Biosynthesis of L -fucose by mammalian tissue

376

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Of all the possible fl-keto-hexonic acids, only fi-keto<-idonate would be expected to give rise to this pair of epimers. Under identical conditions, fi-keto-L-gulonate was shown to form L-gulono- and L-galactonolactone.

National Institutes o/Health, Bethesda, Md. (U.S.A.)

JULIAN GILBERT

1 2 a 4

j. j. j. p. Z. 6 Q. 7 F. s E.

KANFER ASHWELL

1). SMILEY AND Cr. ASHWELL, J. Biol. Chem., 236 (1961) 357. WINKELMAN AND G. ASHWELL, Biochim. Biophys. Acta, 52 (1961) 17o. KANFER, G. ASHWELL AND J. J. BURNS, J. Biol. Chem., 235 (196o) 2518. C. CHAR, R. R. BECKER AND C. G. KING, J. Biol. Chem., 231 (1958) 231. DlSCHE .~ND E. BORENFRI~ZUND, J. Biol. C]'~em., 192 (1951) 583 . ASHW'ELL, A. J. W&HBA AND J. HICKMAN, J . Biol. Chem., 235 (~96o) 1559. LIPMANN AND L. C. TUTTLE, jr. Biol. Chem., 159 (I945) 21. F. L. J. ANET AND T. M. REYNOLDS, Natu'/e, 174 (1954) 93 o.

Received June 26th, 1961 Biochim. Biophys. Acta, 54 (1961) 3 7 3 - 3 7 6

Biosynthesis of L-fucose by mammalian tissue The pathway of L-fucose biosynthesis in Aerobacter aerogenes (ATCC 12657) has been partially delineated by the demonstration that an enzyme preparation from this organism converts guanosine diphosphate D-mannose to guanosine diphosphate L-fucose1. This conversion, which requires T P N H and is stimulated by DPN+, appears to involve a GDP-4-keto-6-deoxyhexose intermediate s. The identification of GDP-Man and GDP-fucose in milk and mammary gland of a number of animals 3-5 suggested that a similar system might be responsible for the biosynthesis of L-fucose in mammalian tissue. The data of the present communication indicate t h a t this is the case. A I : I (w/v) homogenate of rabbit lung was prepared in 0.05 M Tris buffer (pH 8.0) and centrifuged at 25000 ~< g for 30 min. The precipitate was discarded and the supernatant solution recentrifuged for 75 rain at ILOOOO x g. To the resulting clear solution I ° o protamine sulfate was added until no further precipitation of nucleic acid occurred. After centrifugation the precipitate was discarded and the :supernatant solution brought to 7 ° ,.o saturation with solid (NHa)2S Q . The precipitated protein was collected by centrifugation and redissolved in Tris buffer to a final protein concentration of 60 mg/ml as determined spectrophotometrically6. The entire procedure was carried out at o% This protein fraction is capable of forming GDP-fucose from GDP-Man as shown by the following experiment. A reaction mixture was prepared with the same components as described for the complete system in Table I. After incubation for 2 h at 37 ° the nueleotides were adsorbed on 4° mg of charcoal. The charcoal was collected by centrifugation and washed twice with 5-ml portions of o.ooi N HC1. The nucleotides were then eluted from the charcoal with 6 ml of 5 o ,o ethanol containing o.I(~i, conc. NH~OH. Paper chromatography of the nucleotides using 95ci,, ethanol-i M ammonium acetate (7.5 : 3.0 ; ref. 7), revealed the formation of an ultraviolet-absorbing compound with the mobility of GDP-fucose. This compound, which was not formed in a control incubation to which no GDP-Man had been added, was eluted from the paper and characterized as GDP-fucose by the following criteria: (I) The isolated Biochim. Biophys. Acta, 54 (I96a) 376 378

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377

1

FUCOSE FORMATION BY RABBIT-LUNG EXTRACT

T h e c o m p l e t e r e a c t i o n m i x t u r e c o n t a i n e d 0. 3 / ~ m o l e G D P - M a n , 0 . 2 / , m o l e D P N +, 0 . 2 / , m o l e T P N + , 4 / , m o l e s glucose 6 - p h o s p h a t e , lO t l m o l e s n i e o t i n a m i d e , a n d lZ m g of p r o t e i n in I m l of o . o 5 M Tris buffer ( p H 8.o). I n c u b a t i o n period w a s 2 h a t 37 °. The n u c l e o t i d e s were a d s o r b e d o n c h a r c o a l and t h e a m o u n t of 6 - d e o x y h e x o s e liberated into t h e s u p e r n a t a n t s o l u t i o n b y t r e a t m e n t of t h e c h a r c o a l w i t h o . o i N HC1 a t IOO ° f o r IO r a i n w a s d e t e r m i n e d c o l o r i m e t r i c a l l y 8. T h e s e c o n d i t i o n s h y d r o l y s e n u c l e o t i d e - b o u n d sugars. R e s u l t s are g i v e n after s u b t r a c t i o n of a z e r o - t i m e control. Syslem

Fucose formed (m/*molc)

Complete Minus G D P - M a n Minus D P N + Minus T P N +, g l u c o s e - 6 - P

34 o "2 t2

material had an ultraviolet absorption spectrum typical of a guanosine derivative. (Calculated as guanosine, o.o23/,mole was obtained.) (2) The compound exhibited the characteristic 4oo-m/, absorption peak when assayed for 6-deoxyhexose 8. The ratio of 6-deoxyhexose (calculated as fucose) to guanosine was o.9: I.O. (3) Treatment of the isolated nucleotide with o.oi N HC1 at IOO° for IO rain liberated a sugar and changed the chromatographic properties of the guanosine derivative from that of a fast-running compound to one which cochromatographed with GDP. The liberated sugar had the same mobility as fucose when chromatographed in pyridine-ethyl acetate-water (1:3.6:1.15; ref. 9), a solvent system in which fucose can be distinguished from the seven other 6-deoxyhexoses. In all experiments the maior portion of added GDP-Man was recovered unchanged. The greatest conversion of GDP-Man to GDP-fucose observed in 2 h under the conditions outlined above was approx. 3o %. The effect of cofactors on the conversion are shown in Table I. No fucose was formed in the absence of GDP-Man. DPN + and a TPNH-generating system (TPN +, glucose 6-phosphate) were both required for maximal activity. These requirements are similar to those obtained with the A. aeroge~,es system s. Table II lists the relative activities of extracts from different organs ot the rabbit. The extracts were prepared as described for the lung enzyme. While large intestine and lung were the most active of the tissues tested, all were capable of forming GDP-fucose from GDP-Man. It has been demonstrated in bacteria ~°-~2 and man ~a that glucose is converted TABLE

1I

FUCOSE FORMATION BY DIFFERENT ORGANS OF THE RABBIT

T h e c o m p o s i t i o n of t h e r e a c t i o n m i x t u r e s a n d t h e c o n d i t i o n s are the s a m e as described in T a b l e 1. T h e c o n t r o l s were i d e n t i c a l m i x t u r e s m i n u s G D P - M a n . Tissue Large intestine Lung Pancreas Salivary gland Small intestine Liver

Fucoseformed (ml~moles/mgprotein) ,3.8 7.6 3.9 2.3 1.9 I .o

Biochim. B i o p h y s . Acta, 54 (1961) 3 7 6 - 3 7 8

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to fucose with the carbon chain intact. A pathway for this conversion which is compatible with known reactions in bacteria and mammals would be: G l u c o s e ---* g l u c o s e - 6 - P --+ f r u c t o s e - 6 - P --+ m a n n o s e - 6 - P G D P - f u c o s e +-- G D P - M a n +-- m a n n o s e - l - P

GDP-fueose can presumably then function as a fucosyl donor in the synthesis of polysaecharides.

National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, t3ethesda, Md. (U.S.A.) 1 V. 2 V. a R. 4 R.

D. W. FOSTER V. GINSBURG

GINSBURG,J. Biol. Chem., 235 (196o) 2196. GINSBURG,J. Biol. Chem., 236 ( I 9 6 r ) 2389. DENAMUR,G. t:AUCONNEAU ANti G. GUNTZ,Compl. rend., 246 (1958) 2820.

DENAMUR, G. FAUCONNEAU AND G. JARRIGE-GUNTZ, A n n . Biol. anita. Biochem. Biophys,. (1961) 74. s D. M. CARLSON AND R. O. HANSEN, Federation Proc., 2o (1961) 84. 6 0 . WARBURG AND ~V. CHRISTIAN, Bioehem. Z,, 31o (1941 194z ) 384 . 7 A. C. PALADINI AND L. F. LELOIR, Biochem. J., 51 (I952) 426. Z. DlSCHE AND L. B. SHETTLES, J. Biol. Chem., 175 (I948) 595. "~ P. COLOMBO, D. CORBETTA, A. PIROTTA, G. RUFFINI, AND A. SARTORI, .]. Chromatog. 3 (196o) 343III J. I2. WILKINSON, Nature, 18o (1957) 995. 11 S. SEGAL ANn Y. J. TOPPER, Bioehim. Biophys. Acta, 25 (I9571 4 t 9 . 12 E. C. HEATH AND S. ROSEMAN, J. Biol. Chem., 23 ° (1958) 511. 18 S. SEGAL AND Y. J. TOPPER, BiocMm. Biophys. Acta, 42 (19(/o) 147. l

Received June 3oth, 1961 Biochim, Biophys. Acta, 54 (19611 3 7 6 - 3 7 8

Purification and some properties of a highly active inhibitor of trypsin and ~-chymotrypsin from soybeans The presence in soybeans of several trypsin inhibitors has been reported ( L A s K O W S K I AND LASKOWSKI1 and LIPKE et al.2), but only one inhibitor has been crystallized and studied in detail ( K u K r T Z 3, 4). The present study comprises an attempt further to purify a crude, acetone-insoluble trypsin inhibitor ( B O W M A N 5) and to compare its properties to those of crystalline soybean trypsin inhibitor. The behaviour of the two inhibitors towards dilute HC1, peptic digestion and heat treatments was studied and their chromatographic and paper-electrophoretic patterns were determined. The comparative effect of the inhibitors on trypsin, c~-chymotrypsin, pepsin and cysteine-activated papain were also examined. Proteolytic and inhibitor?, activity were generally determined by the casein-digestion method (Kt;Nrr#), but in the case of pepsin ANSON'S6 method was used. Inhibitor (0.5 ml) was preincubated with enzyme for 3° rain at 37 ° and the remaining enzymic activity was then determined. One unit of inhibition was expressed, according to K U N I T Z 4, a s the decrease of one unit absorbancy at 280 mt, in I min. The enzymes as well as the soybean inhibitor were commercial crystalline preparations obtained from Worthington Biochemical Corporation. Crude acetone-insoluble trypsin inhibitor was prepared from ether-extracted soybean flour (Lee var.) according to B O W M A N 5, excluding the treatment with Biochim. Biophys. Acta, 54 (t9611 3 7 8 - 3 8 1