CRNF, a molluscan neurotrophic factor that interacts with the p75 neurotrophin receptor

Science

Volumn 274, Issue 5292, 1996, Pages 1540-1543

  Fainzilber, M ^a,b   Smit, A B ^b   Syed, N I ^c   Wildering, W C ^c   Hermann, P M ^c   Van Der Schors, R C ^b   Jimenez C ^b   Li, K W ^b   Van Minnen, J ^b   Bulloch, A G M ^c   Ibanez C F ^a   Geraerts, W P M ^b  

^a KAROLINSKA INSTITUTE   (Sweden)

^b VU UNIVERSITY AMSTERDAM   (Netherlands)

^c UNIVERSITY OF CALGARY   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

COMPLEMENTARY DNA; CYSTEINE; MESSENGER RNA; NEUROTROPHIC FACTOR; NEUROTROPHIN RECEPTOR; RIBONUCLEASE;

AMINO ACID SEQUENCE; ANIMAL TISSUE; ARTICLE; BINDING AFFINITY; CALCIUM CURRENT; CELL LINE; GENE EXPRESSION; HUMAN; HUMAN CELL; MOLECULAR CLONING; MOLLUSC; MOTONEURON; NERVE CELL DIFFERENTIATION; NERVE CELL PLASTICITY; NERVE FIBER GROWTH; NONHUMAN; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; PROTEIN BINDING; PROTEIN PURIFICATION; RECEPTOR BINDING; SNAIL;

AMINO ACID SEQUENCE; ANIMALS; BINDING, COMPETITIVE; CALCIUM; HEMOLYMPH; HUMANS; LYMNAEA; MOLECULAR SEQUENCE DATA; MOTOR NEURONS; NERVE GROWTH FACTORS; NEURITES; RECEPTOR, NERVE GROWTH FACTOR; RECEPTORS, NERVE GROWTH FACTOR; RECOMBINANT PROTEINS; RNA, MESSENGER; TUMOR CELLS, CULTURED;

ANIMALIA; GASTROPODA; LYMNAEA STAGNALIS; MOLLUSCA;

EID: 10544221631     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5292.1540     Document Type: Article

References (31)

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15. Hemolymph was milked from adult Lymnaea by collecting fluid ejected after tapping the foot. The final 7-liter pool of hemolymph represented approximately 16,000 animal milkings. Aliquots of 1 liter were processed as follows: (i) Ultracentrifugation for 2 hours at 120,000g to precipitate hemocyanin. (ii) Supernatant was applied to Polybuffer 94 (Pharmacia) equilibrated and washed with 20 mM tris (pH 7.9). Bound proteins were eluted by 1 M NaCl, and desalted and concentrated with Poros 20 R1. (iii) Eluted fractions were applied to linked Beckman SW2000-SW3000 molecular exclusion columns, equilibrated, and eluted with 0.5x phosphate-buffered saline/ 15% acetonitrile. (iv) Fractions eluting at apparent masses of 20 to 30 kD were loaded onto fast-flow reverse-phase Poros 20 R1 equilibrated at 4 ml/min in 15% acetonitrile/0.1% trifluoroacetic acid (TFA). After a 2-min wash, the bound proteins were eluted with a linear gradient from 15 to 60% acetonitrile in 2 to 24 min. (v) Fractions eluting from Poros R1 at 32 to 36% acetonitrile were refractionated on wide-pore Vydac C4 (250 mm by 4.6 mm. 5 μm particle size) at 1 ml/min, in a gradient of acetonitrile in 0.1% TFA (Fig. 1B). Amino acid sequence analysis was performed on blotted Coomassie stained bands, or reverse phase purified peptides from Endo-LysC digest, by automated Edman degradation on an Applied Biosystems 473A system. Methods for MALDI-MS were as described (17).
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29. Immobilization of NGF or CRNF to BIAcore CM5 sensor chips was done by amine coupling in 20 mM acetate buffer (pH 5.6 or 3.6). Binding of Sp75 to immobilized ligand was monitored in a BIAcore 2000 Biosensor (Pharmacia) at 20°C, with a flow of 5 ml/min, in Hepes-buffered saline. Kinetic analyses were done with BIAevaluation software, version 2.0 (Pharmacia).
30. Riboprobes for RPA and in situ hybridization were generated from linearized 380-bp subclones of CRNF cDNA in pCDNA3 (Stratagene). RPA was performed with equal amounts of total RNA, using RPAII reagents (Ambion). Equal loading was verified on ethidium bromide-stained gels and by parallel RPA with riboprobes for ubiquitously expressed Lymnaea mRNAs [fructose 1,6-biphosphate aldolase (Genbank accession number U73114) for the experiment of Fig. 3A and a CNS tyrosine kinase (A. G. M. Bulloch, unpublished data) for Fig. 3B].
31. Dedicated to the memory of Professor Håkan Persson (1952-1993), who was one of the most enthusiastic initiators of this project. We thank G. Hauser, R. van Elk, A.-S. Nilsson, E. van Kesteren, C. Popelier, and A. Ahlsen for technical support; L. Johanson and T. Laan for secretarial help; A. Vlamis and A. Holmgren for their generous assistance with the BIAcore; K. Dreisewerd and F. Hillenkamp for sharing expertise in MALDI-MS; and all members of the Molecular Neurobiology group at Vrije Universiteit Amsterdam for cheerful assistance in snail milking, Sp75 baculovirus and antisera were the generous gift of G. Weskamp and L. Reichardt. Supported by grants from the Swedish Medical Research Council (MRC), the European Neuroscience Program, the Canadian MRC, the Canadian Neuroscience Network, the Canadian National Science and Engineering Research Council, and a special equipment grant from the Netherlands Organization for Research. M.F. was supported by a long-term fellowship from the European Molecular Biology Organization and subsequently by the Swedish MRC. A.G.M.B., N.I.S., and W.C.W. were supported by the Alberta Heritage Foundation for Medical Research.