Conformational stability and in vitro bioactivity of porcine luteinizing hormone.
Temperature-dependent dissociation of porcine luteinizing hormone (pLH) and of two of its glycoforms was studied by a combination of SDS-PAGE and micro-scale size-exclusion HPLC in parallel with the study of co-operative folding by high-sensitivity differential scanning calorimetry (HS-DSC). The transition temperature of dissociation of pLH at pH 7.0 as quantified by SDS-PAGE, HPLC and residual activity in radioreceptor assay was found to match exactly the transition temperature of its unfolding as measured by HS-DSC. Free alpha- and beta-subunits did not exhibit any unfolding transition in the same conditions. The microcalorimetric data for two pLH isoforms exhibiting different glycosylations were identical to those of a preparation of non-separated isoforms. It is concluded that: (a) free subunits exhibit no co-operative folding (i.e. no stable three-dimensional structure) and co-operative folding occurs only in alphabeta heterodimers; (b) the co-operative folding is responsible for the stability of the association of subunits; and (c) the heterogeneity of carbohydrate chains does not affect the stability of folding and association of subunits. The fastening of the "seat-belt" of the beta-subunit embracing the alpha-subunit by the Cysbeta26-beta110 disulfide bridge had been postulated to play a role in the preservation of the dimeric structure of gonadotropins. The present work shows that dissociation of subunits is directly related to their loss of common co-operative folding.
Burova
T
T
Laboratoire d'Etude des Interactions des Molécules Alimentaires, Institut National de la Recherche Agronomique (INRA), B.P. 71627, 44316 Nantes Cedex 03, France.
Lecompte
F
F
Galet
C
C
Monsallier
F
F
Delpech
S
S
Haertlé
T
T
Combarnous
Y
Y
D000818:Animals; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015292:Glycoprotein Hormones, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007986:Luteinizing Hormone / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D013552:Swine
D004220:Disulfides; D015292:Glycoprotein Hormones, alpha Subunit; D021122:Protein Subunits; D007986:Luteinizing Hormone