Open AccessSpin‐Labeling Studies of Urea‐Treated Leucine Aminopeptidase
Author(s) -
LASSMANN Günter,
DAMERAU Werner,
SKLENAR Gisela,
SCHWARZ Dieter,
FROHNE Marlies,
LUDEWIG Manfred,
HANSON Horst
Publication year - 1975
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1975.tb04156.x
Subject(s) - chemistry , urea , aminopeptidase , leucine , site directed spin labeling , conformational change , piperidine , crystallography , monomer , stereochemistry , enzyme , biochemistry , organic chemistry , amino acid , membrane , polymer
1 Leucine aminopeptidase (EC 3.4.11.1) from bovine eye lens was spin‐labeled at the most reactive thiol groups with 2,2,6,6‐tetramethyl‐4‐[2‐iodoacetamido]‐piperidine‐1‐oxyl. 2 Electron spin resonance spectra show two spectral parts corresponding to two local conformational states in the environment of bound label. One state (A) exhibits a strong immobilizing effect on the mobility of the bound label whereas the other one (B) immobilizes weakly. Independently on the degree of labeling a ratio of A:B ∼ 4:1 was estimated. In B a hydrophobic environment of label was observed. 3 Treatment of leucine aminopeptidase by 6.2 M urea leads to the following structural changes. a) An additional weakly immobilizing conformational state (B′) with reduced hydrophobic interactions and increased mobility representing an unfolded conformational state appears. B′ shows a time‐dependent increase of its extent at the expense of B and A′ (half conversion time about 0.5 h). The extent of this conformational change is larger, if the enzyme is additionally complexed with Mn 2+ . b) Mn 2+ complexed with the protein is partly released producing hydrated Mn 2+ . c)After withdrawal of urea the observed conformational changes in leucine aminopeptidase are fully reversible, giving the initial ratio of A:B ∼ 4:1 even after long incubation. 4 6.2 M urea is not able to destroy the strongly immobilizing conformational state A completely.