E. M. Shooter ~ Some Aspects of Molecular Neurobiology (continued) presence of 8M urea, The y Subunits. The enzymatic activity of the 75 NGF complex resides in the y subunits. The three individual subunits, y', y? and y3, normaily iso- lated from fresh 7S NGF preparations all have the same specific activity. While the rate of hydrolysis of typical substrates by the y enzyme is linear from the time of addition to substrate, 7S NGF displays a lag phase before reaching maximal velocity. The extent of the lag phase is diminished by in- cubating the diluted 7S NGF solution by itself prior to addition of substrate, by high pH or high ionic strength. These are effects which would be anticipated provided that the incubation conditions produced a shift in the 7S NGF equil- ibria toward more subunits and also provided that the only enzymatically active species are y subunits which are dissociated from the 7S NGF complex. The lag phase would then reflect the time required to achieve the new dissociation equilibrium and produce the relevant concentration of "free" y subunits. Sup- port for this idea comes from the fact that the lag phase is restored if incu- bated dilute solutions are concentrated or the pH and ionic strength brought back into the range of 7S NGF stability. Also, addition of an excess of the enzymatically inactive subunits, a and 8, before dilution of 7S NGF into the assay system decreases the observed specific activity of the latter to about 10% of its value in the absence of those subunits. Since these are conditions which suppress dissociation of 7S NGF, they measure more accurately the in- trinsic specific activity of the 7S NGF complex. The latter is sufficiently low to suggest that the y subunit bound in the 7S complex is inactive. Sup- pression of the y activity requires interaction with both a and 8, either subunit alone having very little effect on the observed activity of the y subunits. These changes in enzymatic activity of the y subunit parallel changes in its physical properties on aggregation and suggest that the two are linked. In spite of the significant difference in net charge (or isoelectric points) which exist between the three individual y subunits, 7S species reformed from them by recombination with one given a subunit and the 8 subunit all have the same net charge, showing that the segments of the y subunits which differ are hidden in the recombination process. Whether this involves a conformational change in the y subunit is not yet known. The association of an esteropeptidase enzyme with a protein (the 8 sub- unit) which stimulates neuroblast differentiation is an intriguing one, espe- cially since enzymes of this type are themselves being implicated increasingly in processes to do with cellular growth and differentiation. Thus estero- peptidase activity is associated with the mesenchymal growth factor while thrombin, itself an enzyme of this type, has an NGF-like activity. Also the thymotropic factor present in extracts of mouse submaxillary gland, and which promotes differentiation of certain lymphocytes, is an esteropeptidase. Grossman, Lele, Sheldon, Schenkein and Levy have recently described the effects of other submaxillary esteropeptidases on the growth of cultured rat hepatoma cells. Of great interest is the recent report that the epidermal growth factor (EGF) can be isolated from mouse submaxillary glands as a 70,000 27. E, M. Shooter - Some Aspects of Molecular Neurobiology (continued) molecular weight complex containing two subunits. One of these subunits is relatively small and acidic and possesses EGF activity while the other is an esterase with properties very similar to those of the y subunits. The exact chemical and physiological relationship of these various enzymes to the y subunit of 7S NGF is unknown but is clearly of interest. It should also be noted that the y enzyme is a glycoprotein. The Metabolic Properties of Certain Synaptosomal Membranes. The methods for the extraction and electrophoretic analysis of at least 90% of mouse brain protein have been described by Grossfeld and Shooter. Using these techniques it has been determined that the half-lives of the proteins of various whole mouse brain fractions increase with increasing solubility; the supernatant and hypotonic extractable proteins had half-lives of about 13 days while the membrane proteins solubilized with Triton X-100 and SLS had half-lives of about 18 days. The proteins of the subfractions of synaptosomes had half-lives tanging from 15 to 19 days; those in the cytoplasm had a half-life of 18.3 days; in the membranes, about 17 days and in the synaptic vesicles, 15.6 days. Although the half-life of the synaptic vesicles was not significantly different from other synaptosomal subfractions, the vesicles gave a different protein pattern on acrylamide gels, which implies that the proteins of the vesicles are qualitatively different from those of other synaptic membranes. The data derived from the relative specific activities of synaptosomal fractions compared with their whole brain analogs supports the contention that a sizeable fraction of the synaptosomal cytoplasmic proteins is transported to the synapse by axo- plasmic flow. The relative specific activity of synaptosomal membrane and synaptic vesicle protein rises much more quickly than for the cytoplasmic material and the alternate possibility of in situ synthesis has to be considered. The Protéins of the Sarcoplasmic Vesicle Membrane. Because of its rela- tively simple protein composition this membrane is ideal for the development of solubilization methods and of chemical probes of membrane structure. The proteins of the sarcoplasmic reticulum isolated from rabbit skeletal muscle have been shown by electrophoresis and other physical methods to be still aggregated when solubilized in either Triton X-100 or in water by the method of prior solubilization in 80% phenol. Total solubilization of the proteins is achieved in sodium dodecyl sulfate and electrophoretic analyses in the presence of detergent showed the presence of a major protein component with a molecular weight of 100,000 daltons. Labeling of this protein with 32p indicated that it was the ATPase present in the sarcoplasmic reticulum mem- branes. This protein was purified on SDS slab geis and its amino acid compo- sition was shown to be identical to that of all the proteins in the reticulum. Electrophoresis of the major protein component on phenol:urea:acetic acid gels indicated that it was in fact composed of two proteins. When the major pro- tein component was chemically modified and analyzed on sodium dodecyl sulfate acrylamide gels two protein components were now observed. The major protein components have either three or four disulphide bonds and cross-linking with dimethylsuberimidate confirmed that the 100,000 units are major constituents of this tissue. E. M. Shooter PERSONNEL Postdoctoral M. Baker J. Bamburg I. Morgan R. Perez T. Schenker Predoctoral K. Borden K. Herrup S. Reed 28. - Some Aspects of Molecular Neurobiology Nerve growth factor - physical properties and relationship of enzymatic and biological activities Nerve growth factor - molecular composition and mechanism of action Synaptosome biochemistry Nerve growth factor — structural studies Nerve growth factor - chemical characterization of differ- ences between subunits and sequence work Specific acidic proteins in nerve cells Nerve growth factor - biological studies and mechanism of action Membrane biochemistry -— isolation of ATPase enzymes W. Mobley (Medical student) - Nerve growth factor - structural and chemical studies 29. W. F. Bodmer Laboratory - Recent Publications Bodmer, W.F. and A.J. Darlington, 1969, Linkage and recombination at the molecular level. In Genetic Organization. E.W. Caspari and A.W. Ravin, eds. Academic Press, New York, Vol. l. Bodmer, W.F., 1968. Demographic approaches to the measurement of differential selection in human populations. Proc. Natl. Acad. Soc. 59:690-699, Bodmer, W.F. and L.L. Cavalli-Sforza, 1968. A migration matrix model for the study of random genetic drift. Genetics 59:565-592. Laird, C.D., L. Wang and W.F. Bodmer, 1968. Recombination and DNA replication in Bacillus subtilis transformation. Mutation Res. 6:205-209. Darlington, A.J. and W.F. Bodmer, 1968. Events occurring at the site of integration of a DNA molecule in Bacillus subtilis transformation. Genetics, 60:681-684, Dec. (1968). Bodmer, W. and G. Gerbrandt, 1968. Short term room temperature storage of human lymphocytes for white cell typing. Vox Sanguinis, 15: 451-455 (1968). Feldman, M., M. Nabholz and W.F. Bodmer, 1968. The evolution of the Rh poly- morphism: A model for the interaction of incompatibility, reproductive compensation and heterozygote advantage. Amer. J. Human Genetics, Vol.21: No. 2, March, 1969, 171-193. Bodmer, W., J. Bodmer, D. Ihde and S. Adler, 1969 Genetic and serological association analysis of the HL~A leukocyte system. Computer Applications in Genetics (Ed. W.E. Morton) Univ. Hawaii Press, p. 117-127. Bodmer, W.F. and A. Jacquard, 1967. La variance de la dimension des familles, selon divers facteurs de la fecondite. Population, n° 5, p. 897. Miggiano, V., M. Nabholz and W. Bodmer, 1969. Hybrids between human leukocytes and a mouse cell line: Production and characterization. Wistar Institute Symposium., Monograph No. 9, "Heterospecific Genome Interaction", Wistar Inst. Préss. Nabholz, M., V. Miggiano and W. Bodmer, 1969. Genetic Analysis Using Human- Mouse Somatic Cell Hybrids. Nature , 923-358-363, Bodmer, W.F., 1970, The Evolutionary Significance of Recombination in Prokaryotes, 20th Symp. Soc. Gen. Microbio, Number XX, H.P. Charles & B.C.J.G. Knight (eds). Cambridge University Press, pages 279-294, Nabholz, M. and W.F. Bodmer, 1970, ‘Cell hybridization". Review article in McGraw Hill Encyclopedia of Science and Technology, Yearbook. Cavalli-Sforza , L.L. and W.F. Bodmer, 1970, The Genetics of Human Populations, Freeman and Company, San Francisco, (in press). 30. W. F. Bodmer Laboratory - Recent. Publications Santachiara, A.S., Nabholz, M., Miggiano, V., Darlington, A.J. and Bodmer, W.F., 1970. Genetic analysis with man-mouse hybrids: linkage between human lactate dehydrogenase B and peptidase B. Nature 227:248-251. Bodmer, J.G. and Bodmer, W.F. 1970, Studies on African Pygmies IV: a comparative study of the HL-A polymorphism in the Babinga Pygmies and other African and Caucasian populations. Am. Jour. Hum. Genet. 22:396-411. Miggiano, V.C., Nabholz, M. and Bodmer, W.F. 1970, Detection of HL-A and other antigens on fibroblast micro-monolayers using a fluorochromatic cytotoxicity assay. p- Histocompatibility Testing, 1970 (Paul I. Terasaki, ed.) Munksgaard, Copenhagen. 623-427. Gabb, B.W. and Bodmer, W.F., 1970. A micro complement fixation test for platelet antibodies. Histocompatibility Testing, 1970. p. 543-547. Bodmer, J., Coukell, A., Bodmer, W.F., Payne, R. and Shanbrom, E., 1970. A new allele for the LA series of HL-A antigens: the analysis of a complex serum. Histocompatibility Testing, 1970. p. 175-185. Bodmer, W.F., Bodmer, J.G. and Tripp, M. 1970, Recombination between the LA and 4 loci of the HL-~A system. Histocompatibility Testing,1970. 187-191. Mattiuz, P.L., Ihde, D., Piazza, A., Ceppellini, R. and Bodmer, W.F., 19/70. New approaches to the population genetic and segregation analysis of the HL-A system. Histocompatibility Testing, 1970. p. 193-205. Hulett, R., Coukell, A. and Bodmer, W.F. 1970 Tissue typing instrumentation using the fluorochromatic cytotoxicity assay. Transplantation 10:135-137. Payne. R., Bodmer, J., Bodmer, W.F. and Shanbrom, E. 1970. Characterization of HL-A antisera produced by planned immunization. Histocompatibility Testing ,1970. p. 207-220. Bodmer, W.F., Cavalli-Sforza, L.L. 1970. Intelligence and Race. Scientific American, 223, No. 4: 19. 31. A. T. GANESAN = Recent Publications 17. 18. 19. 20. 21. 22. Laipis, P.J., B. M. Olivera and A. T. Ganesan, 1969. Enzymatic cleavage and repair of transforming DNA. Proc. Nat. Acad. Sci. 62: 289-296. Polsinelli, M., G. Milanesi and A. T. Ganesan, 1969. Short fragments from both complementary strands in the newly replicated DNA of bacteriophage SPP-1. Science 166: 243-245. Gillin, F.D. and A. T. Ganesan, 1971. Control of chromosome replication in Bacillus subtilis. I. Studies of a strain with multiple growing points. (in prep) Gillin, F.D. and A. T. Ganesan, 1971. Control of chromosome replication in Bacillus subtilis. II. Physical studies. (in prep) Ganesan, A. T., 1971. In vitro replication and repair of B. subtilis chromosome. Proc. Nat. Acad. Sci. Yehle, C. 0. and A. T. Ganesan, 1971. DNA synthesis in bacillus subtilis infected with bacteriophage SPQ1. Bacteriological Proc. p. 196. _L. A. Herzenberg Laboratory 7 Recent Publications 32. 54, 55, 56. 57. 58, 59. 60. 61. 62. 63. 64. 65. 66. 67. Tyan, Marvin L. and Leonard A. Herzenberg. 1968. Immunoglobulin production by embryonic tissues: thymus independent. Proceedings of the Society for Experimental Biology and Medicine 128: 952-954. Lanzerotti, Richard M. and Leonard A. Herzenberg. Population of antibodies recognizing distinct allotypic specificities in mouse immunoglobulin. V. _(in preparation). Herzenberg, Leonard A., B. Pernis and A.S. Kelus. A second locus controlling rabbit heavy chain allotypes on the Fd fragment of a second-class of immuno- globulin. (in preparation). Herzenberg, Leonard A., A. Carbonara, R. Tosi and B. Pernis. Comparison of a locus allotypic specificities in IgG, IgA and IgM in the rabbit (jn preparation). Herzenberg, Leonard A. 1969. Rabbit Aa locus allotypes: quantitative comparisons in IgG, IgA and IgM by inhibition of precipitation. Federation Proceedings, p. 435. (abstract). Hulett, H.R., W. A. Bonner, J. Barrett and L. A. Herzenberg. 1969. Cell sorting: automated separation of mammalian cells as a function of intracellular fluorescence. Science 166: 747. Tyan, Marvin L., Leonard A. Herzenberg and Priscilla R. Gibbs. 1969. Lymphoid precursors: thymus independent antibody production. Journal of Immunology 103: 1283. L'age-Stehr, Johanna, and L. A. Herzenberg. 1970. Immunological memory in mice: I. Physical separation and partial characterization of memory cells for different Ig classes from each other and from antibody producing cells. Journal of Experimental Medicine 131: 1093-1108. Jacobson, E. B., Johanna.Ltage-Stehr and Leonard A. Herzenberg. 1970. Immuno- “logical memory in mice: II. Cell interactions in the secondary immune response studied by means of Ig allotype markers. Journal of Experimental Medicine 131: 1109-1120. $ Hulett, H.R., L. A. Herzenberg, W. A. Bonner and P. L. Wolf. Rapid cell sorter-- a new tool for cell study with clinical applications. Laboratory Investigations (abstract in press) Presented at 59th Annual Meeting of International Academy of Pathology, St. Louis, Missouri, March, 1970. Riblet, Roy J. and Leonard A. Herzenberg. 1970. Mouse lysozyme: production by a monocytoma, isolation, and comparisons with other lysozyme. Science 168: 1595, Herzenberg, Leonard A. Gene interactions in immunoglobulins (in press). Presented at Symposium on Anti Human Gamma Globulins, Lund, Sweden, Oct. 1969. . Herzenberg, Leonard A. 1970. From cell biology to immunology--a short trip. Journal of Cellular Physiology #6:~ .303-310. Merrill, J. T., N. Veizades, H.R. Hulett, P. L. Wolf and L. A. Herzenberg. 1970 An improved cell-volume analyzer. (submitted to Review of Scientific Instruments). n,: 10. “Vi. 33. Publications out of L. A. Herzenberg's Laboratory without his Authorship Herzenberg, Leonore A. and Bertha Gonzales. 1962. Appearance of H-2 ‘agglutinins in outcrossed. female mice. Proceedings of the National Academy of Sciences 48: 570-573. Wortis, Henry H. 1965. A gene locus concerned with an antigenic serum substance in mus musculus. Genetics 52: 267-273. Ozer, Harvey L. 1965. Purine pyrophosphorylase as a selective genetic marker in a mouse lymphoma P 388 in cell culture. Journal of Cell Physiology 68: 1-7. Woods, Roy. 1969. The detection of novel antigenic specificities common — to mouse IgG and IgA molecules (submitted to Journal of Experimental Medicine). Warner, Noel L. and Leonore A. Herzenberg. 1970. Tolerance and immunity to maternally derived incompatible IgG, - globulin in mice. Journal of Experimental Medicine 132: 440. Chan, Eva Lee, Robert I. Mishel] and Graham F. Mitchell. 1970. Cell interaction in an immune response in vitro: requirement for theta carrying cells. Science 170: 1245-1217. Mitchell, G.F., F. Carl Grumet and Hugh 0. McDevitt. 1971. Influence of thymectomy of the primary antibody response of mice to the synthetic polypeptide antigen (T,G)-A--L. Mitchell, Graham F. 1971. An hypothesis concerning T-cell mediated regulation of antibody production. . Melchers, Fritz. 1971. Biocynthesis of the carbohydrate portion of immunoglobulins. Incorporation of radioactive fucose into immunoglobulin G, synthesized and secreted by the mouse plasma cell tumor MOPC 21. Biochemlcal Journal 119: Melchers, Fritz. 1971. Biocynthesis, transport and secretion of immunoglobulin in plasma cells. (submitted to Histochemical Journal). Melchers, Fritz. 1971. Catabolic properties of myeloma proteins with different carbohydrate content. - 34. J. Lederberg Laberatory - Recent Related Publications Ciferri, 0., S. Barlati and J. Lederberg, 1970. Uptake of synthetic poly- nucleotides by competent cells of Bacillus subtilis. J. Bact. 104:684-688. Barlati, S. and 0. Ciferri, 1970. Incorporation of 5-methyl- and 5-hydroxyl- tryptophan into the protein of Bacillus subtilis. J. Bact. 101:166-172. Barlati, S., 1970. Incorporation of uridine into Bacillus subtilis and SPPl bacteriophage deoxyribonucleic acid. J. Bact. 101:330-332. Majerfeld, I., S. Barlati and 0. Ciferri, 1970. Tryptophanless death in Bacillus subtilis. J. Bact. 101:350-354. Barlati, S. and I. Majerfeld, 1970. Partial characterization of the factor responsible for tryptophanless death in Bacillus subtilis. J. Bact. 101:355-360. Barlati, S., 1970. -Polyribosomes from Bacillus subtilis during amino acid starvation in the presence and in the absence of actinomycin. J. Bact. 101:925-930. Buchs, A., A.B. Delfino, A. M. Duffield, C. Djerassi, B. G. Buchanan, E. A. Feigenbaum, and J. Lederberg, 1970. Applications of artificial intelligence for chemical inference. VI. Approach to a general method of interpreting low resolution mass spectra with a computer. Helvitia Chimica Acta 53:1349-1417. Lederberg, J., 1970. "Orthobiosis: The Perfection of Man" in Nobel Symposium XIV, The Place of Value in a World of Facts, held at Stockholm, Sweden, September 1969. (A. Tiselius & S. Nilsson, eds.) John Wiley & Sons, Inc. New York. p 29-58. 35. E. M. Shooter Laboratory - Recent Publications 69. 70. 71. 72. 73. 74. - 75. 76. 77. 78. 79. 80. Varon, S., Nomura, J. and E. M. Shooter, 1968. Reversible dissociation of the mouse nerve growth factor protein into different subunits. Biochemistry 7, 1296-1303. Shooter, E. M., Smith, A. P., and S. Varon, 1968. Heterogeneity of the nerve growth factor protein and its subunits. Fed. Proc. 27, 464. Herschkowitz, N., McKhann, G. M., Saxena, S. and E. M. Shooter, 1968. Characterization of sulfatide-containing lipoproteins in rat brain. J. Neurochem. 15, 1181-1183. Smith, A. P., Varon, S. and E. M. Shooter, 1968. Multiple forms of the nerve growth factor protein and its subunits. Biochemistry 7, 3259-3268. Greene, L. A., Shooter, E. M. and S. Varon, 1968. Enzymatic activities of mouse nerve growth factor and its subunits. P.N.A.S. 60, 1383-1388. McKhann, G. M. and E. M. Shooter, 1969. Genetics of Seizure Susceptibility in Basic Mechanisms of the Epilepsies, H. H. Jasper, A. A. Ward, Jr., and A. Pope, Ed., Boston, Little, Brown and Company, Chapter 24. Herschkowitz, N., McKhann, G. M., Saxena, S., Shooter, E. M. and R. Herndon 1969. Synthesis of sulfatide-containing lipoproteins in rat brain. J. Neurochem. 16, 1049-1057. Goodall, P. T. and E. M. Shooter, 1969. Changes in Heme Environment due to subunit interaction in hemoglobin. J. Mol. Biol. 39, 675-678. ' Smith, A. P., Varon, S. and E. M. Shooter, 1969. Equilibria of the Nerve Growth Factor proteins and their subunits. Fed. Proc. 28, 897. Greene, L. A., Shooter, E. M. and Silvio Varon, 1969. Subunit interaction and enzymatic activity of mouse 7S Nerve Growth Factor. Biochemistry 8, 3735-3741. Shooter, E. M., Smith, A. P., Greene, L. A. and S. Varon, 1969. Aspects of the dissociation equilibria between 7S NGF and its subunits. Abstracts of Second International Meeting of the International Society for Neurochemistry, p. 366. Waehneldt, T. V., Grossfeld, R. M. and E. M. Shooter, 1969. The solubili- zation and electrophoretic analysis of membrane proteins from mouse brain. Abstracts of Second International Meeting of the International Society for, Neurochemistry, p. 411. + 36. E. M. Shooter Laboratory - Recent Publications él, 82, 83. 84, 85. 86. 87. 88. 89, 90. 91. Smith, A. P., Greene, L. A., Fisk, H. R., Varon, S. and E. M. Shooter, 1969. Subunit equilibria of the 7S Nerve Growth Factor protein. Biochemistry 8, 4918-4926. Shooter, E. M. and S. Varon, 1970. Macromolecular aspects of the Nerve Growth Factor proteins in Protein Metabolism of the Nervous System. A. Lajtha, Ed., New York, N.Y., Plenum Press, 419-438. Varon, S. and E. M. Shooter, 1970. The Nerve Growth Factor proteins of the mouse submaxillary gland in Biochemistry of Brain and Behavior, R. E. Bownan and S. P. Datta, Ed., New York, N.Y., Plenum Press, 41-64. Shooter, E. M., 1970. Some aspects of gene expression in the nervous system in The Neurosciences: Second Study Program, F. 0. Schmitt, Ed., New York, N.Y¥., The Rockefeller University Press, 812-827. Shooter, E. M. and S. Varon, 1971. Biological activities of the subunits of the 7S Nerve Growth Factor protein in cellular aspects of growth and differentiation in nervous tissue in Cellular Aspects of Neurla Growth and Differentiation, D. C. Pease, Ed., UCLA Forum in Medical Sciences, No. 14., 269-272. Shooter, E. M. and Elizabeth R. Einstein, 1971. Proteins of the Nervous System in Annual Review of Biochemistry: Vol. 40, R. Fried, Ed., Marcel Dekker, Inc., New York, N.Y. Grossfeld, R. M. and E. M. Shooter, 1971. The quantitative extraction of mouse brain proteins. J. Neurochem., submitted. , Grossfeld, R. M. and E. M. Shooter, 1971. The electrophoretic analysis of mouse brain proteins and a study of the changes in the protein composition of mouse brain during ontogenetic development. J. Neurochen., submitted. Morris, S. J., Ralston, H. J. and E. M. Shooter, 1971. Studies on the turn- over of mouse brain synaptosomal proteins. J. Neurochem., submitted. Greene, Lloyd A., Varon, S., Piltch, A. and E. M. Shooter, 1971. Sub- structure of the 8 subunit of the mouse 7S Nerve Growth Factor. Neurobiology, 1, 1. Louis, Charles and E. M. Shooter, 1971. The Protein of Rabbit Skeletal Muscle Sarcoplasmic Reticulum. J. Biol. Chem., submitted.