1106
BIOCHEMISTRY
FALST,
HEIM,
AVD
UOORE
Murine Myeloma Immunoglobulin Heavy-Chain mRNA. Isolation, Partial Purification, and Characterization of y l , y2a,7 2 b , y3, p, and a Heavy-Chain mRNAst Charles H. Faust, Jr.,* Isabel Heim, and Joan Moore
Seven mouse immunoglobulin heavy-chain mRNAs have been partially purified from several myeloma tumors. Each of the individual, partially purified yl, yza,yzb, y3, or cy heavy-chain m R N A migrated as a 1 7 s species on sucrose velocity sedimentation gradients. However, these heavy-chain mRNAs migrated slightly slower than 18s ribosomal RNA, by use of denaturing acrylamide or agarose gel analyses. Each different heavy-chain mRNA has a molecular weight of 750 000, equivalent to 2200 nucleotides. The partially purified p heavy-chain mRNA migrated as a 2 0 s species on sucrose gradients and ran significantly slower than 18s r R N A on denaturing formamide polyacrylamide gels, with a molecular weight equal to 950 000, or 2800 nucleotides. This R N A fraction also contained a small and distinctly separate 18s R N A moiety-not coding for detectable p heavy chain. The 1 7 s or 20s m R N A fractions coded for the cell-free syntheses of several detectable proteins i n the expected size range of the various authentic heavy chains. In all cases, only one size class of cell-free synthesized protein was serologically reactive with the respective monospecific antiserum. These immunoprecipitated proteins accounted for as much as two-thirds of the cell-free synthesized, [35S]methionine-labeled product. All in vitro generated y heavy chains have an apparent molecular weight of 52 000, as determined by denaturing sodium dodecyl sulfate slab gel analyses. This compares with an apparent molecular weight of 57 000 for the authentically secreted y 1heavy chain of MOPC-3 1 C and 52 000 for each of the other authentically secreted y,,yZdr yzb, and y3 heavy chains studied here, as determined in the same slab gel. The a heavy chain 1 7 s m R N A fraction coded for the i n vitro synthesis of only one major serologically reactive protein component with an estimated molecular weight of 56000. A minor one (99%) probe were required, this could only be achieved from H-chain m R N A prepared by either above method through limited back-hybridization with the appropriate c D N A or through the newly developed cloning procedures of recombinant D N A technology (Rougeon et al., 1975; Maniatis et al., 1976; Rabbits, 1976). At this point a caution must be issued. The untranslated protion of all subclasses of the y H-chain m R N A is 600-700 bases, exclusive of poly(A), and their function and location are presently unknown. Similarly, that of a and j i H-chain m R N A are about 500 and 800 bases, respectively. Therefore, all cDNA initiated conventionally with oligo(dT) will start a t the poly(A) tail on the 3' end of the H-chain mRNA and could contain from 500 to 800 of these untranslated bases before including any portion of the H-chain structural coding region. So, any cDNA probes used for the purpose of assessing CH gene function must be significantly longer than 500-800 bases to include at least part of the CH structural gene and essentially full length to ensure inclusion of the VH structural gene. Otherwise, it is conceivable in light of the new segmented gene data for both rabbit (Jefferies & Flavell, 1977) and mouse /3-globin (Tilghman et al., 1977), and more appropriately mouse X L chain (Tonegawa et al., 1978), that, while untranslated C H gene will be measured, C H structural gene segments will not. Finally, it must be pointed out mat certain questions concerning comparisons of these y H-chain subclasses cannot yet be answered. For example, while each y H-chain mature cytoplasmic mRNA and primary translational product are the same size, their gene templates or primary transcriptional products may not be equal. However, these questions could now be pursued with cloned H-chain probes in a manner already used for preliminary K L-chain studies (Gilmore-Hebert & Wall, 1978). After submission of this manuscript for publication, a report appeared describing the isolation, partial purification, and characterization of a YZb, j i , and CY H-chain m R N A from mouse myelomas (Marcu et al., 1978). Our results are a t variance with some of theirs. Firstly, we obtained similar degrees of m R N A purification with only sucrose gradient fractionation. Secondly, rapid isolation and extraction procedures were not essential, since some of our polysome preparations were stored a t -20 "C for over 2 years before extraction. The yields in both cases are about 0.1%. Thirdly, we find only one major serologically reactive cell-free product for all isotypes studied. Finally, there are significant differences in reported H-chain m R N A sizes. We report lengths for y H-chain m R N A (r,, yza,7 2 b , including unpublished data on MPC- 1 I , and y3),j i H-chain mRNA, and CY H-chain m R N A of 2200,2800, and 2200 bases, respectively. These results were obtained with three different denaturing gel systems. They are significantly larger than the values reported by Marcu et al. (1978) for only a Y2b H-chain mRNA, j i H-chain mRNA, and CY H-chain mRNA-1800, 2 150, and 1800 bases, respectively. These latter values must be viewed with reservation, since the y H chain (-440 amino acids) requires about 1600 bases total for presumed precursor, coding sequence and a maximum of 200 bases for the poly(A) sequence, leaving only about 200 unaccounted bases in their model. Similarly, the CY H chain (-475 amino acids) requires about 1700 bases, leaving only about 100 unaccounted bases, and most significantly, the j i H chain (-575 amino acids) requires about 2000 bases, leaving only about 150 unaccounted bases-all according to the models of Marcu et al. (1978). These models may be untenable, since they leave so little noncoding sequence [only
1118 B I O C H E M I S T R Y
5-IO%, excluding poly(A)] compared with what is known OF other m R N A in general. For example, the m R N A coding for cy- and @-globin(Gould & Hamlyn, 1973), Ig K and X L chain (Mach et al., 1973;Farace et al., 1976), and also ovalbumin, conalbumin, ovomucoid, and lysozyme (Buell et al., 1978) all have about 30-40% of their length as noncoding sequence. Our data for all the y H-chain m R N A subclasses, p H-chain mRNA, and cy H-chain mRNA are consistent with these other previous observations. Therefore, while all classes of H-chain mRNA contain the same percentage of coding sequences, they do not contain the same amount (number of bases) of noncoding sequences. Acknowledgments The authors thank Dr. W. S. Fletcher for his continued support and encouragement throughout this project and Dana McNaught for her expert assistance in the preparation of this manuscript. We also acknowledge the generous gift of avian myeloblastosis virus reverse transcriptase from Dr. J . Beard under the Virus Cancer Program. Supplementary Material Available Supplemental Figures 1-29 which show experimental detailed results for the y,,yIa,?2b, y3,cy, and p H-chain rnRNA showing sucrose gradient profiles, cell-free translational assays, acrylamide gel analyses of total cell-free synthesized protein products and those serologically reactive with their respective antiserum, tryptic peptide maps, and H-chain m R N A rnolecular weight determinations (29 pages). Ordering information is given on any current masthead page. References Adams, J. M., & Cory, S. (1975)Nature (London) 255, 28. Adetugbo, K., Poskus, E., Svasti, J., & Milstein, C. (1975) Eur. J . Biochem. 56, 503. Aviv, H., Packman, S., Swan, D., Ross, J., & Leder, P. (1973) Nature (London),New Biol. 241, 174. Bailey, J. M., & Davidson, N. (1976)Anal. Biochem. 70, 75. Bedard, D. L., & Huang, R. C. C. (1977)J . Biol. Chem. 252, 2592. Blomberg, B., Geckeler, W., & Weigert, M. ( 1 972) Science 177, 178. Bonner, W. A., & Laskey, R. A. (1974)Eur. J . Biochem. 46, 83. Brawerman, G . ( 1 974) Annu. Rec. Biochem. 43, 62 1. Brownlee, G. G., Cartwright, E. M., Cowan, N . J., Jarvis, J . M., & Milstein, C. (1973)Nature (London),New Biol. 244, 236. Buell, G. N., Wickens, M. P., Payvar, F., & Schirnke, R. T. ( 1978) J . Biol. Chem. 253, 247 1. Cooper, M.D., Lawton, A. R., & Kincade, P. W. ( I 972) Clin. Exp. Immunol. 11, 143. Cowan, N . J., & Milstein, C. (1 973) Eur. J . Biochem. 36, 1. Cowan, N . J., Secher, D. S., & Milstein, C. (1976)Eur. J . Biochem. 61, 355. Diggelmann, H., Faust, C. H., Jr., & Mach, B. (1973)Proc. Natl. Acad. Sci. U.S.A. 70, 693. Elder, J . H., Pickett, R. A,, Hampton, J., & Lerner, R. A. (1977) J . Bioi. Chem. 252, 6510. Farace, M. G., Aellen, M. F., Briand, P. A., Faust, C. H., Jr., Vassalli, P., & Mach, B. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 727. Faust, C. H., Jr., Diggelmann, H,, & Mach, B. (1973) Biochemistry 12, 925. Faust, C. H., Jr., Diggelmann, H., & Mach, B. (1 974) Proc.
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Natl. Acad. Sci. U.S.A. 71, 2491. Gilham, P. T. (1964)J. Am. Chem. SOC.86, 4982. Gilmore-Hebert, M., & Wall, R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 342. Gould, H . J., & Hamlyn, P. H. (1973)FEBS Lett. 30, 301. Green, M., Zehavi-Wilner, T., Graves, P. N., McInnes, J., & Pestka, S. (1976)Arch. Biochem. Biophys. 172, 74. Hirs, C . H.W. (1967) Methods Enzymol. 2, 198-199. Honjo, T., Packman, S., Swan, D.,Nau, M., & Leder, P. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3659. Honjo, T.,Packman, S., Swan, D., & Leder, P. (1976a) Biochemistry 15, 2780. Honjo, T., Swan, D., Nau, M., Norman, B., Packman, S., Polsky, F., & Leder, P. (1976b) Biochemistry 15, 2775. Jaffe, B. M.,Simms, E. S., & Eisen, H. N. (1971) Biochemistry 10, 1693. Jefferies, A. J., & Flavell, R. A. (1977)Cell 12, 1097. Jilka, R. L., & Pestka, S. (1977)Proc. Natl. Acad. Sci. U.S.A. 74, 5692. Kacian, D. L., & Myers, J. C. (1 976) Proc. Natl. Acad. Sei. U.S.A. 73, 3408. Kessler, S. W. (1975)J . Immunol. 115, 1617. Kochwa, S., Terry, W. D., Capra, J. D., & Yany, N. L. (1971) Ann. N . Y . Acad. Sci. 190, 49. Kohler, G., & Milstein, C. (1975)Nature (London) 256, 495. Laemmli, U. (1970)Nature (London) 227, 680. Lehrach, H . , Diamond, D., Wozney, J . M., & Boedtker, H. (1977)Biochemistry 16, 4723. Leslie, G . A,, Clem, L. W., & Rowe, D. (1971)Immunochemistry 8 , 565. Lingrel, J. B. (1972)Methods Mol. Biol. 2, 231-261. Lui, Y . 4 . V., Low. T. L. K., infante, A., & Putnam, F. W . (1976)Science 193, 1017. Mach, B., Faust, C., & Vassalli, P. (1973)Proc. Natl. Acad. Sci. U.S.A. 70, 451. Maniatis, T., Kee, S. G., Efstratiadis, A,, & Kafatos, F. C. (1976) Cell 8, 163. Marcu, K. B., Valbuena, O., & Perry, R. P. (1978) Biochemistry 17, 1723-1 733. McDonnell, M.W., Simon, M. N., & Studier, F. W. (1977) J . Mol. Biol. 110, 119. Milstein, C., Brownlee, G. G., Harrison, T. M., & Mathews, M. B. (1972)Nature (London), New Biol. 239, 117. Milstein, C. P., Richardson, h’. E., Deverson, E. V., & Feinstein, A. (1975)Biochem. J . 151, 615. Nossal, G. J. V., Szenberg, A , , Ada, G . L., & Austin, C. M. (1964) J . Exp. Med. 119, 485. Ono, M., Kondo, T., Kawakanii, M., & Honjo, T.(1977)J . Biochem. ( T o k y o ) 81, 949. Pelham, H.R. B., & Jackson, R. J. (1976)Eur. J . Biochem. 67, 247. Putnam, F. W., Florent, G., Paul, C., Shinoda, T., & Shimizu, A. (1973)Science 182, 287. Rabbits, R. H . (1976)Nature (London) 260, 221. Randerath, K . (1 970) Anal. Biochem. 34, 188. Rougeon, F., Kourilsky, P., & Mach, B. (1975)Nucleic Acids Res. 2, 2365. Spohr, G., Mirault, M. E., Irnaizumi. T., & Schemer. K . (1976) Eur. J . Biochem. 62, 313. Stavnezer, J., & Huang, R. C. C. (1971)Nature (London), New Biol. 230, 172. Staynov, D. Z., Pinder, J. C., & Gratzer, W. B. (1972)Nature (London), New Biol. 235, 108. Stevens, R. H., & Williamson, A. R. (1973)Proc. Natl. Acad. Sci. U.S.A. 70, 1127. Stevens, R. H., & Williamson, A. R. (1975)Proc. nlatl. Acad.
MICELLE CONCENTRATIONS OF GANGLIOSIDES
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Critical Micelle Concentrations of Gangliosidest Silvestro Formisano,$ Michael L. Johnson, George Lee, Salvatore M. Aloj,t and Harold Edelhoch*
ABSTRACT:The micellar properties of mixed, bovine gangliosides and purified galactosyl-N-acetylgalactosaminyl(Nacetylneuramin yl)galactosylglucosylceramide were studied by gel filtration, equilibrium dialysis, and band and boundary centrifugation in sucrose gradients. The dissociation of micelles is very slow (days) in water and required us to approach equilibrium by association of monomers rather than by the dissociation of micelles. The gangliosides were therefore first converted into very low molecular weight aggregates (1-3 molecules) by dissolving them in Me,SO. Galactosyl-Nacetylgalactosaminyl(N-acetylneuraminyl)galactosylgluco-
sylceramide was then diluted into aqueous sucrose gradients and sedimented by the boundary centrifugation technique. This gave a sedimenting micelle and a nonsedimenting monomer concentration of (1-2) X M (or less) which corresponds to the critical micelle concentration value. The mixed gangliosides revealed two micellar sizes (i.e., 10 and 4.5 S), the slower sedimenting species being formed from the larger one with time (days). The critical micelle concentration of the mixed gangliosides was found to be approximately M by gel filtration, equilibrium dialysis, and band centrifugation.
G a n g l i o s i d e s were originally described as acylsphingosyl oligosaccharides containing sialic acid (Klenk, 1942). It was later shown that higher order gangliosides (GMI,GD,,, GDlb, GT1, etc.)’ have a common basic structure, Le., N-acylsphingosineglucosegalactose-N-acetylgalactosaminegalactose, with one or more molecules of N-acetylneuraminic acid (Svennerholm, 1962). G M Zand G M 3are biosynthetic precursors with deletions at the terminal galactose and galactosaminegalactose residues, respectively. Gangliosides have been implicated in the binding or function of many biologically important molecules, Le., cholera toxin (Sattler et al., 1977), tetanus toxin (Helting et al., 1977), botulinum toxin (Haberman & Heller, 1975), thyrotropin (Mullin et al., 1976), human chorionic gonadotropin (Lee et al., 1976), luteinizing hormone (Lee et al., 1977), serotonin (Ochoa & Bangham, 1976), interferon (Vengris et a]., 1976), bilirubin (Kahan et al., 1968), and wheat germ agglutinin (Redwood & Polefka, 1976).
Table I: Literature Values for the cmc of Gangliosides cmc (M) ganglioside technique reference
From the Clinical Endocrinology Branch (S.F., M.L.J., and H.E.) and the Laboratory of Biochemical Pharmacology (G.L. and S.M.A.), National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, Bethesda, Maryland 20014. Received July 27, 1978. ‘Present address: Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R., Naples, Italy. Abbreviations used: cmc, critical micelle concentration; Me2S0, dimethyl sulfoxide; ganglioside nomenclature is according to Svennerholm ( 1964)-G,,, galactosyl-N-acetylgalactosaminyl(N-acety~neuraminyl)galactosylglucosylceramide; G,,,,, N-acetylneuraminylgalactosylN-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylcerimide; G D l b , galactosyl-N-acetylgalactosaminyl(~-acetylneuraminyl-~acetylneuraminyl)galactosylglucosylcerimide; G T I , N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl(N-acetylneuraminyl-~acetylneuraminyl)galactosylglucosylceramide; GM,, N-acetylgalactosaminyl(N-acetylneuraminyl)galactosylglucosylcerimide; GM), N acetylneuraminylgalactosylglucosylcerimide.
1X 1 X 10” 1X 7.5 X lo-’ 8.5 X lo-’ 9.5 X lo-’ 1X
mixed
surface tension mixed conductance GM, neuraminidase G M ~ triiodide method G M ~ triiodide method G D , ~ triiodide method triiodide GT, method
Gammack (1963) Howard & Burton (1964) Rauvala (1976) Yohe & Rosenberg (1972) Yohe & Rosenberg (1972) Yohe & Rosenberg (1972) Yohe & Rosenberg(l972)
The interpretation of studies involving gangliosides depends on a knowledge of their state of aggregation since the observed biological effects occur, in many cases, in the concentration range of the reported cmc values, i.e., 10-4-10-5 M (see Table I). Unfortunately, none of the cmc values was obtained by use of a technique which directly measured either the size or the molecular weight of the gangliosides. It has also been observed that at concentrations below these values purified preparations of G M land GM2were unable to cross a dialysis membrane (Kanfer & Spielvogel, 1973; Sattler et al., 1977). In an attempt to resolve this apparent contradiction, we undertook a study of the molecular behavior of typical gangliosides. Materials and Methods Mixed, bovine brain gangliosides were purchased from ICN Pharmaceuticals. The composition was measured by thin-layer chromatography (TLC) on silica gel plates and is shown in Figures 1 and 2 . Purified G M , was either purchased from Supelco or obtained by a purification of the mixed gangliosides
This article not subject to U S . Copyright. Published 1979 by the American Chemical Society
