Functional and misfolded NTS1 during purification.

Three different ways (columns A–C) were used to estimate the amounts of functional NTS1, misfolded NTS1 and contaminants in the NiE, NTFT and NTE fractions. All values in columns A–D are given in mg quantities. Abbreviations: NiE, Ni-NTA column eluate; NTFT, flow-through of NT column; NTE, NT column eluate; NA, not applicable.1The amount of functional NTS1 in the NiE, NTFT and NTE fractions was determined by [3H]NT binding (see Tables 3 and 4). For example, the NiE contained 3678 pmoles or 0.355 mg functional NTS1.2The total amount of NTS1 protein in the NiE, NTFT and NTE fractions was determined by ImageJ analysis of Coomassie-stained gels (Table 4).3The amount of functional NTS1 in the NiE and NTE fractions was derived from the respective specific [3H]NT binding values (in pmol/mg, Table 3) compared to the theoretical value of 10363 pmol/mg. For example, the NiE fraction has a specific binding value of 3724 pmol/mg and hence 36% (3724/10363) of the total protein in NiE (0.92 mg) is functional NTS1 (0.331 mg).4The amount of functional NTS1 was estimated from data in Table 4.5The diluted NiE is 91% functional after 6 hrs (Table 1). This decrease of the amount of functional NTS1 is indicated by a left pointing arrow. The corresponding amount of misfolded NTS1 is listed.6The amount of functional and misfolded NTS1 was calculated from the total NTS1 amount in the respective fractions considering the proportion of functional receptors (Table 4). For example, the NiE contained 0.443 mg total NTS1 which is 80% functional4 i.e. 0.355 mg functional NTS1 and 0.088 mg misfolded NTS1.7The amount of contaminants was calculated by subtracting the total NTS1 amount from the total protein content of a given fraction (Table 4).8The total protein content of the NiE, NTFT and NTE fractions was determined by the Amido Black method.