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810 Table I. Vasopressin Binding to Soluble and' Purified Ftactions Age of tissue Type of assay at assay (days) Soluble Protein: 1 Void Volume with Mg 2 Void Volume with Ni 2 Purified Protein: Filtration assay 3 PEG precipitation 3 Gel Filtration 3 Addition to membranes 3 Coupling to membranes S Junig and Abood Vasopressin bound Fmoles/mg protein Degree of purification 117 1 59 18 175 1.5 203 1.7 1218 10.4 S,230 44 18,U00 150 Binding of I nM (3H1AVP to soluble and purified fractions. Fractions were prepared and assays were performed as described in Experimental Procedure. Assays of soluble and purified ma- terial were performed in the presence of 5 mM Ni2', with the exception of the addition and coupling to membrane experiments, which were performed in the presence of 1 mM Ni='. Values represent means of triplicate determinations within representative experiments. band. The 62 kdalton band was also specifically eluted, in lower yield, when the column was run in the presence of 10 µM ZnZ' (not shown). EDTA in the column buffer presumably functioned to remove endogenous ions from the solubilized protein. When 1 mM EDTA was not included in the column buffer during sample application and washing, both 55 and 62 kdalton bands were obtained in the presence of either Mg'- or Ni'` (not shown). j3HJAVP Binding Properties of Purified Frac- tions. The binding activity of protein not retained by the column ('void volume')~ was measured and compared to the binding activity of the starting ma- tcriali abl'e . Binding to non-retained'protein was decreased by 48% when applied in the presence of Mg2+ and by 85% in the presence ofNiz-. Eluted column fractions were assayed for AVP binding activity in the presence of Mg2' or NiZ'.. The fraction eluted from the column by AVP in the presence of MgZ', which contained mainly the 55 kdalton protein, did not bind vasopressin in the presence of Ni2' or Mg2 `, as measured by filtration on PEI-treated filters or PEG precipitation. Binding was also not demonstrable to fractions eluted with EDTA in ion-free buffer. The fraction eluted with A 8 C D E 71 K 68KL 62K- 55K~ ~_ ®s -66K ~. -15 K 36 K , Yiiq~ ~r~ --29K ^ ~ ~ -2•tK "W 02 0 Fig. 7. Absorbance at 220 nm of column fractions of a typical column run, In this example. the column was run in the presence of 5 mM Vi=-. and 5 ml fractions were collected and their ab- sorbance measured, 'AVP' represents the addition of 10 µM AVP to the buffer: 'vaCl' represents the addition of 100 mM NaCl. For each run. fractions 15-24' were combined for gel! electro- phoresis or for binding assays. ~ r.Ottt/ «.+ ~ , L L L 0 5. 10 1S. 20 25 30 FRACTION NUMtjER •w Fig. 8. Gel electrophoresis of eluted'column fractions. A: Protein was applied in the presence of 5 mM Ni='. and eluted with Nf- free buffer containing 5 mM EDTA. B: Protein was applied in the presence of 5 mM Mg='. and eluted with Mg-free buffer con- taining 5 mM EDTA. C: Protein was applied in the presence of 5 mM Mg='. and eluted with the same buffer plus 10 µM AVP. D: Protein was applied in the presence of 5 mM Ni=-. and eluted' with the same buffer plus 10 µM AVP. ErMoleeular weight stan- dards: BSA (55 kdaltons). ovalbumin (45 kdaltons). glyeeralde- hyde-3-phosphate dehydrogenase (36 kdaltons). carbonic anhv- drase (29 kdaltons). and trypsinogen (24 kdaltons).