) (11) ‘1 9H s 9H LiAl claum-cw-cn-cn-cagou ——>— Clsfizy-CEC-CH-CH-CHZOH y H 11112 NH2 (1) H2/Pd- 3% on (12 ). clang-c-g- 8:11- 911- cuaon 11 NH 2 (13) A different approach for the synthesis of sphinganine was re- al. (1958). Acylation ported by Shapiro and Segal (195h) and Shapiro st of ethyl acetoacetate anion (15) with palmitoyl chloride yielded diketo- acid ester (16). Diazotization of diketo-acid ester (16), in the pre- sence of ammonium salt, gave hydrazone (18). Reductive acetylation of hydrazone (18) with zinc in acetic acid and acetic anhydride mixture gave quantitative yield of 2-acetamido-5-keto-acid ester (19). This keto acid ester (19) could be reduced to N-acetylsphinganine (21) in one or two steps, as shown below. Zn \ ACEO/fi SYch I: m (ShaF t0y1 xYSpt 15 Na* Na “ c H -c001 (Ea-CO-CHZ-COZEI‘: —> Gila-CO-CH-CogEt —l§JJ—p clsnal-co-cH-cozsc coca3 (1h) (15) (16) O'NiN. C1- COCHS ——> Clsfisl-w-‘C'C02Et ——* Clsusl'CO-E'COZEt NW N- (17) 0 (18) Zn NaB 9H flclsfial-OO-CH-CogEt ———l‘A——> clsnal-csng-coasc Ac20/Ac H fiH—Ac NH-Ac (19) Link Am, (20) H C15H31-CH-CH-CH20H (21) NH-Ac This synthetic procedure has been used repeatedly for the synthesis of radioactive sphinganine for biological studies (DiMari _£'§l., 1971; Stoffel and Sticht, 1967b). Substitution of palmitoyl chloride with 2-hexadecenoy1 chloride (Shapiro and Segal, 195k; Shapiro £3 31., 1958a) and 2-methoxypalmi- toyl chloride (Kisic 53.31., 1971), yielded h-sphingenine and h-hydro- xysphinganine, respectively. A stereospecific synthesis of thggg- and erythro-sphinganine was first reported by Jenny and Grob (1955). The cis- and trans- 2,5-epoxide (22) were treated with ammonia, and threo- and ery- thro-2-hydroxy-5-amino acids were formed in equal amount. Specific introductic using benz: C15331' Hg/Pd The rEported 5,6-1Sopr Oxida tiOn tetradeey (Wittig r °btained. tratiOn O 60% Yield last Step pr”Getin 16 introduction of the amino group on C-2 could be accomplished by using benzylamine (Sisido £5 51., 196%). O / \ 0'22““ 9“ clsflal-CH'CH-COZH —_> C15H31'CH'9H'002H NH'CH2 (22) (25) OH H s /Pd 311 2 Cisfiai‘bfi'gfl'coan Mb C15H31' 'gg'maon NH2 2) LiA1H4 2 (2h) (2) The synthesis of g-erythro-sphinganine and h-sphingenine was reported by Reist and Christie (1970). Selective removal of the 5,6-isopropylidene blocking group of (25) yielded (26). Periodate oxidation of (26) gave aldehyde (27) which was allowed to react with tetradecyltriphenyl phosphonium bromide in strong basic solution (Wittig reaction). A mixture of cis- and trans-olefins (28) was obtained. Varying the reaction conditions, especially the concen- tration of the phenyl lithium, the trans-isomer could be obtained in 60% yield. The mixture (cis and trans) might be hydrogenated, in the last step, to saturated derivative with simultaneous deblocking of the protecting (benzyloxycarbonyl) group. C,0»?! |\o-CH (CHSJE (so) rv'v— 1"?“ 17 c,o—cn 1120}: c110 |\o-cn O HO-CH O N IO 0 A .A OR a (Cfis)2 \\ q c 4 O 0 O ‘\ 0——C(cu3)2 0—C(CH3)2 o..__c NH-Cbz NH-Cbz NH-Cbz (CH3)2 (25) (26) (27) CH: CHO( CH2)120H3 CH3( CH2) P113131» Br- Aq- AcOH &(CH3)2 NH—Cbz (28) c11=c11(cu2)12c113 ———+OOH__—’ Nan4 NH-Cbz (29) CH- c11( c112 )12c11;3 on on on CHNana4 I I I ——> amazes: cs- ('31- ('211- c112 ——+c151131c11- (‘31- C112 (50) “Chg“ on NH— Cbz Pd' C N112 (51) (2) Stereospecific synthesis of[215122-h-hydroxysphinganine from Q-glucosamine was described by Gigg gt 21- (1966). The aldehyde (52) which is a derivative of 2-amino-2-deoxy12-allose, was synthesized fromIQ-glucosamine. This aldehyde (52) was condensed with Wittig reagent (triphenyltridecylphosphonium bromide or -pentadecylphos- phonium bromide). The olefin product (55) was saturated by catalytic hydrogenation. Hydrolysis, reduction with NaBH4 and deblocking yielded 2-ribo-h-hydroxysphinganine derivative (55). —,‘. Ir CHt Pd- C L Alt nine fro“ E and Warren a 1“tennis 1: nine fI‘Om I cl 41125 18 CH- CH( CH2 )120113 c110 OCH 0 3 + _ 0 00113 CHs(C32)12P'Ph34§£_ PhLi ° N (52) 0 N ( 55) \Cfi’h \C/{Ph C14H29 O OCHS 1) CELgOH-HCI OH on NH-co-Ph —H2—/P§:c—’ 2) Ho + cl4H29CH'CH'CH'CH2OH ( u) 5) H30 5 o N 1+) “83114 (35) \cf/ph Alternate routes for the synthesis of =D- and L—h-hydroxysphinga- nine from D-galactose were described by Gigg and Gigg (1966) and Gigg and Warren (1966). Sisido et a1. (1970), on the other hand, synthesized a racemic mixture of ribo-, arabino-, ly_g— and xy_g-h-hydroxysphinga- nine from the same intermediate, ethyl-2-acetamido-5-octadecynoate (56). H H 014H29-(ZC-CH-002Et ——p. C14H29-(FC-CH-002Et NH-Ac -Ac (56) (57) 1) 1100311 “All“ 1) Iz-AgOAc 2) KOH 2) LiA1H4, 3) Liam, H (58) curiae-#9434022: alg-Arabino-T QL—Lyxo-T 1) H0031! - '— 1) Iz-AgOAc 2) son (39) (1+0) 2) 1.1.1111. ”Lulu" g-Xylo-T DL-Ribo-T + DEL-Arabino-T (#1) (3) (59) 'r c14H29-cs(ou)cu(on)qH-cn2011 NH-Ac E.Biosynt Stur and Coulo ate were Hansenula thesis of (1958) su base bios With the 1 arrived a for the m and Stoff substrate brain how 3.7%: i sequerltly rat liver (Brat1n 3 tion of 3 and only this 8112: HYdeEn 1968c). underStOO stereospe 19 B. Biosynthesis. Studies 19.3139 by Zabin and Mbad (1955 and 195%) and Sprinson and Coulon (l95h) provided the first evidence that serine and palmit- ate were the two precursors of h-sphingenine in animals. The yeast, Hansenula ciferri, also utilized serine and palmitate for the biosyn- thesis of h-hydroxysphinganine (Green 21 51., 1965). Brady and Koval (1958) successfully isolated an active cell-free system for long-chain base biosynthesis. Enzymatic activities were found to be associated with the microsomal fraction of rat brain. These authors, however, arrived at a wrong conclusion, that palmitaldehyde was the substrate for the microsomal enzyme. The later work of Braun and Snell (1967) and Stoffel _1 £1. (1968a) indicated that palmitoyl CoA was the actual substrate for the condensing enzyme. h-Sphingenine formed with rat brain homogenate was shown on TLC to be identical with the natural egzthro isomer (Fujino and Zabin, 1962). 5-Ketosphinganine was sub- sequently shown to be an intermediate with the microsomal system of rat liver (Stoffel 51‘51., 1968a), rat brain (Kanfer and Bates, 1970), H. ciferri (Braun and Snell, 1968; Brady‘s; 31., 1969), mouse brain (Braun £1 51., 1970) and oyster (Hammond and Sweeley, 1975). Reduc- tion of 5-ketosphinganine was catalysed by an NADPH-dependent enzyme and only the 3 isomer, but not the 1 isomer, was the substrate for this enzyme, forming erythr -sphinganine (Stoffel 55‘51., 1968a). Hydrogen was transferred from the B-side of NADPH (Stoffel £1 21., 1968c). The biosynthesis of h-hydroxysphinganine is still not well understood. Conversion of h-sphingenine to h-hydroxysphinganine by’ stereospecific hydration was postulated by Weiss (1965) but this hypothesi by severa porated i teropalmi of only 1971), 81 fomatior the Prime (Thorpe a sWingern‘ C‘L) Has ] Sphingam' experimer Swine my [1‘14C,3. that 33/: 2O hypothesis was ruled out by experimental data reported subsequently by several authors: that 1) h-sphingenine itself could not be incor- porated into h-hydroxysphinganine (Stoffel 21.21., 1968b), 2) perdeu- teropalmitate was converted to h-hydroxysphinganine with loss of only one (obligatory) denterium atom on C-2 (Polito and Sweeley, 1971), suggesting that there was no unsaturated intermediate in the formation of h-hydroxysphinganine from palmitate, and 5) H150 was not the primary source of the hydroxyl group on C-h of h-hydroxysphinganine (Thorpe and Sweeley, 1967). With yeast grown in the presence of [h,5§H]- sphinganine, Weiss (1965) found that about 50% of the radioactivity on C-h was lost relative to that on 0-5. Thus, it was concluded that sphinganine was directly transformed to h-hydroxysphinganine. This experiment, however, does not exclude the possibility that 5-ketosphin- ganine might be the immediate precursor of h-hydroxysphinganine. Using [1-14C,5-3H:]sphinganine as the substrate, Stoffel _1 £1. (1968b) found that aH/l‘C ratio of h-hydroxysphinganine dropped to 1/7 that of the sphinganine substrate and it was concluded that 5-ketosphinganine was the immediate precursor of h-hydroxysphinganine. A contradictory result was reported from the same laboratory (Stoffel and Binczek, 1971), that yeast converted [5-3H,5-14C ]sphinganine to h-hydroxysphinganine without loss of tritium on C-5. The isotopic ratio in h-hydroxysphinganine was the same as that of sphinganine substrate, suggesting that sphin- ganine (but not 5-ketosphinganine) was the immediate precursor of h- hydroxysphinganine. Regardless of whether 5-ketOSphinganine or sphin- ganine was the immediate precursor of h-hydroxysphinganine, Polito and Sweeley (1971) and Stoffel and Binczek (1971) independently reported that conversion of palmitate to h-hydroxysphinganine involved the loss of the p_t whereas t with the authors t tention c is still ruled out poorly in The troversia shown to 2) at the and Sweel. nine inte. StereOcheI 3% 9111 °f palm:a minat]'.0n j Sen 0n C_3 CUrz Fi8.1. F. Degrada1 Stud Banine intt duct in 111 21 of the EEQ'R hydrogen atom on 0-2 of palmitate or C-h of sphinganine, whereas the 312-8 hydrogen was retained. These findings together with the known stereochemistry of h-hydroxysphinganine led these authors to conclude that the hydroxylation step proceeded with re- tention of configuration. The origin of the hydroxyl group on C-h is still an elusive problem. H180 and molecular oxygen were both ruled out as the source of this hydroxyl group, although water was poorly incorporated (Thorpe and Sweeley, 1967). The sequence of steps in h-sphingenine synthesis is still con- troversial. Introduction of the double bond between C-h and C-5 was shown to occur at 1) fatty acid level, in yeast (DiMari £1 £1., 1971), 2) at the 5-ketosphinganine level (Fujino and Nakano, 1971; lhuunond and Sweeley, 1975) and 5) after the reduction of the 5-ketosphinga- nine intermediate (Stoffel £1 21., 1971a; Dag and Brady, 1975). The stereochemical course of dehydrogenation was shown to proceed via a 11251 elimination in yeast, where the £11-R hydrogens on 0-2 and C—5 of palmitate were removed (Polito and Sweeley, 1971) and via a £11 eli- mination in rat, in which the EEQ'R hydrogen on C-2 and the 219—5 hydro- gen on C—5 of palmitate were removed (Stoffel £1 £1., 1971). Current knowledge of long-chain bases biosynthesis is summarized in Fig.1. F. Degradation. Studies on the biodegradation of long-chain bases were initiated by Barenholz and Catt (1967), who injected [9,10-3HIJ-h-hydroxysphin- ganine into the tail vein of rats and characterized the degradation pro- duct in liver. Pentadecanoic acid was the major radioactive component. _ <00 T3333 + ocauom 22 .A0H93#Ho>u uon nu neon Husvw>avca as now oocova>o opwmaaocoo when: new: one nonfia wouuouv woman sauna-waou mo Hawaonsuua ansavqsuounu .a.wam ocficowcwnnaazuahoafz b... I I 1 I ocacswcwsazhu..n_._.uhoe.). O . .. . . ., ., .... ......Tiu...“ .... .~..nun"~ah+uua. ..mpiyvfi .-. . .. .....l t .m a. 1 . a e -_ . . _. .2 . . . . ..ca. i m . m m . . . . . .....1. u 1 .u _ u .. . . 3 . .... ... .m . n 1 . . f i. O 1 u x. n .w . o B I u t ..m - t . u u p 1 . m .m, a a .4 m ... a. . .D D. .m. A a . m w. p._ I a u ..m .m so.” . . i- .: Juan“, r n r F 8 y I n .m o V .1 _ .H vs. . -m. a .. _ 1 .. .. m 1 D. O h - . C e 8 u r n I- . u .. o a , . ., B y ‘0. H . u. . . . D n L a m v 1‘? a u an . I; c L r n a . “:4 . v . e h P . . .....w.\...1nd».“ h.” ..V‘ .“ fl “ a.“ . . ”EU-.. 374.5166 1 . .... ......uyfi .Mu “A. . . : I. fatter: no (a: v . . A. - .. .9 u w . — n .u . auom haOSu «a munomqu amass mo uaumfiuwuoauuso one scans assumes esu wcauwuuusaaa «avamaaowcandw xuaaaoo mo ceauwvauwwa .N.w«h o uoudaflvomv umoédééczofi Aznmouummvoxaoa owumaousuuuozv no Axoam-eauauazv emanauuwasm omucwumaaowaannm n V um you fine smegmouuuaeoufl o Aupnmumv Auoaoacuv uwugmoosaon Q noouoaw AnamovwacuouH5mouocAv emmvfiwouueauolu <2<¢ “8-05-18 V “8-0848 Awnocmuhuav Raunchy unavaeaacmoxmm «encamOuouHmUIB noooUHonawUnov a new < owwuacaasmoxom uuUuu~U-HQUIHuUno0 / N m ow \ / . m mommo/ mommo-w-2umo- +7; + 3.736.. .m 1:. m smmmso-w H£306.50 o a «VG w Hammao-oflmwo AHHHV AHV N :moo/ A. m ow \ / HE \ouzéo. -m All. mommoéééo- 2.: l + momuo _ III ocfiuom m “'1‘. nur- A- Ha1 Chemie Palmi' Ethyl Hanno N-ACe es DiEthl MATERIALS AND METHODS A. Materials . Chemicals Palmitoyl chloride Pfaltz and Bauer Inc., Flushing, New York. Palmitoyl chloride was double redistilled under reduced pressure. Ethyl acetoacetate Aldrich Chem. Co. Inc., Milwaukee, Wisconsin. Ethyl acetoacetate was re- distilled before use. Mannosamine-HCl Sigma Chem. Co., St. Louis, Missouri. N-Acetylglucosamine Sigma Chem. Co. Ilfi-Egythro-sphinganine Sigma Chem. Co. IIl=)_ll.'-Erythro-le-sphinganine sulfate Sigma Chem. Co. Hexmmethyldisilazane and trime- Analabs, Inc., North Haven, Connecti- thylchlorosilane cut. Yeast extract, malt extract and Difco Laboratories, Detroit, Michigan. peptone Solvents General solvents All solvents were redistilled before use. Diethyl ether IMallinckrodt Chem, Works, St. Louis, Missouri. Diethyl ether was stored over small pieces of sodium metal. 35 Stable 0130211. We, anhydr Bis(tr oroace 3330 e 5% SE mesh) Silic Ambe' Sili Mesh Rats LiVe *4 m [9’ g? / Stable Isotopes 36 CHGOZH, 2H20, N§H4.2H20, LiA12H4, Merck Sharp and DOhme of Canada, Ltd., NaBZH‘, d3-serine and de-acetic anhydride Bis(trideuteromethylsilyl)triflu- oroacetamide-dla Hfiao (normalized) Chromatographic Supplies 5% 83-50 on Supelcoport (80-100 mesh) Silica gel G Amberlite MB-5 Silicic acid (Unisil, 100-200 mesh) Animals Rats (lO-lh day-old) Live oysters Yeast Hansenula ciferri (mating type F-60-10) Montreal, Canada. Distributed in the United States by Merck and Co. Inc., Rahway, New Jersey. Regis Chem. Co., Morton Grove, Illi- nois. IMiles Labs., Inc., Elkhart, Indiana. 'Monsanto Research CorP- Supelco, Inc., Bellefonte, Pennsyl- vania. EM Reagents Division, Brinkmann Ins- truments, Inc., Westbury, New York. Mallinckrodt Chem. Works. Clarkson Chem. Co., Inc., William- port, Pennsylvania. Spartan Research Animals, Haslett, Michigan. City Fish Co., Lansing, Michigan. Gift of Dr. Kurztman,C.P. Yeast Media Liquid medi LiQuid mee Liquid me Chrom 57 Yeast Media Liquid medium-l (UM-l) yeast extract (0.5%), malt extract ’(0.5%), peptone (0.5%) and glucose (1.0%) in water. The medium was sterilized by millipore (0.8 u) filtration. Liquid medium-2 (LM-2) the same as liquid medium-1 excepted that sterilization was accomplished by autoclaving at 120°C for 25 min. Liquid medium-5 (EM-5) yeast extract (0.5%), MgSO4.7H20 (0.07%), (N11,)2504 (0.12%), NaCl (0.05%), KH2P04 (0-596)- B. Methods. 1. Gas-Liquid Chromatography (GLC) Gas chromatography was carried out on a Hewlett-Packard Model F and M #02 gas chromatograph equipped with a flame ionization detector. A 6 ft. glass column packed with 5% SE-5O on SupeICOport, 80-100 mesh (Supelco Inc., Bellefonte, Pa.), was used throughout the studies. Nitrogen was the carrier gas. The flash heater and detector were set about 20°C above the column temperature, which was set as described elsewhere. 2. Mass Spectrometry (MS). Mass spectra were recorded with an LKB 9000 combined gas chromatograph-mass spectrometer. Conditions for gas chromatography were the same as those described for gas chromatography except that helium was at 70 eV, 1. current of computer in described labeled co nator (AVA 5. [2* by treati Procedure CWPOUndJ hydrochl. solution l’eached Glacial f0: 31101 58 helium was used as the carrier gas. Mass spectra were recorded at 70 eV, with an accelerating voltage of 5.5 KV and filament current of 60 uA. The mass spectrometer was interfaced to a PDP-8/I computer for on-line, real-time data collection and reduction as described by Sweeley £5 21. (1970). The deuterium content of labeled compounds was analysed by the accelerating voltage alter- nator (AVA) technique as described by Holland t al. (1975). 5. Preparation of [2-150] Glucose [2-180] Glucose was synthesized from mannosamine hydrochloride by treating the aminosugar with nitrous acid in H580 according to the procedure of Horton and Philips (1972) for synthesis of the unlabeled compound, with a slight modification as described below. ‘Mannosamine hydrochloride (216 mg) was dissolved in 5 ml of séeo (8.8%). The solution was stirred magnetically in an ice-bath. When the temperature reached 0°C, sodium nitrite (278 mg) was added in small portions. Glacial acetic acid (0.5 ml) was added slowly and stirring was continued for another h hours at 0°C. Nitrogen was then bubbled through the solu- tion for 5 min. and the solution was deionized by passing through a column packed with 10 g. of amberlite MB-5 mixed-bed resin. The column was allowed to run dry and Héao was recovered by lyophilization. The column was washed once with 20 ml of distilled water. The second eluent and the lyophilized glucose were combined and then passed through a second column packed with 50 g. of the mixed-bed resin. The column was washed with 20 ml of distilled water and eluent was concentrated by lyophilization to give a pale yellow syrup. TLC of this syrup on silica gel G (developed with butanol-pyridine-water, 70:15:15) showed a minor by-product at the origin and glucose was at Rf;: (3.2 (mannose inset Rf : hpurity. 11 for growing Yield, dete C-2 of gluc h. E l (20% enric‘ Vhich time residue we “‘03 (10 1 Excess Na] Bot-1c sci. eater by residue. vithOut ‘ 50 m1 of temperat prEcipit half of “no 4W4 The pre rizing 39 was at Rf = 0.28). GLC analysis of TMSi derivative showed about 1% impurity. This syrup was diluted to a proper concentration and used for growing yeast. No growth inhibition of yeast was observed. The yield, determined by GLC,‘was 95 mg (52%) of 6.h5% 180 enrichment on C—2 of glucose. h. Synthesis of [5-180]Serine NAAcetylglucosamine (2 g.) was dissolved in 5'ml of H580 (20% enrichment). Thosolution was left at 14°C for 10 days, after which time fléeo was recovered by lyophilization, The ‘minoaugar residue was redissolved in 20 ml of cold water and 1M Nth in 0.05N NaOH.(lO‘ml)‘was added. The mixture was kepted at h°c overnight. Excess NaBH4 was removed by careful acidification with 0.1N HCl. Boric acid so formed was removed under reduced pressure as its methyl ester by repeated addition of absolute methanol to the dried solid residue. The NAacetylglucosaminitol residue was used in the next step without purification. Water (50'ml) was added to the solid residue, followed by 50 ml of NaIO4 (pfith.5). The solution was kept in the dark at room temperature overnight. Methanol (50 ml) was added and the inorganic precipitate was removed by filtration. The filtrate was reduced to half of the original‘volume- KMnO4 (ho mmoles in 80 ml of water) was added and the solution was kept at room temperature overnight. Excess rune, was removed by addition of a dilute solution of oxalic acid. The precipitate was filtered and the filtrate was treated with decolo- rizing carbon, refiltered and lyophilized. The solid residue was extracted three tin dried und 95% pure gram co-c of the Ti THSi-N-ac at 80°C 1 and evapc three tin under reel 6-3. TLC 60:20:20 328041308 Supplemen ngth of S. enriChmEn TritOn X- 15 Inin. 11 days. Fate of nin-oge acid "as t 10% methan "ream 0f I LO three times with 10 ml of absolute methanol. The combined extracts were dried under reduced pressure and the residue was determined to be about 95% pure by GLC of its TMSi derivative. The major peak in the chromato- gram co-chromatographed with di-O-TMSi-N-acetylserine. The mass spectrum of the TMSi derivative of this residue was identical to that of di-O- TMSi-N-acetylserine (Fig.7). The solid residue was dissolved in 20 ml of 5N HCl and heated at 80°C for 6 hours. The solution was neutralized with dilute Na2C03 and evaporated to dryness under reduced pressure. Serine was extracted three times with 10 ml of 90% methanol. The combined extracts were dried under reduced pressure, yielding 0.5 g. with 6.9h% of 18O enrichment on C-5. TLC on silica gel G (developed with butanol-acetic acid-water, 60:20:20 and with phenol-water, 75:25) showed only one nihydrin- and H2804-positive spot at the same Rf value as that of reference serine. Supplementing yeast medium with this labeled serine did not inhibit the growth of the yeast, Hansenula ciferri. 5. Preparation of [l-leoflPalmitate. Palmitoyl chloride (100 mg) was added to 2 m1 of aéao (9.5% enrichment). The mixture was sonicated for 5 min. and two drops of Triton x-100 were added. The mixture was then sonicated for another 15 min. The tube was sealed and left at room temperature for three days. Fatty acid was extracted with chloroform and dried under a stream of nitrogen. For analysis of 18O enrichment, about l‘mg of the fatty acid was treated with freshly prepared diazomethane in ether containing 10% methanol. Excess diazomethane and solvents were removed under a stream of nitrogen, hexane (5 ml) was added to the residue, and the mixture canted, 6 from pal Shapiro CH? 00-C (11. 1“ 50 ml was cooled then adde d. 80dimm hde and the aqu 1+1 mixture was sonicated briefly. The hexane extract was carefully de- canted, concentrated and analysed by GLC-AVA. 6. Synthesis of Deuterated Sphinganines. Ethyl 2-acetamido-5-ketooctadecanoate (19) was synthesized from palmitoyl chloride and ethyl acetoacetate as described by Shapiro 25.21. (1958) and Shapiro (1969) as outlined below. co-CH3 ’ 01511310001 ———_yclsaslco-c-coeat ans-co-cna-coasc &[CH:,— 00- CH- C02E]Na (1h) (15) (16) a ,_ ' 15H31-C0- 9' C02E N: (17) (5 041:1? 01" qocfia ] [c . Zn in W 0151131-00- figjgem ( 19 ) [1,1,5-2H3]NHAcetylsphinganine. Ethyl 2-acetamido-5-ketoactadecanoate (500 mg) was dissolved in 50 m1 of anhydrous diethyl ether-tetrahydrofuran (1:1). The solution was cooled in an ice-bath and lithium aluminum.deuteride (15 mg) was then added. The solution was refluxed gently for one hour, after which sodium hydroxide (0.2N, 50 ml) was added; the phases were separated and the aqueous phase was extracted with ether (10 ml). The combined organic phases advdrous sodi ofnitrogen. b. [5-21 Ethyl 2 10ml of meth. borodeuteride room for 2h h flu solvents that formed 1 ted addition residue left hydrofuran (: sOllicated br erials, The and N'acetyl °f [113.2% c. [1, [1.1-2 described ft and lithium teride and d.[ Sodi (10 m1). h2 organic phases were washed twice with 5 m1 of water, dried over anhydrous sodium sulfate and evaporated to dryness under a stream of nitrogen. b. [5-2H] NsAcetylsphinganine. Ethyl 2-acetamido-5-ketooctadecanoate (100 mg) was dissolved in 10 ml of methanol. The solution was cooled in an ice-bath and sodium borodeuteride (10 mg) was added. The solution was left in the cold room for 2% hours after which time it was acidified with dilute HCl. The solvents were evaporated under reduced pressure. Boric acid that formed in the reaction was removed as its methyl ester by repea- ted addition and removal in 12222 of anhydrous methanol to the dry residue left after each evaporation. Diethyl ether (5 ml) and tetra- hydrofuran (5 ml) were added to the dry residue and the suspension was sonicated briefly in a sonic oscillator to help dissolve organic mat- erials. The solution was then reduced with lithium aluminum hydride and N-acetylsphinganine was recovered as described in the synthesis of [1,1,5-2H3] N-acetylsphinganine. c. [1, 1-21-12 JN-Acetylsphinganine. [1,1-2H2] N-Acetylsphinganine was synthesized by the procedure described for [5-2H:]N-acetylsphinganine except that sodium borohydride and lithium aluminum deuteride were used in place of sodium borodeu- teride and lithium aluminum hydride, respectively. d. [1,1,2,5,h,h-2H6;]NeAcetylsphinganine. Sodium metal (about 0.2 g) was added in small pieces to CH302H (10 m1). When the evolution of deuterium gas had subsided, deuterium oxide (0.5 m1 canoate (100 ‘ hours, after (20 m1) and u centrifugatic Folch’s uppe reduced press absolute eth. deuteride as Safline. e. [11- N-Ace Cave: and 8» added to thel ‘ Slight sh. tion Was 1e (2 m1) and Phase was w and the 1m. 8m. Hie 1+3 oxide (0.5 ml) was added, followed by ethyl 2-acetamido-5-ketooctade- canoate (100 mg). The solution was left at room temperature for 2h hours, after which time acetic anhydride (2 ml) was added. Chloroform (20 ml) and water (5 ml) were added, the two phases were separated by centrifugation, and the lower phase was washed once with 10 ml of Folch's upper phase (Folch 25‘21., 1957) and then evaporated under reduced pressure. Traces of water were removed by azeotropization with absolute ethanol. The residue was then reduced with lithium aluminum deuteride as described in the synthesis of [1,1,5-233:]N-acetylsphin- ganine. e. [N-2H31-Acetylsphinganine. N-Acetylation of sphinganine was carried out as described by Gaver and Sweeley (1966). Hexadeutero-acetic anhydride (0.1 ml) was added to the solution of sphinganine (1 mg in 1 ml of methanol) with a slight shaking to help distribute the anhydride evenly. The solu- tion was left at room temperature for 10 min., after which chloroform (2 m1) and water (0.5 ml) were added with thorough mixing. The lower phase was washed once with Folch's upper phase (Folch‘££.gl., 1957) and the lower phase was evaporated to dryness under a stream of nitro- gen. f. [h,5-2H2]Sphinganine. This compound was a biosynthetic product isolated from oyster microsomes incubated with serine and [2,5—2H2]palmitate as described by Eamond and Sweeley (1975). 7. Preparation of Microsomal Enzymes. ZMicrosomes were prepared from liver of lO-lh day-old rats as described by 1 volumes of 0.1 homogenate was ultracentrifu: was suSpended ring 1 mti PLP giual voltune. 8. Isol At the (1 N, 1 ml) to m1 of diethyl with 10 m1 of P°rated to d! dissolved in Silica gel G the side Ian 65:25;4 (Bra briefly and scraped off. Portions (1( Potated in 9. P1- SPhin bases. The was added. 44 described by Brady‘g£.gl. (1965). The tissues were homogenized in 5 volumes of 0.25 M sucrose with (a teflon homogenizer at 2-4°C. The homogenate was centrifuged at 8600 x g for 15 min. Supernatant was ultracentrifuged at 100,000 x g for 45 min. The microsomal sediment was suspended in 0.1 M.potaasium phosphate buffer (pH 7.5), contai- ning 1 mM.PLP and 1 mM.dithiothreitol (DTT), equal to 0.2 of the ori- ginal volume. 8. Isolation and Purification of Sphinganine. At the end of incubation (1 hour at 37°C), sodium hydroxide (1 N, 1 ml) was added and the lipids were extracted 3 times with 10 m1 of diethyl ether. The combined ether extracts were washed once with 10 ml of water, dried over anhydrous sodium sulfate, and eva- porated to dryness under a stream of nitrogen. The dry residue was dissolved in a minimal volume of chloroform and then applied on a silica gel G thin layer plate with a sphinganine standard applied on the side lane. After developing with chloroform-methanol-water, 65:25:4 (Braun and Snell, 1967) the plate was exposed to iodine vapor briefly and the area at the same Rf value as that of the standard was scraped off. Sphinganine was extracted from the silica gel with three portions (10 ml each) of chloroformemethanol 1:1. Solvents were eva- porated _i_.p_ £5932 . 9. Preparation of Volatile Derivative of Sphingolipid Bases. Sphingolipid bases were N-acetylated by the procedure of Gaver and Sweeley (1965). Methanol (2 ml) was added to the dry long-chain bases. The mixture was sonicated briefly and acetic anhydride (0.2 ml) was added. The reaction mixture was left at room temperature for 10 min. L 1.3.1 “ grvza Chloroform (1+ were mixed th lower phase a Disi-donor a; trimethylchh about 10 min 10- Gr Sphinganine. Easier: rent liquid Yeast previo C9118 were h Philization. dry cells w hS Chloroform (h ml) and water (1.0 ml) were then added. The two phases were mixed thoroughly and then separated by centrifugation. The lower phase was evaporated to dryness under a stream of nitrogen. TMSi-donor agent, consisting of pyridine-hexamethyldisilazane- trimethylchlorosilane, 10:2:1 (Gaver and Sweeley, 1965), was added about 10 min. prior to analysis by GLC. ILL Growth of Yeast and Isolation of Tetraacetyl-h—hydroxy- sphinganine. .gansenula ciferri was grown aerobically at 26-28°C in diffe- rent liquid media (10 m1). Growth was initiated by adding 0.1 m1 of yeast previously grown to stationary phase and kept at hOC overnight. Cells were harvested h8 hours after growth by centrifugation or lyo- philization. Tetraacetyl-h-hydroxysphinganine in the cell paste or dry cells was extracted twice with 10 ml of acetone, that from the medium was extracted two times with 10 ml of petroleum-ether. The combined extracts were dried under reduced pressure. 11. Hydrolysis of Sphingolipids. Complete hydrolysis of sphingolipids was accomplished by the procedure described by Gavarand Sweeley (1965) with a slight modifica- tion. The sphingolipids were dissolved in 10 ml of methanol followed by the addition of HCl (6 N; 2 ml). The mixture was heated at 80°C for 16 hours in a Teflon-lined screw-capped tube. Chloroform (20 m1) and water (h ml) were added, the lower phase was washed once with Folch's upper phase (Folch £3 21., 1957), and was dried‘in.!§ggg. Partial hydrolysis was used to remove O-acetyl groups selectively. The crude tetraacetyl-h-hydroxysphinganine was dissoved in 2 ml of 0.1 N sodium hydroxide in methanol and the solution was kept at 60C) for #6 1 hour (Thorpe and Sweeley, 1967). Chloroform (h m1) and water (1 ml) were added, the chloroform layer was washed once with Folch's upper phase (Folch gt 31., 1957) and dried under a stream of nitrogen. RESULTS A. Mass Spectrum of Tris-O—TMSi-N-acetyl-h-hydroxysphinganine. The mass spectrum of tris-O-TMSi-N-acetyl-h-hydroxysphinganine (Fig.h) was reported previously by Thorpe and Sweeley (1967). The molecular ion (EAE 575) was not present in the spectrum but could be deduced from the ion at M915 (mflg 560), which is probably derived from the loss of a methyl residue from one of the TM31 groups. Direct clea- vage between C-5 and C-h yields ions at ng 276 and 299 with the posi-' tive charge retained on C-5 and C-h, respectively. The ion at mfig 218 is possibly derived from further cleavage of the C-N bond of the for- mer ion with the loss of nitrogen containing fragment, as shown below. tfl'MSi £02481 t0TMSi CH3(CH2)13(I:H-CH-t|:H-CH20TMSi —-+ CH-(fH-CHeoTMSi -—> CIi=CH-CH20mSi o'mSi NH-Ac CNl-I-Ac 51/3 276 31/ g 218 Cleavage between C-2 and C-5 gives rise to ions at m[§ h01 and 17h, depending on which fragment charge retention occurs. Homolytic cleavage of C-1 and C-2 bond yields two ions at m[g 105 and mfig #72. The ion at g[g 105 was shown to be derived from both C-1 and C-5 of TMSi-N-acetylsphinganine, and may be derived from other fragments of TMSi-N-acetyl-h-hydroxysphinganine. Supporting evidence for the above statement is the presence of ions at both m[g 105 and m/e 105 in the spectrum of TMSi-[1,1-2H2]N-acetyl-h-hydroxysphinganine (Fig. 20). La h8 .mcwcmwcwLambs»vxnuzuaxuoomnziwgiobfiuu mo Esuuooam mom: .: .wfim omm oom one 00¢ 41 4 1 0072 91.5. 8n n_lz 5? mum u >22 _ .108 1.192% 055: mEorodxoioloaifeg SE. lama. kg 0mm mx me oom 0mm mmN mhm 9N com #9 wt 8/ cod 2. om TON V.ow OOH (%) MSUGJUI aAuolag LL9 The ion at EAE #72 is unstable and loses TMSiOH to give a more stable ion at m[g 582. The ion at‘m[g 299 is useful for the analysis of labeled oxygen on C-# (Thorpe and Sweeley, 1967) since substitution of 180 will shift this ion to m[g 501. It contains only one oxygen atom, thus, the increment of isotope ratio of m[g 501/299 from the reference (natural isotOpe) value can be used directly for the calculation of isotopic enrichment on C-#. The origin of this oxygen is of considerable interest, and can be studied using oxygen on C-1 and C-5 as experimental controls in case of negative incorpora- tion into C-#. Therefore, analysis of 18O on C-1 and C-5 is usually necessary. Analysis of 180 on C-1 and C-5 was therefore carried out by stepwise subtraction. The ion at ng #01 consists of oxygen on C-5 and C-#. Subtraction of isotopic enrichment on C-# (from mflg 501/299) will give the value of isotopic enrichment on C-5. The intense ion at.m[g 218 is suitable for the analysis of 180 on C-1 and C-5. Isotopic enrichment on C-l is then obtained by sub- traction of that calculated for C-5. B. Studies on The Incorporation of [1,1,5-2H8]Sphinganine into #-Hydroxysphinganine. [1,1,5-2H3]Sphinganine (100 ug) and Triton X-100 (#0 mg) were dispersed in 10 ml of LMF2. The medium was innoculated with 0.1 m1 of viable yeast and was shaken at 26-28°C for #8 hours. Tetraacetyl-#- hydroxysphinganine was isolated and converted to the TMSi-N-acety1-#- hydroxysphinganine as described in Methods. Deuterium contents in #- hydroxysphinganine were analysed by GLC-AVA, using intensity of the ions at m[g 218, 220 and 221 for the analysis. The ion at m[g 218 is 50 Table 2. AVA Analysis of [1,l—2H2]-#-Hydroxysphinganine and [1,1,5-2H3]-#-Hydroxysphinganine from Yeast Grown in the Presence of [1,1,5-2H318phinganine. .gzg 220/218 311/2 221/218 Determinations Reference Sample Reference Sample 1 0.0919 0.1590 0.0227 0.1#1h 2 0.0915 0.1588 0.0225 0.1#05 5 0.0926 0.1589 0.0226 0.1590 Average 0.0920 0.1589 0.0226 0.1#05 * - 0.0669 - 0-1177 * is the difference between sample and reference values. g/g 218, cu: CH—CH20TMSi; OTMSi m/e 221, co=ca-cn20m3i. OTMSi .972 220, cases-CDZOTMSi; OTMSi 51 derived from C-1, C-2 and C—3 with two TMSi groups (Results, A). Substitution of deuterium on these three carbons would be expected to increase the mass of this ion by an amount corresponding to the number of deuterium atoms incorporated. Therefore, direct hydroxy- lation of sphinganine to #-hydroxysphinganine will be expected to involve retention of all three deuterium atoms an C-1 and C-5 in #-hydroxysphinganine and the ion at m[g 218 should be shifted to ‘EIS 221. On the other hand, if the process occurs via a 5-ketosphin- genine intermediate, an obligatory loss of deuterium on C-5 will be expected and the ion at m[g 218 will be shifted to m[g 220. Re- sults are normalized to the ion at m[g 218 and are summarized in Table 2. An increase in the isotope ratio of m[g 221/218 (11.77% above the reference sample) suggested that sphinganine was incorpo— rated into #-hydroxysphinganine without loss of deuterium; thus it is inferred that hydroxylation proceeded at the sphinganine level rather than 5-ketosphinganine. An increase in isotope ratio of;m[g 220/218 (6.7%) is of interest since it suggests that 5-ketosphinga— nine is also a direct precursor of #-hydroxysphinganine. Loss of deu- terium due to degradation and resynthesis by utilization of the degra- dation product is unlikely. Degradation of sphinganine would result in loss of all the three deuterium atoms rather than just one of them, Hence it was concluded that hydroxylation of sphingolipid bases proba- bly occurs at both 5-ketosphinganine and sphinganine level (Fig. 5). C. Incorporation of [1,1,5-2H3]Sphinganine into #-Sphingenine. Although sphingolipid bases are not soluble in water, several 52 .mwmonucmmOfin unansweanammxouvmsu: mo amacoaooa vomoaoum .m .mam ocwcmmcfizawhxouummu: 1‘ 9::menumbnouvanuzuououum a . ocqcowawnam ‘ w ocwcmwgnamouoxnn 53 methods can be used to disperse them in water. Kanfer and Gal (1966) dissolved #-sphingenine in 0.1 M acetate buffer, pH 5.0 (20 ug/ml). Barenholz and Gatt (1968) dispersed the free bases in saline (1 mM), but #-sphingenine did not yield a clear solution at this concentra- tion. Egg lecithin was then added (10 times the sphingolipid bases weight). Bovine serum albumin (5% solution) was used as a dispersing agent by Stoffel and Sticht (1967), who obtained a clear solution of 5 mg/ml. Keenan and Okabe (1968), on the other hand, dissolved the sphingolipid bases in dimethyl sulfoxide (1 mM) instead of water. Stoffel _5 21° (1971) used 17% Triton WR-l559 in saline to solu- bilize the sphingolipid base (100 mM) for intracerebral studies. This is the highest dispersing power (to my knowledge) for in vivo studies of sphingolipid bases metabolism, [1,1,5-2H318phinganine was therefore prepared in this solution at a concentration of 100 mM. Each rat received 5 ul by injection into the brain via the frontal sagittal suture. Brains were removed by decapitation after 2# hours. #-Sphin— genine was isolated and converted to TMSi-N-acetyl derivative as des- cribed in Methods and deuterium content was analysed by GLC-AVA. The mass spectrum of TMSi-N-acetyl-#-sphingenine (Fig.6) was reported previously by Gaver and Sweeley (1966). Cleavage of the bond between C-2 and C-5 gives ions at.m[g l7# and 511. Substitution of deuterium on C-1 and C-5 will shift the mass of these ions to m[g 176 and 512 respectively. 5# FII .ocflcowcfisamuaiamuoomuziamzaionmwn mo Esuuooam mmmz .w .wwm m\ E . . t b0min... _ e .omN . 00¢ 0mm 00m 0mm CON om" 00“ cm > P > P p » b4h> P >~P >~bbphh<fl>h>~spr>pbpr bibbrbr-bbpbbrfidthbburhhu- bb ON? A d 375: 05? Tom 3.. .3 owe .32 E 8 3-12 0.65; fiEofoflzoaxo Ioioeiazomxo .8 no.8» m 2.5.» =m 00“ (0/0) KigsueiuI aAgi0|aa 55 CTMSi C1 31127 - CH= CH- CHLCH- CH20TMS i ____.5 CH- CHEOTMS i l n :NH-Ac +NH-Ac 52/2 17# i+0TMSi OTMSi ' I!) II C1 31127-011: CH- CH CH- CH20TMS i ————p C13H27- CH: CH— CH l NH-Ac 3.1/2 5 1 1 Analysis of ions at m[§ 17# and 176 indicated that there was no deuterium enrichment in #-sphingenine. GLC analysis did not indi- cate any accumulation of sphinganine. The failure to detect deutera- ted #psphingenine in rat brain might be due to rapid degradation of injected sphingolipid bases (Stoffel gt 31., l968b) and a high endo- genous #-sphingenine in rat brain. Attempts to inject higher doses (25 ul each) into rat brains were not successful. Severe bleeding was observed in several rats and those which were not bleeding only survived for a few hours. The dead frozen rats and the survivors were pooled. There was no deuterium detected in the isolated #-sphingenine. This problem was not further investigated. D. Studies on Yeast Grown on [2-180]G1ucose. 1. Determination of 180 Enrichment on C-2 of Glucose. The mass spectrum of TMSi-methoxime of [2-180]glucose synthe- sized from mannosmmine-HCl was identical to that of TMSi-methoq xime glucose which was reported by Laine and Sweeley (1971). The 56 spectrum is easily interpreted and isotopic substitution on each carbon can be distinguished. The ion at m[g 160 is derived from direct cleavage between C-2 and C-5 with the charge retained on C-2 fragment. Since oxygen on C-l is removed during the formation of the methoxime derivative, this ion can be used directly for deter? mination of isotopic abundance on C-2 without complication. Substi- tution of 180 on C-2 will shift this ion to m[g 162. Therefore, the increment of ratio at m[§ 162/160 from the natural isotopic abundance will be the net incorporation of 180 into this position. The result from AVA analysis (Table 2) shows that isotopic enrichment at this position is 6.#5%. 1 ................ cu- nocns CH=OTMSi 33/2 160 2. #-Hydroxysphinganine Produced by Yeast Grown on [2-180]G1ucose. It was speculated by Thorpe (1968) that phosphoenolpyruvate might be an oxygen donor in #-hydroxysphinganine synthesis. Experiments with phosphoenolpyruvate itself may suffer from rapid hydrolysis and com- plication by poor transport across cell membrane. In this experiment it was h0ped that some answers might be obtained about whether phospho- enolpyruvate is a donor or not, using [2-180]glucose, since the hydroxyl 57 Table 5. AVA Analysis of 180 on Carbon-2 of Synthetic Glucose. ‘m[g 162/160 Determinations Reference Sample 1 0.0520 0.1156 2 0.0508 0.1155 5 0.0510 0.1155 A 0.0507 0.1151 5 0.0512 0.11#9 6 0.0505 0.115# Average 0.0510 0.1155 [5* \ - 0.06#5 1:5 is the difference between sample and reference values. 111/3 160, ('ZlFN-OCHs ; 33/3 162, CHIN-OCI-Ia , 1 0118013131 CHI-20mm 58 group on C-2 of phosphoenolpyruvate can be derived from hydroxyl groups on either C-2 or C-5 of glucose from glycolytic pathway (Thorpe, 1968). Yeast was grown on LM-5 in the presence of 1% [2-180]glucose (6.#5% isotopic enrichment). Tetraacetyl-#-hydroxysphinganine obtained from the medium and intracellular sources were combined_and partially deacetylated with 0.1 N methanolic NaOH. After trimethylsilylation it was analysed by GLC-AVA. The ratio of ions m[g 501/299 was the same as that of the reference sample, indicating that there was no isotopic enrichment into C-# of #-hydroxysphinganine. It was concluded that the hydroxyl group of C-# of #-hydroxysphinganine is not derived from oxygen on C-2 of glucose, glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-diphosphate. It is important to note that partial or complete loss of 180 on C-2 of glucose may happen at the following steps. 1) This hydroxyl group was transformed into -the carbonyl oxygen in fructose-6-phosPhate, fructose-1,6-diph03phate and dihydroxyacetone- phosphate, hence loss of some 180 by exchanging with water in the medium might occur. Heron and Caprioli (1975) reported loss of about 50% of the isotope from C-2 of fructose-1,6—diphosphate at room tem- perature for 20 hours. However, the actual loss of 180 on C-2 of glucose might be much less than the value given by Heron and Caprioli, since fructose 1,6-diphosphate formed in the cell might be subjected to further metabolism.and the time of exposure to the medium should be less than 20 hours. S9 2) Aldolase, which catalyses the cleavage of fructose-1,6-di- phosphate to two molecules of triose phosphate, may cause a complete loss of isotope on C-2. Such a mechanism requires PLP-Schiff's base formation, as shown with rabbit muscle aldolase (Horecker _e_e_t_:_ §_1_., 1961). Yeast aldolase, on the other hand, does not require PLP for catalytic activity (Rutter, 196#). Accordingly, yeast aldolase converts fructose-1,6-diphosphate to dihydroxyacetone phosphate with- out loss of isotope on C-2 (Heron and Caprioli, 1975). E. Studies on Yeast Grown in the Presence of [5-180]Serine 1. Determination of 180 on C-5 of Serine. Isotopic abundance on C-5 of serine was determined by GLC- AVA of the N-acetyl-TMSi derivative. A mass spectrum of this deri- vative is shown in Fig.7. Molecular ion (31/3 291) is not present in the spectrum but can be deduced from the ion at M-15 (ml; 276) which may be derived from the loss of a methyl group from one of the T1181 groups. Loss of trimethylsilanol from this ion and that from the molecular ion give ions at m/g 186 and 201, reSpectively. Cleavage between C-1 and C-2 with charge retention on the nitrogen containing fragment gives an ion at 31/5 17#. Cleavage between C-2 and C-5 with the charge retained on the latter fragment gives an ion at 31/5 105. This ion contains only one oxygen of C-5 and its intensity is strong, thus it is used for the analysis of isotopic enrichment in this posi- tion. Results in Table # show an enrichment of 6.9#%. The ion at El; 261 (ll-50) is probably derived from loss of formaldehyde from C-5 by transfer of Mi to the nitrogen atom as outlined below. 60 ma .ocwuomaaumom-ZuwmzHuouan mo Bouuooam mam: .N .me l m\E _ b bO-mm- _ - nobomb - b 0%“ p b boom . - _ . a. . . . . . mwm are 6N smug: 8+3. 536635on652» use. We. ‘fr’ nxufi Q. Aum AXVH (°/o) MSUGJU' eAgiolaa 61 Table #. AVA Analysis of 180 on C-5 of Synthetic Serine. Determinations -969 105/103 Reference Sample 1 0-0520 0.1212 2 0-050# 0.1201 5 0.050# 0.1207 n 0-0518 0.1205 Average 0-0512 0.1206 Zfif - 0.069# * is the difference between sample and reference values. .gzg 105, CH2=OTMSi; .979 105, CHEQOTMSi. 62 c112- 011- 0021115 i . 011- 00 ms i l HI '(,1 2 (pr? NH- Ac 4 +NH-Ac TMSi CHSCSi'WHsla 93/3 26 1 nu-sficas)2 Ac 59/3 116 This type of mechanism has been illustrated for the frag- mentation of the sphinganine derivative (Hamarstri'am _e_t _a_1., 1970). This ion (g[g 261) may then undergo 1,2-elimination, involving the loss of a methyl group of TMSi and C-1 and C-2 fragment as shown above, to give an ion at m[g 116. 2. #-Hydroxysphinganine from Yeast Grown in the Presence of [5-180]Serine. Yeast grown in LM-5 in the presence of 1% glucose and 0.5% [5-180:]serine. Tetraacety1-#-hydroxysphinganine from.medium.and intra- cellular sources were combined and N-acetylated. GLC-AVA analysis of 180 enrichment in #-hydroxysphinganine is summarized in Table 5. The ion at‘g[g 299, which is derived from cleavage of C-5 and C-# bond, is used for analysis of isotope abundance on C-#. Substitution of 180 'will shift the ion to 9A2 501. Subtraction of the ratio of m£g 501/299 from that of the reference sample gave a value of 0.005 or .5%. Since [5-180]serine used in this study was 6.9#% enriched with 180, the 65 amzeo» . “assohmoéoumo £8 is memzaoumo-mw-smmaau .Hoa mym. x 2028 -5 Lhmaao + . amzaohmuéo "mo «mZHomH a game a mac n. was 392825-586 Jon mE + . I 1 EB aefioasmmoumo 68 3a AmZHw egommo .5 .886 £3 «E + megméohmmflo .mmm 3m . emzao .$:.m® we #10 was $0 me mic a&®.: mg :10 coca cos we cowumuoauoocH .mQDafl? GOGOMOHOH Una waaadm d003u0£ OOGflHOWMHU 050 m« LAN memo.o - nmoo.o - mnoo.o - *AV mmaa.o ammo.o mmma.o oHnH.o eaeo.o aaeo.o eweee>< mmaa.o ommo.o mam~.o Hmna.o seeo.o aaeo.o a Hmaa.o ommo.o oHnH.o oHnH.o aaeo.o naeo.o n mmae.o ammo.o nena.o smm~.o oneo.o aaeo.o m moaa.o mnmo.o omnn.o mHnH.o mneo.o naeo.o a oHanm ooaouomoa oaaaom mucouomom oHaaom oosouomom maOauocaauouon 280%.. ME 8.18: we mmmzon we. .Afidm.mv oaguomnomaund *m.o use mucosao RH mo ooeomoum use as n-2u ea cacao yams» scum deficmmcfisammxouwzw.a a“ one mo mamaflwc< s>< .m magma 6# theoretical percent incorporation was (.5/6.9#)x100 or #.8%. Total incorporation of isotOpe into C-5 and C—# was (.25/6.9#)x100 or about 5.5%, which is slightly less than that of C-#, thus it is concluded that there is no net incorporation of isotope into C-5 of #-hydroxy- sphinganine. Analysis of isotope on C-1 and C-5 (the ratio of m[g 220/218) shows an incorporation of 81.5%. Since there is no incor- poration of isotope into C-5, this incorporation is then attributed to that of C-1 only. This is slightly less than the theoretical value (100%), probably due to dilution from synthesis from non-isotope endo- geneous serine. Although a #.8% incorporation into C-# of #-hydroxy- sphinganine has been observed, this value may be insignificant in this study because the precision of the instrument has been reported to be 1% (Holland 35 21.,1975) and the net isotopic enrichment on C-# was only 0.5% which is probably beyond the precision of the instrument. There- fore,[5-180]serine and [1—180]sphinganine, which can be derived from [5-180]serine‘i_ situ, are ruled out as the hydroxyl donors. IF. Studies on Yeast Grown in the Presence of [l-lBOJPalmitate. Isotopic enrichment on C-1 of palmitic acid was analysed by GLC- AVVA as its methyl ester derivative. The mass spectrum of methyl palmi- tate has been reported elsewhere (Budzikiewicz _t_e_t_: _a_1_.,l967). The mole- <>ular ion is relatively intense and has been used for the analysis of total 180 in the molecule. The ion at gi/g 259 (ll-51), which arose from the loss of the methoxyl group was used for determination of iso- topic abundance on the carbonyl oxygen. The result in Table 6 shows that enrichment on the whole molecule was 11.1%, and that on the car- bonyl group was 8.5%. 65 Table 6. AVA Analysis of 180 in Methyl Palmitate. g/g 272/270 2/5 2#1/239 Determinations Reference Sample Reference Sample 1 0.0257 0.1552 0.5625 0.##26 2 0.02h0 0.1568 0.5622 0.##65 5 0.0255 0.1555 0.5655 0.##6# # 0.0252 0.1560 0.5682 0.##67 Average 0.02#6 0.1559 0.5625 0.##55 [5* - 0.1115 - 0.0850 £§*'is the difference between sample and reference values. 311/2 270: C15H31'C'0CHS; 09' 180 f I C15H31' C a 0? 04* I l cisnei-claocue; in/e 239. ciaHai-C; 180+ fl 39/2 272, Clsnal-C’OCHS and 3/3 2#1, 66 Table 7. AVA Analysis of 180 in #-Hydroxysphinganine from Yeast Grown in LM-l in the Presence of [$80]— Palmitate (1 mg/ml). Determinations .E(£ 301/299 'EAE LOB/hOI Reference Sample Reference Sample 1 0.0681 0.0672 0.12#6 0.1779 2 0.0680 0.0669 0.12h9 0.1766 5 - - 0.1269 0.177# t - - 0.1278 0.1786 Average 0.0681 0.0671 0.1260 0.1776 A* - - - 0.0516 13f is the difference betwee sample and reference value. 180 on C-5 of #-hydroxysphinganine is 62.17%. 4 + :9/5 299, c14n29-ca=0rM3i; m/e 501, C14H29- egorMSi; pIMSi + orMSi + 9/3 1101, C14H29-CH-CH=O'ndSi; 31/5 1105, 014n29-cn-cnléomsi leprMSi + and Glaze-ca-cnmmm. 67 Adding 1 mg of this [1-180]palmitate, dispersed in 20 mg of Triton X-100, into LMrl (10 ml) resulted in an incorporation of 180 into C-3 of 4-hydroxy3phinganine after incubating with viable yeast for 48 hours at 26-28°C. About 62% (100 x 5.2/8.2) enrichment was found on C-3 (Table 7). None was observed on C-4 of 4-hydroxy- sphinganine. It was concluded that [1-180Jpalmitate and [3-180]3phin- genine, which can be derived from [1-180]palmitate‘ig situ, were not the oxygen donor on the hydroxyl group on C-4 of 4-hydroxysphinganine. G. Incorporation of H580 into 4-Hydroxysphinganine by Yeast Grown on Differrent Media. 1. Yeast Grown on LMél and LMEZ. Table 8 shows the incorporation of Héso into various hydroxyl groups of 4-hydroxysphinganine by yeast grown on LMhl, containing 0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 1% glucose in 10 ml of H580 (30.13%). Incoporation of 180 into C-4 of 4-hydro- xysphinganine (16.5%) was comparable to that reported by Thorpe and Sweeley (1967). When the medium was autoclaved at 120°C for 25 min- utes (EM-2, Hgao used was 19.1%), incorporation of 180 from.H;80 into C-4 of 4-hydroxysphinganine was not affected by heat (Table 9, 13.9%). The slightly lower value (2.6%) is probably due to experimental errors in the determination of low isotopic abundance in 4-hydroxysphinganine product or due to slight variations of harvesting time which is not known whether it is a critical factor for isotopic incorporation. The hydroxyl group on C-3 of 4-hydroxysphinganine was fully derived from water in both experiments (LM-l and LM92). This hydroxyl 68 amzyowa amXHOn _ . I I amxeo-nm0ixonmo nan n xuonanmonmo .omm a\e amino» dawns Haw _ -1 .1.1 memzao.mmo-monmu .mam axe mamxHOnmu-mo-nnma.o sea amzaommmo.mu-ewmaao .moa a\e + + a. messenmo-mm-hmano Joe ME 329038-886 Jon we. magm.mo-fimeao .mmm we: . amzeo a .&:N.Nm ma Hnu cam $mm.mo~ me new «Kw:.wa we :10 ouch oma mo cowumuoauoocH .mosao> oocouowou paw masses eooauon monouomwwv onu ma LAN aaom.o - oomn.o - mmao.o - *AV wmmm.o memo.o oHom.o onn.o Pena.o ammo.o emana>< mmmm.o oamo.o mnem.o oeHH.o seaa.o mmmo.o a wmmm.o mnmo.o Hama.o aHmH.o omHH.o memo.o n Emma.o mamo.o nHOm.o Hama.o nmaa.o wwwo.o m mamm.o samo.o maOm.o 6Hm~.o span.o nemo.o H oaaamm ooaouomom magnum oocouomou oaaaom mucouomou 936mm ME. Simon 3m amazon 3m 3235833 .A&na.onv omamm ca #124 a“ macaw onus» Bonn ocucowcwnammxouvhmu: ca omH mo manmfioa< <54 .m manna «mayomn emzwo» . I I see amzaonsmmonmu nomm axe amzeo-nmui=onmu amzeo» amzeona emzao . . . ...N 3.. a . 1.1:. mamzao-mmo-monmu awam n\s mamzHOnmo-mo-smmeeo eea enzyommmo.mo- m o no: d\ . + 4 + 52826-6-Rm36 :3 3m 392636-536 Jon 3m ”229951.230 .mmm flea . emzao . .flofi.m> we #10 use emm.m0a me new a$>®.ma we :10 one“ omH mo eofiumuoauoocH .mOSHG? flocwhflwwh VG“ OHn—Ewm Cflwsfiwwn— woawhmwwwv Q5“ mH *q 69 aaem.o - mamm.o - memo.o - LAN mama.o mamo.o mean.o cama.o mamo.o ammo.o emane>< mome.o oamo.o Hmmm.o mman.o nemo.o mmmoio a amma.o mnmo.o Hmmn.o aHmH.o mamo.o memo.o m Nema.o memo.o mamm.o onH.o mamo.o ammo.o m mama.o samo.o damn.o mHmH.o mnm0.o nemo.o H ogaaom oocouomom oaaaom oocouomom oaasom ooaouomom ncOauoeHauouoa @838 me». Since 3m. amazon we .A$o~.mav 00%: ca mean ca csouu umoow scum osaamwcasawmxouvmmud an om.H mo maozamc< «>4 .m manna 70 group was derived from palmitate; exchange with water in the medium somewhere after palmitate synthesis cannot account for complete exchange and therefore the exchange with water must take place prior to the incorporation into palmitate. Incorporation of HfiBO into the primary hydroxyl group on C-1 of #-hydroxysphinganine was increased from 57.7% in LM-l to 79.1% in LM-2. This increase is expected because an increase in temperature would increase the rate of oxygen exchange between water and the alde- hydic oxygen on C-1 of glucose. This oxygen is transformed into the hydroxyl group on C-5 of 5-phosphoglyceric acid during glycolysis then to the hydroxyl group on C-5 of serine (Umbarger and Dmbarger, 1962). This hydroxyl group of serine was shown in the earlier study to be the precursor of the hydroxyl group on C-1 of #-hydroxysphinganine. 2. Yeast Grown on Ethanol as the Principal Carbon Source. Since over 95% of the dry weight of malt extract was found to be anthrone-positive compounds (calculated as glucose), it was necessary to omit this nutrient in order to minimize the source of carbon other than ethanol. Peptone contained about 1.5% of anthrone-positive material and was also omitted in this study. Yeast extract, however, contained about 7% of anthrone positive substances, but could not be eliminated without affecting growth of the yeast. Yeast was grown on LM-5, containing 0.12% amonium sulfate, 0.07% magnesium sulfate, 0.05% sodium chloride, 0.5% potassium phosphate (dibasic), 0.5% yeast extract and 2% ethanol in 10 ml of H580 (15.7%). To utilize ethanol as the building block for other biological substances, yeast must oxidise it to acetate. Oxygen of acetate is assumed to be 71 senor use? . I 1. amzeo-mmo-monmo eea amzHonsmmUHmo .omm a\e “mayo“ amzewne emzaw . .I I mamzno-mmo-m6nmo .mam a\a mamzHOnmu-mu-smmeeo ene awesommmu-mu-emmaao anon e\e + + m 1 1 IN dd « M. clue “A IFN #H a M W! m ”A ISOIENma‘HU «mam ”\m emzH0-mo mm m u Hoe \ .mzaonsmo m o no \ .mzam . amzeo a .&mH.N® we are one emb.mm we n10 nfimm.ma me :10 Once Gm.fl mo cowuouoauooca .mo3~o> mucouomou one cameos coozuon oocouommgo onu ma *AV oaom.o - amaa.o - Osmo.o - *AV mmmm.o memo.o annm.o onH.o mome.o mmmo.o amnao>< mmmm.o mmmo.o mmmm.0 aHmH.o mamo.o moeo.o a nemm.o oamo.o menm.o mmHH.o camo.o eoeo.o n mmmm.o memo.o momm.o Hama.o momo.o mmoo.o m nmmm.o eamo.o nanm.o memn.o oomo.o Hmwo.o H magaom oocouomom oHaaom oocououom oaaaom monogamom 2368. ME Since ME. amnion ME 2333538 .Aee.mav came as Hosanna fim ocm nix: cw cacao ammo» scum ocacuwcqnamhxonohmu: cu omH mo mamhaoc< <>< .oH wanna 72 Table 11. AVA Analysis of 180 on C-2 of Glucose from Yeast Grown in LM—3 and 2% Ethanol in H580 (15.7%). Determinat ions 52/2 162/ 160 Reference Sample 1 0 0529 0.1#75 2 0 0529 0.1#77 5 0.0530 0.1#75 “ 0-0535 0.1#60 ‘Ver‘ge 0-0551 0.1471 A* - 0.09110 A" is the difference between sample and reference values. Incorporation of 180 into C-2 of glucose is 68.61%. .g/g 160, cn=N-ocns; ‘m/e 162, oxen-0033. + + CH-OTMSi caiSorMSi 75 .oouoom conuoo Haaaoaaua sea as Hoconuo so anon» unmom soum wouofioma Am one mucouomou A< moeqxoSuoa owoosawnwmza mo ouuooam new: .w .wwm 3|9 205 2l7 350 400 450 BB 300 250 73 205 2|? 200 I47 ISO 150 l03 100 I47 ISO IO3 73 SO 100 80-1 604 20* 1 O (D 100 (‘70) issued Saunas sol 40-4 20" 150 200 250 300 350 400 450 100 $0 m/e 75 easily exchanged with water in the medium and most biological com- pounds would, therefore, be expected to bear oxygen isotope in the molecule. Table 10 shows that incorporation of H580 into C-# of #-hydroxysphinganine was not increased compared to the results with yeast grown in LM—l and LM-2. The only increase was that on the primary group on C11 which originated from serine. By either path- way, glyceric acid (Umbarger and Umbarger 1962) or glycine and formaldehyde (Kislink and Sakami, l95#), an increase in the 180 abundance on C-5 of serine, relative to that of LM-l and LM-2, would be expected. It was a surprise that the isotopic abundance of the hydroxyl group on C-5 of #-hydroxysphinganine was less than that in yeast grown in 111-1 and 124-2. This difference in 180 incorporation might be due to the fact that acetyl CoA, the oxidation product of ethanol, might be used directly for the synthesis of palmitoyl CoA. Thus, unhydro- lysed acetyl CoA might be responsible for incorporation of the ethanol oxygen and would give a lower level of 180 from water. Analysis of glucose isolated from the cell paste (Table 11) indicates that 68.6% of oxygen on C-2 was derived from water in the medium. The spectrum of TMSi-glucose methoxime from the cell paste (Fig.8B) indicated the incorporation of 180 into various positions of glucose, thus glucose was tentatively ruled out as the possible oxygen donor to the hydroxyl group of #-hydroxysphinganine. Interpretation of the mass spectrum of TMSi-glucose methoxime (Fig.8A) has been published (Laine and Sweeley, 1971). The major ions are shown below. 76 The ions at.g/g #09 and 507 are unstable and subsequent loss of TMSiOH yields an ion at m/g 519 and 217, respectively. cum-00113 CH-OTMSi 160 moi; """"""" 99 teens """" 36'? hang; """" is n.) cease; """" 163 H. Mass Spectra of Bis-O-TMSi-N-Acetylsphinganine. The mechanism of electron-induced fragmentation of TMSi-N- acetylsphinganine was studied with the aid of deuterium labelling and exact mass measurement. The ions formed on electron impact ioni- zation at 70 eV were divided into two main categories with respect to electron abstraction from one of the oxygen atoms or the nitrogen atom of sphinganine. l. Ions Derived by Electron Abstration from Oxygen Atoms. The mass spectrum of TMSi-N-acetylsphinganine (Fig.9) was previously reported (Gaver and Sweeley, 1966). The molecular ion was not detected, but could be deduced from other ions in the spec- trum, especially the ion at Mr15 (m/g #72). This ion, atlm/g #72 (C25H54N038i2; calc. #72.56#l; observed, #72.5651), is derived from loss of a methyl group from one of the TMSi groups. Substitution on TMSi with dg-TMSi on N-acetylsphinganine resulted in a shift from the 77 loss of 15 amu to 18 amu (Fig.10), suggesting the loss of a da- methyl from.one of the dg-TMSi groups. However, which of the two TMSi groups (on C-l or C-3) the methyl was lost from was not ascer- tained. It is assumed to be a mixture of the following two structures: OTMSi | + clsusl-CH-ca-CH20=Si(CH3)2 NH-Ac +?=Si(CH3)2 C15H31-CH- c.111- CH20mSi NH-Ac It is possible to differentiate these two ions, if selective derivatization of one of the hydroxyl groups with dg-TMBi is achieved. This technique has been used successfully with steroids which contain a hindered hydroxyl group on C-17 and a nonhindered hydroxyl group on C-5 or C-2O (Vouros and Harvey, 1975). The two hydroxyl groups of sphinganine appear to be equally reactive to TMSi-donor agent, however, and they are probably trimethylsilylated instantaneously. Attempt was not made to pursue this problem, therefore. The ion at'm/g 105 (C4H11081; calc., 105.0579; observed, 105.0579) is probably formed by cleavage of C-1 and C-2 bond. 9% . . C15H31-CH-CH-CH2-OTMSi ‘--——4> CH2=OTMSi NH-Ac 52/ g 105 Substitution of two deuterium atoms on C-l shifted the ion to;m/e 105 (Fig.12 and 15) regardless of isotopic substitution at other positions 78 (Fig.11 and 15) and it was observed at m/g 112 in the spectrum of d9-TMBi derivative (Fig.10), indicating that the ion is composed of one TMSi group and the methylene group on C-l. This ion also derived partially from C-5 of sphinganine by a more complicated mechanism, however, since substitution of deuterium on position-5 shifted a significant prOportion of the intensity at‘m/g 105 to m/g 10# (Fig.11). Cleavage of both C-C bonds of C-5, accompanied by transfer of hydrogen to the positively charged ion, is necessary to account for this component of m/g 105; the origin of the hydrogen transfer cannot be ascertained in this study. The ion at m/g 217 (C9H21028i2; calc., 217.1079; observed, 217.1085) may be derived from the following two pathways. OTMSi 11 OTMSi ' 9 t Q! m 1' clsnal-ca-tlm- —0TMSi 4 C15H31-CH-CH=CH——O'DiSi NH-A {7’ c .9/2 #28 Q 0H=rcacn=omsi Comm else 217 Reason... C 011131 3.1/3: 217 torus| i 11 clsnal-cn-cnf'cn—omsi 4. clsasl-cH-cmca—o'mSi (NH-Ac 3011151 .9/5 #28 79 It is assumed that this ion can be derived from the molecular ion which has charge retention on the oxygen atom of either C-l or C-5. The initial loss of the elements of neutral acetamide by 1,2 elimi- nation gives an ion at m/g #28, which is very weak in the spectrum of the protium species (Fig.9) but for an unknown reason is enhanced (at 9/2 #29) in the spectrum of [5-2H]-sphinganine (Fig.11). The intensity of this ion is suppressed to an undetectable level by further substitution of deuterium atoms at other positions. Homolytic cleavage of the ion at 9/2 #28 gives a more stable ion at gMg_2l7. A reversal of the sequence leading to this ion, involving initial cleavage of the bond between C-5 and C-#, followed by loss of elements of neutral acetamide, is also possible. However, the ion at.g/g 276, which is presumably derived from cleavage of the C-5 and C-# bond initially, has never been detected in the spectra of protium or deuterium forms of sphinganine. The ion at'm/g 217 is, therefore, more likely to be derived from ion at m/g #28 rather than from m/g 276. Substitution of two deuterium atoms on C-1 of sphinganine shifted the ion by only one mass unit (Fig.12), suggesting that one of the deuteriums is lost during the formation of the ion at'g/g #28. As would be predicted from.the mechanism,the ion at‘m/g 217 is not affected by deuterium.atom on C-# and C-5 (Fig.15) or on the acetyl group (Fig.l#). It is shifted by 18 mass units in the spectrum of the bis-O-dg-TMSi derivative (Fig.10). Deuterium substitution on C-1, C-2, C-5 and C-# shifted the ion by three mass units (Fig.16). Since 80 substitution of deuterium.on C-1 and C-3 shifted the ion by two mass units (Fig. 15) whereas substitution on C-4 had no effect (Fig. 13), it can be inferred that deuterium on C-2 could contribute one mass increment to this ion. The structure and mechanism of the formation _ of this ion, shown above, is therefore consistent with the spectra shown in Figs. 9-16, and the composition of the ion (given above) is exactly the same as that of ion at 9/2 217 derived from penta- O-TMSi-Ob-p-g-glucopyranose (DeJongh gt a_l., 1969), which was observed in the spectra of TMSi derivatives of a variety of sugars (DeJongh gt ‘21., 1969; Laine and Sweeley, 1973). The ion at 9/2 313 (019H41081; calc., 313.2926; observed, 313.2934) is believed to be derived directly by simple cleavage between C-2 and C-3 with charge retention on the trimethylsilyloxy group, as shown below. Substitution of deuterium on C-3 (Fig. 11), C-4 and C-5 (Fig. 13) and on the trimethylsilyl residue (Fig. 10) shifted the mass of this ion by 1,2 and 9 mass units, respectively. Substitution of deuterium on C-1 and on the acetyl group did not affect its mass, as predicted by the proposed mechanism. Further fragmentation of this ion with loss of elements of tridecane, by a mechanism.shown below, gives an ion at‘m/g 129 (C6H13081; calc., 129.0735; observed, 129.0736). This ion was affected, as predicted by the proposed pathway, when deuterium was substituted on C-3 (mfg 130; Fig. 11), C-4 and C-5 (g/g 131; Fig. 13) and on the TMSi groups Qm/g 138; Fig. 10), whereas substitution of deuterium.on C-l (Fig. 12) and on the acetyl group (Fig. 14) had no effect. 81 ??E?Si '+OTMSi n C15H31'CH' (IIH- CHQOTMSi + C13H27-CH2191-CH NH-Ac H LIVE. 5 13 CH2= 011- 011- 01113 i 9/2 129 2. Ions Derived by Electron Abstration from the Nitrogen Atom. The ion at.m/g l7# (C5H16N028i; calc., 17#.0950; observed, 17#.O9#5) arises by direct cleavage between C-2 and C-5 with charge retention on the nitrogen containing fragment, as show below. Substitution of deuterium on C-l (Fig.12), on the acetyl group (Fig.1#), and on the TMSi group (Fig.10) shifted the ion to 9/9 176, 177 and 185, respectively. 0TMSi {V C15H31 - CHECH- CH2- OTMS i 4 013012- OTMS i ll 1- NH-Ac + NH-Ac .Ie/s 171+ CH=CH2 f NH-Ac 3/3 85 82 Substitution of deuterium on C-5 (Fig.11) and on C-# and C-5 (Fig.15) did not affect the mass of the ion but deuterium substitution on C-1, C-2, C-5 and C-# shifted the mass by three mass units (Fig.16), which is assumed to be accounted for by the two deuterium atoms on C-1 and that on C-2, rather than the deuteriums on carbons-5,# and 5. Further fragmentation, with a loss of TMSi radical, yields an ion at m/g 85 (C4H7N0; calc., 85.0528; observed, 85.0550). This ion is expected to be affected by the same deuterium substitution pattern as the ion at 'm/g 17# except for that due to the TMSi residue, which is lost in the conversion of‘m/g l7# to 85. The ion at g/g 58# (022H4N028i; calc., 58#.5297; observed, 58#. 5275) is presumably derived from covalent bond cleavage between C-1 and C-2 with loss of the C-1 containing fragment as shown below. Deu- terium substitution on any position other than C-l would therefore be expected to affect the mass of the ion, and the results of deuterium labelling on various positions (Figs.9-l6) are consistent with the structure. CTMSi OTMSi l Clsflgl'CH-c'ny CHE-Mi —'——-. C15H31-CH-EH 1. NH-Ac +NH-Ac 32/2 5811 A cyclic transition mechanism has been proposed by Hammarstrdm ‘g£'_l. (1970) to account for the formation of an equivalent ion of .g/g 2#7 in the mass spectra of long-chain fatty acyl sphinganine ceramides. Data obtained here on the spectra on N-acetyl derivatives 85 of sphinganine labeled with deuterium at various positions agree with this type of fragmentation mechanism, which is shown below. The ion (C10H25N02812; calc., 2#7.l#25; observed, 2#7.l#2#) is at.m/g 2#7 in the protium species (Fig. 9) and occurs at.g/g 2#9, 250, 250 and 265 in [1,1-2fle]sphinganine (Fig. 12), [1,1,2,5,#,#-2Hg]sphinganine (Fig. 16), N-[2H31acetylsphinganine (Fig. 1#) and ds-TMSi-sphinganine (Fig. 10), respectively. 051131-531; (Im- cngomsi .. -c11- 0112011151 ?) fNH-Ac + NH-Ac ms 1’ ms i 93/3 2117 The ions at m/g 157 and 116 are presumably derived from.further fragmentation of the ion at 9/2 2#7, as shown beldw. Loss of TMSiOH gives a companion ion at 9/9 157 (C7H15NOSi; calc., 157.0925; observed, 157.0922). The sphinganine labeled with deuterium on C-1 and C-2 (Fig. 16) and on the acetyl moiety (Fig.l#) did not loss 91 amu instead of 90, indicating that the hydrogen atom involved in this elimination process is from a source other than C-1, C-2 or on the acetyl group. The hydrogen atom involved in this elimination process was tentatively assigned to that attached to the nitrogen atom. A concomitant loss of one methyl radical and a carbene from the ion at m/g 2#7 is suggested to account for the ion at'm/g 116 (C4H10NOSi; calc., 116.0551; observed, 116.0552). This ion is not affected by deuterium substitution on the sphinganine moiety, but shifts to'm/g 119 and 122 in N-[ZHBJacetyl sphinganine (Fig. 1#) and dg-TMSi species (Fig. 10), respectively, 8# 00m 0m¢ 00¢ 0mm bbLbrerlhlbrbbbhhrbibrrth hLIblbbbh bbbbb it m\:e 00 . 000.35 .108 17.. @088 4808.16 5410.36? 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W a no . a _ Sm ow M . m . 8v .3: .8 .Ne 59 a .5872 9%: mi . .mefiorufim 8.32.10 .8 3 v as Q. con m\c._ com one 8.» on» ,8». mm... -.LLonmt r on. 8. .on -r>r>.p r .4 p L A“ d d 441 9.: En EN m8 TON a 4 m. m. com H: A I8 W m mm mum an m . omen?! .om u. (A d o m . 99:2 053: .8 agongcxoaxfife 5 n33...» 8. n .2 co“ .cgcuwcananamuouunmmmunzuamvfiuoédn no .553on and: A3325 .2 .wam % .acacuwcafimauoomuzmmmm$rm $2-053 mo 65.53% mun: A26an 3 .wrm own 0? 03 on» com omm com on. 02 on a _ _ _ . 8n _ . 9-2 .on no ‘ _ m. NmN EN .9 _ _ IIW. L 13 m w w: 8 M w .. we“; 0.” ..OG MW (A {0812 0.92.. w . m2»o£w.d%+x869&f26 .8 . _ 3......» 5. £5 m oou 87 00m one. 00¢ own 000 0mm com on" 00— on . a P w m. .. a N M . .3 W 833: A .6812 0.92» ...... W J. .8 _ E25 $0449.70 ow. .. 8.8m _ 2...!» Q. 09 mr .ocgcuwgnamahumumquosmu:a:«mam;«HM—uwmzauoéz mo asuuuoam and: 4330.5 3 .wwm .acacuwcanamahumonuZHmmmun..."«5 ...mmzauoéan mo Hangman and: AQSAQV ma .wrm 8.... on.» 8.» on... com on... oo~ . on. 8. on 9.: man ma . on. .3 H. p mvu 2. m... A Km 18 m w in W. a you "W. 4 omv 32 [A 685. w . .mzpodwqflmw .... 06$? 8. .8 . Q. 8. N" 88 indicating the presence of an acetyl group and a dimethylsilyl residue. The structure and the fragmentation mechanism for its formation from the ion at gflg 2h? are as follows. (V . - 'c'm— CH§0m51 (.JH- CH2 Hfl'wiAc $. tu-Ac 11151 TMSi 311/2 21+? 311/2 157 CIéH- cng-oms i + NH- Ac ' l + NH-AC >- Si-Cns + [:CH-CH-OTMSi + CH3. .I | \J CHa'Si'(CHs)2 CH3 m/g 21+? _u_1/_e_ 116 enema-0mm I. Incorporation of [2,3,5-2H3:]Serine into Sphingolipid Bases. 1. Characterization of [2,3,5-2H3JSerine by lass Spectrometry. [2,5,5-2H3:]Serine was characterized by GLC-MS as the TMSi-N-benz- alidine derivative. This derivative was prepared by adding TMSi-donor agent (Gaver and Sweeley, 1965) to the dry solid serine (1 mg/ml). When all the solid had dissolved, benzaldehyde (0.2 volume) was added. The solution was left at room temperature for 30 min. before analysis by GLC-MB. The mass spectrum of TMSi derivative of benzaldehyde adduct of Relative Inimsity (°/o) Reloflve Intensity (7.) Relative Intensity (%) 89 100 I 73 234 b. ., o n 804 I47 220 mqgtyiguicoom 'r f E 601 IO3 c 0"0 MW =33? . 40‘ m5 * "7 |29 :90 247 201 s M'30 * V'YVAVL'tfiuf‘i'Il'lu‘v 'hT‘T—‘jjT'V'I'Y'1'F‘T'Vfrf1fi 50 100 150 200 250 300 350 m/e 100 73 b~. ..o :47 ; . 80‘ 222 Tuggngqn‘coow ‘ 236 ff 1 CH . . 60 '05 c 0 MW 339 40- ' m5 . :3: 20« "7 '90 249 M3 . so 100 150 200 250 300 ‘ 350 m/e 100 73 .47 b: (0 mo *co-Lcoomst P 801 .... .0 223 [c . 60- '05 237 “*0 mm 340 . 40‘ u-ns ‘ 204 . Fig. 17. Mass spectra of bis-O-TMSi-N-benzylidineserine; A) serine 100 150 200 250 "Va 300 350 B) [5,5-2H2]serine and C) [2,3,5-2H3]serine. 9O serine is shown in Fig. 17A. The molecular weight could be calcu- lated from ion at M—lS (311/3 522) and M-9O (gt/g 21W), which resulted from loss of a methyl group and TMSiOH, respectively. These two ions were shifted to m[§ 325 and 250 in the spectrum of the [2,3,5-2H3]- serine derivative (Fig. 170), proving there are three deuterium atoms in the molecule. The ion at‘m[§ 32% is probably derived from loss of a methyl group from one of the TMSi groups of dideuterioserine deri- vative impurity. The intensity at m[§ 32% was about 10% at mflg 525. The base peak at g[§ 25% is probably derived from loss of ion 2 or ion g-l by the following mechanisms. CHE-CH- cogmsi CH- cogmm l LI 9 u M-b TMSi-O fN-‘CH-Ph +N=CH-Ph - 9.1/2 251+ [011151 IOTMSi CH -CH-C\ CH -CH=C u 2 v (‘0! 1, , 2 ‘35s TMSi-O N\\n’H oms1 M' {9'1} 6 I Ph 93/3 2314 These two ions were split into two ions at m[g 235 and 257 in the spectrum of the [2,5,5-2H3:]serine derivative (Fig. 176), indicating that two deuterium atoms are on C-3 and one on C-2. The ion at MEa-b (g[g 190 in Fig. 173; 191 in Fig. 17C) provides unequivocal evidence that there is one deuterium atom on C-2. The ion at M930 (m[g 307; Fig. 17A) is probably derived from a cyclic transition by cleavage of C-2 and C-5 bond with a simultaneous transfer of TMSi on C-3 to the 91 functional group C-2, accompanied by expulsion of formaldehyde: c.2112?" -COZTMSi 'oH-congi (prfN-CH-Ph + +I:J=CH—Ph TMSi TMSi 9/2 307 The loss of 52 amu from [2,5,3-2H3:]serine (Fig. 170) and [3,5-2H3]serine derivatives (Fig. 17B) is consistent with this type of mechanism. A cyclic transition with transfer of TMSi to the vicinal nitrogen atom has been observed in the spectrum of TMSi-N- acylsphinganine (Hammarstrbm gt 31., 1970) and TMSi-N-acetylsphinga- nine. 2. Incorporation of [2,5,5-2H5:]Serine into Sphinganine by Rat Liver Microsomes. The first step of sphingolipid base biosynthesis involves the PLP-dependent condensation of serine and palmitoyl CoA, yielding 5-ketosphinganine. There are at least two broad mechanisms for the formation of j-ketosphinganine (Braun and Snell, 1968), depending on (whether the initially formed serine-PLP Schiff's base complex I under- goes decarboxylation to furnish complex II or loss of the abhydrogen atom to form complex III (Fig.3). To differentiate between these two me- chanisms [2,5,3-2H3]serine was incubated with a crude rat liver microsomal system, in the presence of the necessary cofactors (Table 12), at 37°C for one hour. Since j-ketosphinganine has been reported to be very unstable (Mendershausen and Sweeley, 1969), it was reduced to sphinganine with NADPH-dependent S-ketosphinganine reductase which 92 Table 12. Composition of Reaction Mixture for the Incorporation of [2,3,5-2H3]Serine into Sphinganine by Rat Liver Microsomes. Palmitic acid 20 umoles Triton X- 100 20 mg Mg612 25 umoles CoA 7.5 umoles ATP 10 umoles NADPH l umole PLP lO umoles .DTT 10 umoles ds-serine 1+0 umoles Microsomal enzymes 2 m1 Phosphate buffer (pH 7.5) 10 ml Final volume 12 ml 93 was present in the microsomal fraction (Stoffel ££.2l-: l968c). The reaction was terminated by adding 1.0 ml of l.ON sodium hydroxide followed by ether extraction. Sphinganine was partially purified by TLC on silica gel G as described in Methods (Section 8). The partially purified sphinganine was converted to the TMSi-N-acetyl derivative and characterized by GLC-MS. It eluted from a 5% SE-EO column at the same retention time as authentic TMSi-N-acetyljBE-erythro- sphinganine, suggesting that the sphinganine formed in the microsomal preparation has the erythro configuration (Gaver and Sweeley, 1966). The mass spectrum of the TMSi-N-acetyl derivative is shown in Fig.18. The locations of the major ions are consistent with the characteristic ions of TMSi-N-acetyl-sphinganine (Fig. 9) except that some of these ions are shifted to higher mass due to the presence of deuterium atoms in these fragments. The ions at g[g h72, 2h7 and 157 are shifted to .glg hTH, 2&9 and 159, respectively, indicating that there are two deu- terium.atoms in these ions. The ion at.m[g h7h arises from the mole- cular ion by loss of a methyl group from one of the TM31 groups. The ions atig[g 2h9 and 159 are derived from cleavage of the bond between C-2 and C-3 with transfer of TMSi on C-5 to the nitrogen atom by a cyc- lic transition as shown in the Section on Mass Spectra of Bis-O-TMSi-N- Acetylsphinganine, suggesting that both deuterium atoms are on C-l and/or C-2. When fragmentation involves charge retention on the C-5 fragment, homolytic cleavage of the bond between C-2 and C-3 results in loss of both deuterium atoms and yields an ion at m/g 313, providing additional indirect evidence that both deuterium atoms are on the C-1 and C-2 fragment. Homolytic cleavage of the bond between C-1 and C-2 9h cowuuwo :oHuowou HoaomOHOHa aoum vouQHOoa wouuASUcH aofiuommu HQEOmouoHa aoum woumHOmw .ommm 5 use ocficnwaasnmamuoomuZuqmza mo asuuuoam and: .daouuonv ma .wam .ofiuoammmmAfié :33 mafiauwcasmmahuooo-Zuam2H mo azuuooam can: .Aaoev m. .mam 95 m\C. 00m 0m¢ 00¢ 0mm 000 0mm 00m OmH OOH cm L .P hm b ..4 <— h h .— 1.41m _ 1» >4- <‘> h .11 nrn— 1.14% b bl >4... 1- - ..1 up: 1-1_ 1 h >1P WL‘ L f. h > .— — . - _ b ...... .... .4. _. 4. ...... 4 _ - a-.. . 8-5.. mew om . 8.132 3. :2 oz? 3 -3 . .mEo 1040.04.01. 54.5 3-..... .8 - 8m-.§m . Mk II OOH 00m 0m¢ 00¢ 0mm 000 0mm 00m om. 0m 4 p w _ n p p - JI- p b — - h P _ — — n p — p - _ — b . _ — h L h b h u — > n“ u » >1 th‘h - p1— . E.» 4 4 4414. .44.... - - own 4m_20 .ON 3-5: mum . mwviss. -0¢ N 3.17.. 05):. m an . .msto 84.104.10.90. :0 3-..... -8 ._ 4 fin . .. o v .13 mm. .om .0. Q. .8. (%) Mzsuew! alums 96 with charge retention on the C-2 fragment and liberation of the C-1 fragment as a free radical yields an ion at glg 384, indicating both deuterium atoms are on C-l. Thus it was concluded that the microso- mal preparation from rat liver converted 2,3,3-trideuteroserine to sphinganine with loss of the deuterium on C-2. J. Incorporation of Deuterium from 2H20 into Sphinganine. To determine whether the hydrogen atom lost from serine during Sphinganine synthesis can be replaced be a proton from.the medium, rat liver microsomes were incubated with serine in 2H20. The composition of the reaction mixture is given in Table 13. The reaction mixture was shaken in a Dubnoff metabolic shaking incubator (Precision Scien- tific Co., Chicaco, Ill) for one hour at 37°C. Sphinganine formed in the reaction carried out in 2H20 was about half of that formed in H20. The mass spectrum.of the TMSi-N-acetyl derivative is shown in Fig. 19. The ion at M—lS was located at 2/3 473, suggesting the presence of deu- terium.atom in the molecule. The characteristic ions of TMSi-N-acetyl- sphinganine, 93/3 247 and 157, are shifted to g/g 248 and axle 158, but the ion at'g[g 313 is unaffected, thus this deuterium should be on either C-l or C-2. Homolytic cleavage of the bond between C-1 and C-2 with charge retention on the C-2 fragment yielded an ion at 9A2 385 which was shifted 'from the ion at‘gfg 384, in the protium.form (Fig.9), by one mass unit, suggesting that deuterium is on either C-2 or on the nitrogen atom. Hydrogen on the N-atom is labile, however, since sphinganine and N-acetylsphinganine obtained after exchange equilibration with deuterium in 2H20 was completely lost during GLC analysis. Thus, it was concluded that the deuterium atom.was on C-2. 97 Table 13. Composition of Reaction Mixture for the Incorporation of Deuterium.from 2H20 into Sphinganine by Rat Liver Microsomes. Palmitic acid 20 umoles Triton X-100 20 mg MgC12 25 umoles CoA 7.5 pmoles ATP 10 umoles NADPH l umole PLP lO umoles DTT 10 umoles Serine 40 umloes Microsomal enzymes 2 m1 Phosphate buffer in 2H20 (pD 7.5) 10 m1 Final volume 12 ml 98 Although isotOpic purity of 2H20 used in this study was over 992, it was diluted by microsomal solution to about 80%. Incorp8ra- tion of deuterium on C-2 of sphinganine was about 55% (judged from comparisons of the ions at mflg 158, 248 and 585 with those at m[§ 157, 247 and 584, respectively). The decrease in incorporation of deuterium into C-2 of sphinganine might be due to a slow rate of exchange between hydrogen and deuterium around the active site of the enzyme, 5-keto- sphinganine synthetase. Thus, a relatively high initial rate of incorporation of hydrogen would be expected. The slow rate of exchange between hydrogen and deuterium may involve the exchange of hydrogen attached to a nitrogen atom (such as an amino or amide nitrogen) in a hindered site or in a relatively hydrophobic region of the enzyme active site. K. Incorporation of Serine/2,5,5-Trideuteroserine into Sphinganine. Substitution of deuterium (an) or tritium (3H) for an ordinary hydrogen often causes appreciable variation in the rate of an enzyme- catalysed reaction, particularly when the reaction involves breaking the bond to the labeled atom (Gould, 1959). This isotope effect is usually expected if 1), the bond to the labeled atom is broken at the rate-limiting step or 2), a rapid and reversible reaction preceeding the rate-limiting step may involve cleavage of this bond. If the bond to the labeled atom is broken after the rate-limiting step, little or not isotope effect will be observed. To determine whether there is any isotope effect involved in the breaking of the 2H-C bond of serine during the conversion to sphinganine, 2,5,5-trideuterioserine and serine were allowed to compete for the microsomal enzymes for one 99 Table 14. Composition of Reaction Mixture for the Incorporation of [2,5,5-2H3:]Serine/Serine into Sphinganine by Rat Liver Microsomes. Palmitic acid 20 umoles Triton X-100 20 mg Mg012 25 umoles 00A 25 pmoles CoA 7.5 umoles ATP 10 umoles NADPH 1 umole PLP 10 umoles DTT 10 umoles [2,5,3-2113 JSerine/Serine (1.17) 0.5 mg* Microsomal enzymes 2 ml Phosphate buffer (pH 7.5) 10 ml Final volume 12 m1 *second experiment employed 5 mg. 100 hour at 57°C. The composition of the reaction mixture is given in Table 14. The exact ratio of d3-serine/serine was determined by GLC- AVA of the TMSi-N-acetyl derivative, using ions at m[§ 174 and 177 for the determination: The ion at m/g 174 is derived from simple cleavage between C-1 and C-2 with charge retention on the nitrogen-containing fragment. Substitution of three deuterium atoms on C-2 and C-5 shifted the mass of this ion to m[§ 177. Results are shown in Table 15. The deuterium content in the sphinganine product was analysed by GLC-AVA as the TMSi-N-acetyl derivative. The ions at m[g 157 and 59 were used for the analysis. The ion at m[g 157 was shown in the previous section (Mass spectra of bis-O-TMSi-N-acetylsphinganine) to be derived from the first two carbons. Substitution of two deuterium atoms on C-l shifts the mass of this ion to m[g 159 (Fig. 18). If there was no isotope effect, the same proportion of 2,5,5-trideuterio- serine and serine would be expected to be converted to[1,1-2H2]sphin- ganine and sphinganine and the ratio of d2-sphinganine/sphinganine should be the same as that of the d3-serine/serine substrate. On the other hand, if there was an isotope effect in breaking the 2H-C bond, incorporation of d3-serine into dg-sphinganine should be less than that of serine into sphinganine. Results in Table 16 show that the ratio of d2-sphinganine/sphinganine is 0.2767 when the concentration of d3- serine and serine mixture is 0.5 mg/l2‘m1. The ratio of dg-sphinganine/ sphinganine is raised to 0.6755 when the concentration of d3-serine and serine mixture is 5.0 mg/12 ml. These two ratios of d2-sphinganine/ sphinganine are still lower than that of d3-serine/serine substrate (1.17). The change in d2-sphinganine/sphinganine ratio is indicative 101 Table 15. Determination of the Amount of [2,5,5-2H3J- Serine/Serine in the Mixture by AVA. 111/33 177/ 174 Determinations Reference Sample 1 .0105 1.1764 2 0.0104 1.1825 5 0.0090 1.1887 4 0.0090 1.1615 Average 0.0095 1.1772 [5* — 1.168 zsfis the difference between sample and reference values. 39/2 174, fiH-Cfig-OTMSi; 111/3 177; fiD-CDg-O'niSi. +NH-Ac +NH-Ac 102 of the presence of endogenous serine. The amount of endogenous serine is then calculated as follows. Assuming that there is no isotope effect and the amount of endogenous serine is X pg, da-serine - 0.2767 X — serine 500 (1.17/2.11 . 0.2767 x - 300/2.17 x . 446 pg. Substitution of this X value into the 5 mg mixture give a value of d3-serine/serine of 0.88 as shown below. da-serine . _3ooo(1.1772.17) x - serine 446 - 3000/2.17 . 0.88 The calculated value of d3-serine/serine (0.88) is still higher than the observed value of d2-sphinganine/sphinganine (0.6755). This difference is ascribed to an isotope effect. The calculated value for endogenous serine (446 pg) might not represent the exact amount of endogenous serine because the calculation was based on the assump- tion that there was no isotope effect, which is not true. Correction for the isotope effect should give a lower value for endogenous serine than 446 pg. Accordingly, the ratio of d2-sphinganine/sphinganine should be higher than 0.88. It was therefore concluded that serine was incorporated into sphinganine faster than da-serine into d2-sphin- ganine. 105 Table 16. Determination of the Amount of Sphinganine and [1,1-2H2]- Sphinganine Formed in the Microsomal Reaction Incubated with [2,5,5-2H3]Serine/Serine Mixture. .QAS 159/157 Determination Reference 0.5 mg Mixture 5.0 mg Mixture 1 0.0608 0.5225 0.7550 2 0.0594 0.5559 0.7585 5 0.0601 0.5285 0.7557 4 0.0586 0.5610 0.7147 Average 0.0597 0.5564 0.7550 [y - 0.2767 0.6755 * is the difference between sample and reference values. 9/3 157, ‘cm-cng- 011481; I 2/2 159, 'cn-cve-onasi. l ‘fNHPAC l TMSi 104 L. Incorporation of [2,5,5-2H3]Serine into Sphingolipid Bases by Yeast. It was demonstrated in the previous section that serine was transformed into sphinganine with the complete loss of the Kehydro- gen atom by rat liver microsomes. The yeast, Hansenulg ciferri, was shown by Stodola and Wickerham (1960) to produce a relatively large amount of acetylated 4-hydroxysphinganine and a minor amount of acety- lated sphinganine (Stodola £5 31., 1962). To study whether this strain of yeast synthesizes sphingolipid bases by the same mechanism as that demonstrated in the mammalial system, [2,5,5-2H3]serine (0.5% weight/- volume) was incubate with viable yeast in LM-5 in the presence of 1% glucose at 26-28OC for 48 hours. Sphingolipid bases from the medium and cell paste were combined, partially deacetylated and trimethyl- silylated as described in Methods. The mass spectra of TMSi—N-acetyl- sphinganine and TMSi-N-acetyl-4-hydroxysphinganine are shown in Figs. 20A and 203, respectively. The spectrum of TMSi-N-acetylsphinganine from yeast grown in the presence of [2,5,5-2H3]serine was identical to that of TMSi-N-acetylsphinganine from rat liver microsomes in- cubated with [2,5,5-2H3]serine (Fig. 18), suggesting that the yeast, .§.ciferri, svnthesizes sphinganine by the same mechanism as the rat microsomal system. The spectrum of TMSi-N-acety1-4-hydroxysphinganine (Fig. 208) is consistent with I},1-2H2]-4-hydroxysphinganine derivative. The ion at M915 was shifted from mflg 560, in the protium form (Fig. 4), to'g[g 562, indicating the presence of two deuterium atoms in the molecule. Loss of fragments 2,12 and a are accompanied by loss of both 105 deuterium atoms, thus both deuterium atoms must be on C-l. This is consistent with the finding that sphinganine isolated from the same source also contained two deuteriums on C-l. 106 .ocauwmmmmmumxmxmg mo oucomoua msu cw caouw umumh scum voumHOmw ABOuuomv ocflcmwcflzamszHoxzazuamuouwuz-wmZH cam Aaoav ocwcwwcfinamazuoouuzuwmzfi mo muuooam mum: .om .me 107 $8 9%. com one 00¢ 0mm 08 omm com own 02 om .. L-»L1r.[lrLLLIr¥LLlfrrr . a _ 8m _ . «on .9... _ u - om M B. .5 mam 3N fl. A Eb 3.2 To.» _ - ESL. o- w a 04.12 O 0mm2m. n mom m 5.258410418an «10:8 m 1 o. u " om . 18.1.9.8 m 4 . 6158 Q. .2 4 - 02 ..-.L , .nmm . E Lomm L em.» 84 omm com omm sow one 09 om . s? can 48 M 6.-.): 3-3 ... mVN TO." m $.32 .8 ._ 04.12 055:. H ... . ... N m an. Q. @240 8 1041i 6:8 - u A. . 3 2. Tom ‘ 0L8» m 218 mm. " at 1,02 (94.) may woes DISCUSSION The yeast, Hansenula ciferri, was found by Wickerham and Stodola (1960) to accumulate a relatively large amount of acetylated base in the medium. The acetylated base can be extracted without prior breaking of the cell and need not be subjected to extensive purification. There were important reasons for the choice of this strain of yeast for extensive studies of the metabolism of 4-hydroxy- sphinganine (Green £5 31., 1965; Braun and Snell, 1967; Thorpe and Sweeley, 1967; Stoffel-25.21., 1968b). Experiments on yeast grown in the presence of [1,1,5-2H3]sphinganine indicated that 4-hydroxy- sphinganine, from the medium and cell paste, consisted of 11.77% trideuterated species, 6.7% dideuterated species and the remainder was non-deuterated species. The presence of trideuterated species suggested that sphinganine can serve as a direct precursor of 4-hydroxy- sphinganine. However, the presence of dideuterated 4-hydroxysphinganine is of interest since this species probably results from loss of a deu- terium atom during the conversion of trideuterosphinganine to 4-hydroxy- sphinganine. Loss of one of the two deuteriums on C-l is unlikely because the experiment on yeast grown on trideuteroserine did not in- dicate any loss of deuterium from that position (Fig. 20). Loss of deuterium on C-5 by degradation and reutilization of the degradation products is also unlikely because this process would result in the loss 108 109 of all three deuterium atoms instead of only that on C-5. One of the de- gradation product would be ethanolamine containing two deuterium atoms; it is not a precursor in the synthesis of sphingolipid bases (Sprinson and Coulon, 1954). It is, therefore, concluded that dideutero-4- hydroxysphinganine must have been synthesized by oxidation of sphin- ganine to 5-ketosphinganine followed by hydroxylation and reduction to dideutero-4-hydroxysphinganine. It therefore appears that 4-hydroxy- sphinganine can be derived by hydroxylation of both sphinganine and 5-ketosphinganine. The data presented in Table 2 indicate that sphin- ganine is converted to 4-hydroxysphinganine much faster than 5-keto- sphinganine; however, these do not represent physiological conditions. Addition of trideuterosphinganine into the growth medium made it much more readily available to the enzyme than 5-ketosphinganine, which must have been present in negligible concentration at the time of addition. Under physiological conditions, on the other hand, 5-keto- sphinganine is the first biosynthetic product and its rate of conver- sion to 4-hydroxysphinganine might be significantly greater than that of sphinganine. The nature of the hydroxylase is still obscure. The two most likely oxygen donors, water and molecular oxygen, have been ruled out as the primary source of the hydroxyl group on C-4 of the 4-hydroxy- sphinganine (Thorpe and Sweeley, 1967). Condensation of 2-hydroxy- palmitoyl CoA and serine seems also to be unlikely (Green'g£.gl., 1965; Braun and Snell, 1968). Another possible oxygen donor is one of a 110 variety of oxygen-containing compounds (Hayaishi, 1969). Some pre- liminary experiments were carried out to test whether oxygen was transferred intramolecularly, from either C-1 or C-5 to C-4, as shown below. 5,4-Epoxysphinganine (46) was listed as one of the possible intermediates of 4-hydroxysphinganine by Thorpe (1968). Attack of water on C-5 would result in transfer of oxygen from C-5 to C-4. Another cyclic ether is anhydro-4-hydroxysphinganine (44). This cyclic ether has been detected in acid hydrolysates during isolation of 4-hydroxysphinganine (Carter.g£.§1., 1954; O'Connel and Tsien, 1959). Attack of water on C-1 of this ether (44) would result in transfer of oxygen to C—4. Using sphinganine labeled with 180 on H gnfi curb-H : on, 313.50 / \CH' N112 + 180 / KCH- N112 R—CH— - 0H R—CH-CH- OH | 44 {‘3 (45) ( ) 18011 on N112 l R-CH—CH-CH-CH20H (47) . £11. 3.1 18 H12? 1:32 /0 “2 a—cu—ca—ca—cuaoa + R—cu-ca—ca—cngoa I H\ 11-0 A (45) )2 (‘16) 111 C-1 or C-5) to evaluate these possibilities has certain disadvantages. The compounds are difficult to synthesize and incorporation of sphin- ganine into 4-hydroxysphinganine is low.[l-180]Palmitate and[5-180]' serine were therefore used for the studies. These two compounds are converted to sphinganine with 180 on C-1 and C-5, respectively. 4- Hydroxysphinganine isolated from yeast grown in the presence of [1-180]' pahmitate and [5-180]serine was found to contain 180 on the expected positions but negligible amounts of the isotope were found on C-4. Thus it was concluded that[1-lao]pa1mitate, [5-180]serine, [5-130]' sphinganine and [1-180]sphinganine were not oxygen donors to the hydroxyl group on C-4 of 4-hydroxysphinganine. Experiments carried out in LMsl (containing 0.5% yeast extract, 0.5% malt extract, 0.5% peptone and 1.0% glucose) in HéBO indicated that incorporation of 180 from water into C-4 of 4-hydroxysphinganine was about 16%, confirming the experiment of Thorpe and Sweeley (1967) and indicating that water is not the primary source of the oxygen donor. The donor must be present in the medium and its oxygen must not be easily exchanged with that of water in the medium. Carbon dioxide, which can easily exchange its oxygen with water, was ruled out as the possible donor (Thorpe, 1968). Yeast which was grown on [2-1803- glucose as the principal carbon source failed to incorporate 180 into C-4 of 4-hydroxysphinganine. Glycolysis of [2-180Jg1ucose would yield several products which bear 180 in the molecule, as outlined in Fig. 21. Weaknesses in this experiment were that exchange of 180 with water in the medium might have caused a partial loss of isotOpe on C-2 of fructose-l-diphosphate, 112 CH0 CH0 0112011 I cal—80H (lull—9011 c1=80 I l Ho-CH 110- CH Ho- CH CH-OH CH-OH c|:H-0H I | CH- 0H CH- 0H _ 011- on _ I | - | 9 _ CH20H one-o-y-o CI-Ig-O-P-O 0 ‘ 0 (48) (49) (50) 9’ - 9' .. CHE-O-P-O GHQ-o-g-o I 0 I 031.90 01.80 | I (52) 9 _ HO" (In! CHQOH C02 - 2' 0 I 0 CH-OH ———> H ——> CH-l—BOH _ ———’> I CH0 | 9 _ CHPOH _ | GHQ-O-P-O I 9 _ CH-OH _ o GHQ-o-g-o I 9 o GHQ-O-P-O (5h) 0 (51) (55) COZH C02}I _ C0211 I I 9 - | 9' - CHE—80H ____, CH-l—BO-lf-O ___.{ CEO—g-o l 9 _ I 0 II one-o-g-o C1120}! CH2 (55) (56) (57) Fig. 21. Glycolysis of [2-180]g1ucose (enzymes and cofactors were omitted). 115 fructose-6-phosphate and dihydroxyacetone phosphate, and complete loss of 180 of fructose-1,6-diphosphate to the medium might have happened during transformation to two moles of triose phosphate (Heron and Caprioli, 1975). If one of the [2-180]g1ucose metabolites listed in Fig. 21 was the oxygen donor and negative incorporation into C-4 of 4-hydroxysphinganine was due to complete or partial loss of isotope in one of the aforementioned steps, however, it would be ex- pected that incorporation of 180 from H580 into this hydroxyl group would be high. Thus it seems to be possible to rule out all of the [2-180]g1ucose metabolites outlined in Fig 21 as oxygen donors. Experiments on yeast grown on ethanol as the principal carbon source indicated no increase in the incorporation of 180 from water into the hydroxyl group on C-4 of 4-hydroxysphinganine. Malt extract and peptone were omitted in this study. The only organic substances in the medium were yeast extract and ethanol. It was tentatively concluded that glucose was not the oxygen donor. In gluconeogenesis from ethanol, yeast must oxidise ethanol to acetate which exchanges its oxygen with water in the medium. Therefore, most of the oxygen in glucose should be labelled. Glucose isolated from the growth medium and cell paste was characterized by GLCsMS as methoxime-TMSi deriva- tive (Fig. 8B) and 180 on C-2 was analysed by GLC-AVA (Table 11). About 68% of the oxygen on C-2 was shown to be derived from water in the medium. The increase in the intensity of various ions, 9!; 521, 219 and 207, in the spectrum of glucose methoxime-TMSi (Fig.8B) indi- cated that assimulation of 180 into these ions occurred to a certain 114 extent. Since peptone and malt extract were omitted in this experiment, it was logical to conclude that the oxygen donor is in the yeast extract. Inorganic phosphate and sulfate oxygen are exchangable with water, particularly at high temperature (Hayaishi, 1969); they were also ruled out, therefore, as the oxygen donor. This was in good agreement with the experiment of Thorpe (1968) that inor- ganic 180 phosphate was not the oxygen donor of 4-hydroxysphinganine. Experiments on yeast grown in autoclaved medium (LM92) did not indicate any increase in the incorporation of 180 from.water into the hydroxyl group on C-4 of 4-hydroxysphinganine, suggesting that the donor is heat-stable (not destroyed and no exchange of its oxygen at elevated temperature). Thus, carboxyl oxygen and carbonyl oxygen which can exchange their oxygen with water, especially at elevated temperature, might be ruled out as the possible oxygen donors. The incorporation of water into the hydroxyl group on C-4 of 4-hydroxysphinganine is not completely negative, however, as found by both Thorpe and Sweeley (1967) and myself. About 11-17% incorpo- ration was observed in most cases, suggesting that the oxygen donor can be synthesized by yeast and that the biosynthetic sequence must proceed through at least one intermediate which can partially exchange its oxygen with that of water or else incorporates water into the mole- cule. Although this problem was not pursued further, it is important to reemphasize Thorpe's comments (1968) on the failure to obtain an active cell-free system for 4-hydroxysphinganine synthesis, that there might be an unidentified oxygen donor absent in the in vitro system. 115 If the comments were correct, adding yeast extract should enable one to obtain an active cell-free system for 4-hydroxysphinganine biosynthesis. It is generally accepted that the initial step in the biosyn- thesis of sphingolipid bases involves PLP-dependent condensation of serine and palmitoyl CoA. 5-Ketosphinganine is formed with the con- comitant release of C02 and CoA. 0 O I II €15H31‘C'COA + Serine Y... c, 5H31-C- CH- CH20H I co2 + CoASH ""2 Activation of serine involves the formation of Schiff's base with PLP (Braun and Snell, 1968). Studies in a nonenzymatic system, by Metzler gg‘al. (1954), suggested that the strong electron with- drawal effect of the nitrogen atom in the heterocyclic ring of PLP is responsible for electron displacement from the bond to the u>carbon atom in the amino acid, thus giving a conjugated system of double bonds extending from the electron attraction group to the site of reaction, as shown below. IiI R COZH Rifizrcozfl 'C' N' IN C/ 4.1 +@ (58) III (59) in -‘“’z{’r/ é} 116 This general mechanism accounted satisfactorily for all of the known nonenzymatic PLP-catalysed reactions of amino acids and also the corresponding enzymatic reactions that were catalysed by PLP- dependent enzymes (Snell, 1958). Thus PLP-dependent condensation of serine and palmitoyl CoA may be approximately represented in Fig. 22. Loss of the deuterium atom on C-2 of 2,5,5-trideuteroserine was obser- ved both in rat liver microsomes and whole cells of yeast. Nonenzyma- tic loss of a deuterium atom on the x-position of a carbonyl group at the 5-ketosphinganine or its PLP Schiff's base II (Fig. 5) is pos- sible but it is unlikely that this phenomenon will account for the complete loss of the isotope in 1 hour at 57°C and pH 7.5. Loss of tritium (28) on the C-2 of glycine due to exchange with protons in the medium during conversion to 5-aminolevulinic acid was reported to be somewhere between 15 and 25% (Akhtar and Jordan, 1968). The bio- synthesis of 5-aminolevu1inic acid from glycine and succinyl CoA (Fig. 26) is analogous to that of 5-ketosphinganine from serine and palmitoyl CoA, thus loss of isotope on C-2 of serine due to exchange should be comparable to that of [28-3Hngycine. If loss of deuterium on C-2 of serine was due to exchange with a proton in the medium at the level of 5-ketosphinganine, both sides of the carbonyl group would be expected to be approximately equally exchanged. Experiments carried out in deuterium oxide indicated that only one deuterium was incorporated, into C-2 of sphinganine. A very small amount of deuterium was detected on C-5 to C-18, but it repre- sented a negligible amount compared to that on C-2 (Fig.19). It is 117 CODCIUdEd: therefore, that loss of deuterium on C-2 of serine is an obligatory step in 3-keto3phinganine biosynthesis. This observation is consistent with the report of DiMari.g§.gl. (1971) that decarbo- xylation of serine in sphingolipid base biosynthesis is palmitoyl CoA dependent. Loss of deuterium on C-2 of serine due to rapid equilibrium of complexes I and III (Fig. 3) on the enzyme surface while the actual sequence of 3-ketosphinganine synthesis goes via complexes II and V is unlikely because such a process requires reorientation of groups on the enzyme surface. Reorientation of groups on the enzyme surface is unlikely since the specificity of PLP-catalysed enzymatic reaction is achieved by binding two of the three groups (M-atom is already co- valently bonded to PLP) bonded to thecxrcarbon atom of amino acid (Snell, 1958). Free rotation around the qt-N bond is then limited to a single orientation in which the bond to be broken lies in a plane perpendicular to that of the conjugated pi system of the heterocyclic ring (Dunathan, 1966). The rate of utilization of 2,3,3-trideuteroserine is slower than that of serine. This isotOpe effect suggests that breaking the H-C bond is probably the rate limiting step (Gould, 1959), unless this selective effect was due to secondary isotOpe effect (Richards, 1970) or to the fact that the two bulky deuterium atoms on C-3 of trideutero- serine cause a steric effect in the enzyme-substrate complex. Whether the aldimine complex (60 in Fig. 22) loses the hydrogen atom before or simultaneously with condensation with palmitoyl CoA cannot be established from the data presented in this thesis. The 118 H _ 0=C(CH2)1L,CH3 V 020\ ,CH20H Y Ozc‘f‘CHZOH fi * 020—p-CH20H N N\ N\\ \\CH CH CH II \ \ */ | .. I | + If N/ H (60) 1% (ffizhtr . =C C 0H CH H \ / H2 ,3 v '6" (052)14 -C'3-CH20H N N \ \\ CH CH ll H \ Eh .. I 'fi I ___...... §/ ——a- CH3(CH2)1nC-i-CIEOH I I NHZ H H (66) (65) (64) Fig. 22. Probable mechanisms of 5-ketosphinganine biosynthesis. 119 latter possibility requires that the incoming (palmitoyl) group approaches from the opposite side of the carbon bearing the (hydrogen) atom, and the reaction would proceed with inversion of configuration. This type of mechanism is contradictory to most of the known examples of stereospecificity of PLP-dependent enzymatic reactions, shown in Table 17. The mechanism of 5-ketosphinganine synthesis proposed in Fig. 22 involves two electrophilic substitutions, by the palmitoyl group for hydrogen and by a proton for the carboxyl group. Each of them may proceed either with retention or inversion of configuration. There- fore, there are four possible sequences: 1) both steps proceed with retention of configuration, 2) both steps proceed with inversion of configuration, 5) the first step proceeds with retention of confi- guration and is followed by inversion of configuration and 4) the first step proceeds with inversion of configuration and is followed by retention of configuration. Since the configuration of the serine substrate and the 5-ketosphinganine product are known (Fig. 25), the consistency of these four reaction sequences can be evaluated. The first two reaction sequences can be ruled out since they lead to the wrong stereoisomer of 5-ketosphinganine. Isolation and characteriza- tion of one of the intermediates (62) or its hydrolytic product (65) will give a straight-forward decision about the remaining possibili- ties. However, in a recent review on the stereochemical aspects of PLP catalysis, Dunathan (1971) noted that "results with a broad spectrum of PLP enzymes reinforce the generalization of 'one-side' chemistry in which all bond making and breaking takes place on one 120 Table 17. Some Examples of PLP-Dependent Enzymatic Reactions which Are Accompanied by Retention of Configuration. Substrates Enzymes Products :g-Serine Serine hydroxymethylase S[2-3H]Glycinel in 3H20 ‘L-Threonine Serine hydroxymethylase S[2-3H]G1ycine1 in 3H20 Aminomalonate Aspartate-fi-decarboxylase S [2-311]G1ycine2 in 3H20 :g-Tyrosine Tyrosine decarboxylase R[l-2H]Tyramine3 [2-2HJTyrosine Glycine RS[2-3H]G1ycine Sphinganine-1- phosphate in 2H20 Tyrosine decarboxylase in H20 Serine hydroxymethylase in 3H20 Serine hydroxymethylase in H20 Sphinganine-1fphosphate lyase in 3H20 S[1-2H]Tyramine3 S[2-3H]G1ycine4 R[_2-3H]Clyc1ne4 R[2-3H]Ethanolamine- 1-phosphateS 1, Jordan and Akhtar (1970); 2, Palekar t (1970 and 1971) 5, Belleau and Burba (1960); 4, Akhtar and Jordan (1969); 5, Akino as 21,- (1974). 121 COZH :1 -020Q NHZ H2NI>4?—- H>C9""H MHZ-- Cane-H —--NH2-—~9-— H—fiu-C-«NHZ I ' I 3 o : - o E CH2 0H CHZOH CHZOH (67) (74) (77) 7‘ °=<= 9 NH. " 2S-5-Ketosphinganine c3203 (70) Fig. 24b. Fig. 24. Possible reaction sequences of 5-ketosphiganine biosynthesis. 124 side of the cofactor-substrate imine." Some of the PLP-dependent enzymatic reactions which proceed with retention of configuration were listed in Table 17. If 5-ketosphinganine synthetase falls into one of the above generalizations of one-side chemistry, replacement of the palmitoyl group for a hydrogen atom will proceed with retention of configuration and intermediate (62) or (65) will have the S configu- ration. Intermediate (62) may be decarboxylated with or without a decarboxylase. In the presence of a decarboxylase the reaction would be expected to proceed with retention of configuration and lead to the improper stereoismer of 5-ketosphinganine. Thus participation of a decarboxylase is not favored by this analysis. The unstable nature of 2-amino-5—ketoacids are, however, well documented. Laver'_£._1. (1959) reported that 2-amino-5-keto-butyric and -adipic acids undergo decarboxylation with a half-life of less than one minute at pH 7.0. PLP is also known to increase the rate of decarboxylation of “Eamino acids (Metzler 25.51., 1954). The reinforcement of the electron with- drawal effect of PLP would, therefore, be expected to accelerate the rate of decarboxylation of intermediate (62) to an extent that a decarboxylase may not be necessary. The reaction would possibly yield a planar intermediate (65) with the palmitoyl group occupying that where the carboxyl group was located. Addition of a proton from the less hindered side would give 5-ketosphinganine after hydrolysis. This type of mechanism would fulfill the stereochemical requirements of the 5-ketosphinganine product as well as the 'one-side' chemistry of the PLP enzyme. However, in the absence of more definitive evi- dence, the fourth reaction sequence (Fig. 24b) and decarboxylation of 125 intermediate (65) cannot be ruled out. The serine-PLP Schiff's base (60) may have several conformations due to 1), free rotation around the qu bond; 2),.gig and trans con- figuration of the Ci=N double bond; and 5), free rotation around the Ci-C4 bond of the cofactor. However, some of these factors have been limited to only a single variable. For example, the Cé=N double bond has been defined as trans since the‘gig geometry results in serious steric hindrance to planarity (Dunathan, 1971); free rotation around the C4-CL bond of the cofactor has been restricted by metal chelation (Metzler 25 al., 1954) or hydrogen bonding (Metzler, 1957) between the phenolic oxygen and nitrogen of the substrate. Free rotation around the GEN bond has been postulated to be limited by binding two of the three groups surrounding the ubcarbon atom (Snell, 1958; Dunathan, 1966). The aforementioned factors limit the several variable conformations of serine-PLP Schiff's base complex to two possible conformations, shown in Fig. 25. Since the hydrogen on C-2 of serine is first labili- zed by the enzyme, 5-ketosphinganine synthetase, the qu bond should lie in a plane perpendicular to the plane of the heterocyclic ring, either on the.£g;gi face (78) (Hanson, 1966) or gi;£g face (79) of the u-CL—C4 plans. It is interesting to point out that in all the five cases where absolute stereochemistry is known: glutamic-aspartic transaminase (Besmer and Arigoni, 1969), pyridoxine-pyruvate transaminase (Ayling t al., 1968), dialkyl amino acid transaminase (Bailey gg‘gl., 1970) glutamic decarboxylase (Sukhareva 25.21., 1971; Voet £5 21., 1973), 126 cowuamamuu on» a“ omen a_mwanom 898.50.--- omom momma - ...o ...-umom — m .mudum oowuoeumam mo mcofiumsuomcoo manmnoum .mm .wwm » m 127 the conformation is similar to that of (79) with the'gi;£g face of the H=C4-C4 being exposed to the medium. The biosynthesis of 5-aminolevu1inic acid from glycine and suc- cinyl CoA (Akhtar and Jordan, 1968 and 1969; Jordan and Shermin, 1972) is analogous to that of 5-ketosphinganine from serine and palmitoyl CoA. Activation of glycine was reported by Akhtar and Jordan (1968) to be accomplished through the loss of hydrogen on C-2 of glycine. Interestingly, the Egg-R hydrogen which is removed by S-aminolevulinic acid synthetase has the same configuration as the H-atom on C-2 of ;- serine. 902H H2NtvC-‘HR (Glycine) HS (80) It was predicted (Dunathan, 1971) that condensation of glycine- PLP complex (81) and succinyl CoA should proceed with retention of con- figuration and that the product (85) should have the R configuration. This is analogous to the proposed mechanism of 5-ketosphinganine syn- thesis. As discussed earlier, the first substitution (palmitoyl group for the hydrogen atom) would lead to a retention of configuration. 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