ENZYMATIC SYNTHESJS O F ~-O-C?-D-GLUCOPYFL~NOSYL-D-GLUCOSE
Feb. 5, 1057
STRUCTURE OF THE PEPTIDES OBTAINEDFROM
TABLE I11 CHYMOTRYPTIC AND TRYPTIC HYDROLYSIS OF 8-MSH
THE
Peptide no. a
Enzyme
Ch-5
Chymotrypsin Trypsin Chymotrypsin Trypsin Chymotrypsin Trypsin Chymotrypsin
T-2 Ch-3 T-4 Ch-2 T-3 Ch-4
Complete amino acid sequence
625
Structure
Asp. Glu. G1y .Pro. T y r Asp.Glu.Gly.Pro.Tyr.Lys Lys.Met.Glu.His.Phe Met.Glu.His.Phe.Arg Arg.Try
Try.Gly.Ser.Pro.Pro.Lys.Asp G1y.Ser.Pro.Pro.Lys.Asp
Asp.Glu.Gly.Pro.Tyr.Lys.Met.Glu.His.Phe.Arg.Try.Gly.Ser.Pro,Pro.Lys.Asp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
See Figs. 1 and 2.
TABLE IV COMPARISON OF THE STRUCTURE OF 8-MSH WITH THE RELATED AREA OF P-MSH I
THE
CORTICOTROPINS
D
Corticotropins
corticotropins is well documented, 28.44 although the magnitude of the activation is open to some question. Inherently the corticotropins possess less than 1% of the melanocyte-stimulating activity of RISH, a fact which may be explained by the nature of the sequences adjacent to the heptapeptide core. Such sequences may be inhibitory, so that treatment of the corticotropins with alkali, by removing some of this inhibitory structure, may consequently potentiate the melanocyte-stimulating activity. Tn our experience, however, even when potentiation has occurred, the melanocytestimulating activity of the corticotropins is less than that found in MSH. This may be a result of the LysSer. interchanges pointed out above. I n other words, i t may be that the undecapeptide discussed above is necessary for full melanocytestimulating activity. Finally, the finding of a partial structure common to two different hormones is highly reminiscent of the findings in connection with oxytocin and vaso-
p r e s ~ i n . In ~ ~ the case of these latter hormones, two variations occur in a basic nonapeptide structure, and each hormone is endowed with some of the major activities characteristic of the other. Both of these hormones are produced by the hypothalamus and stored in the posterior pituitary. The corticotropins are produced by a group of the cells of the anterior lobe of the pituitary, whereas MSH is produced from an entirely different group of cells, those of the intermediate lobe of the pituitary. This lobe, however, is derived embryologically from the same anlage as the anterior lobe. A full discussion of the biological activity of P-MSH and the biological significance of the structure will be presented elsewhere. Acknowledgments.-This work has been supported in part by grants from the National Institutes of Health of the United States Public Health Service (Grant No. G-2907) and the Albert and Mary Lasker Foundation.
(44) E. B. Astwood, in “The Hormones,” Vol. 111, Ed. by G. Pincus and K. V. Thimann, Academic Press, Inc., New York, N. Y., 1955.
BERKELEY4, CALIF.
[CONTRIBUTION FROM
THE
(45) V. du Vigneaud, The Harvey Leclures, Series L, 1 (1956).
DEPARTMENT OF BIOCHEMISTRY AND NUTRITION, COLLEGEOF AGRICULTURE,UNIVERSITY OF NEBRASKA]
The Enzymatic Synthesis and Disproportionation of 3-0-CY-D-Glucopyranosyl-D-glucosel S 2 BY JOHN H. PAZUR,TANIA BUDOVICH AND CARLL. TIPTON RECEIVED JUNE 25, 1956 3 - ~ - ~ - ~ - ~ ~ u c o p y r a n o s y l - D - g(nigerose) ~ u c o s e is synthesiied during the action of the transferring enzyme of Aspergillus oryzae on glucose and maltose. It was characterized by its specific rotation, its paper chromatogram mobility and its crystalline phenylosazone derivative. Evidence from isotope studies indicates t h a t the compound is synthesized by a transfer of the glucosyl moiety of maltose t o the three position of cosubstrate glucose molecules. On further treatment of nigerose with the transferring enzyme, i t is disproportionated t o new glucosyl oligosaccharides.
The a - ~ - ( l + 3 ) glucosidic linkage is a character(1) Published with the approval of the Director as Paper NO. 762, Journal Series, Nebraska Agricultural Experiment Station. Supported in part by a grant from t h e National Science Foundation. (2) A preliminary account of a portion of this work has been publisbed in abstract form in Federation Proc.. 16, 325 (1956).
istic structural feature of a number of glucosyl polysaccharides and comprises approximately half of the glucosidic bonds in nigeran,3 a variable frat(3) S. A. Barker, E. J. Bourne and (1953).
M.
Stacey, J. Ckem. Soc., 3084
tion of the bonds of bacterial dextrans4 and a small The oligosaccharide possesses a free reducing group but significant fraction of the linkages of waxy reactive with several of the reducing sugar reagents. maize ~ t a r c h . ~Little ,~ information is available Paper chromatographic examination of partial and on the mechanism of biosynthesis of the WD- prolonged acid hydrolysates of the oligosaccharide (1-3) glucosidic linkage. A4number of years ago, revealed that glucose and unhydrolyzed compound tlie enzymatic synthesis of a glucosyl disaccharide were present in the former and glucose only in the ior which an CY-D-(~+?) structure was suggested latter. rkterinination of glucose in tlie hydrolLWS observed in digests of maltose and the transysates showed that the oligosaccharide \v;ts ferring enzyme of Aspergillus oryzize.7,s Slore re- quantitatively converted to glucose on hydrolysis. cently such a compound has been detected chro- SIelting point nnd X-ray diffraction patter11 (lata oi' niatographically in digests of nialtose with a vari- the crystalline phenylosazone of the enzymatically ety of other fungal enzymesg and with some yeast synthesized nigerose are in agreement with values enzymes. The oligosaccharide produced by the previously reported for the osazone of the c o n A.ovyzae enzyme has now been isolated in chroma- p o ~ n dand~ ~for~ the ~ osazone of turanose,' i ~ ' : tographically pure form and in sufficient amount which, of course, yields the same osazone as i i i for structural characterization. The compound gerose. is ~~-O-a-D-glUcOpyranOSyl-D-glUCOSe (nigerosej The nigerose which was synthesized in :t digest Nigerose on further treatment with the transfer- of glucose-C14 and maltose was found to be rxlioring enzyme is disproportionated to three reducing active. This result, together with the earlier oligosaccharides, one of which is a new glucosyl findings on the mechanism of enzyme action on in:tlttrisaccharide with an a-~-(l--t3) and an 01-11- 0se7 and on point to a two-step trms( 1 - 4 ) linkage. glucosylation mechanism for the synthesis of i i i It has been reported by Peat, et a1.,12and by gerose. The two-step mechanism is shown tlinjirairi Shibasaki13 that nigerose is produced in small matically in the accotnpanying equations. aiiiounts during the action of enzyme preparations (Step 1 ) G'--GI' iE g? G'.E + G fro111ilspergillus niger on concentrated solutions of (Step 2 ) G'.E - G* G'--G'* + I? glucose. I n our study, it was found that the A . The notations are as follows: G, glucose; G*, ouycae enzyme synthesized considerably more nigerose from a mixture of glucose and maltose than glucose-C1*; G', glucosyl unit; G " glucose unit from glucose alone. A transglucosylation reaction substituted a t position 4; G'*, C'Qlucose unit subi n which the glucosyl unit of maltose is transferred stituted a t position 3, and E, enzyme. Evidence to the three position of glucose is probably respon- for the formation of a glucosyl-enzyme complex sible for the increased synthesis. Evidence from (G'.E) in this sequence of reactions was obtained isotope experiments supports the transglucosyla- from the isotope experiments. From glucose-C I tion niechanism for enzyme action. In view of the and non-radioactive maltose the transferring e l l similarity in action patterns of the A . oryzue zyme produced three radioactive oligosaccharides, cnzyrne and of those enzymes responsible for the namely, maltose, isomaltose and nigerose. Since synthesis of glucosyl polysaccharides, transglu- under our experimental conditions little synthesis cosylation may also be the type of mechanism in- of oligosaccharides occurred from g1ucose-Cl4, a volved in the synthesis of the a - ~ - ( 1 + 3 ) linkages reversibility of step 1 of the reaction sequence is indicated. Further, the incorporation of free glu-i i i the glucosyl polysaccharides. In the isolation of 3-O-a-~-glucopyranosyl-~-cose-C14into maltose indicates a separate transitorq. glucose (nigerose), use was made of its non-fer- existence of a glucosyl-enzyme complex. I n anmaltoseiiientability by bakers' yeast and of its fast migra- other isotope experiment, i~omaltose-C'~, tion rate on paper chromatograms. From an en- CI4 and nigerose-C1 were produced by the erizynie zymatic digest of 10 g. of glucose and 10 g. of malt- from glucose-c'? and nigerose. X disproportionose, 0.2 g. of chromatographically pure oligosac- ation of the nigerose and the participation of glucose-C14 as a cosubstrate in the reactions is probcharide w;is obtained ; specific rotation, +SOo ably the mechanism of synthesis of labeled maltosc, (4) A . Jeanes, W. C . Haynes, C . A. Lvilharn, J . C. Rankin, I
