Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions.
The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.
Cotgreave
I A
IA
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Weis
M
M
Berggren
M
M
Sandy
M S
MS
Moldéus
P W
PW
D000818:Animals; D001643:Bridged Bicyclo Compounds; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes; D008099:Liver / Q000032:analysis*; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D051381:Rats; D013347:Subcellular Fractions / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis*
D001643:Bridged Bicyclo Compounds; D005456:Fluorescent Dyes; D011506:Proteins; D013438:Sulfhydryl Compounds; C028381:monobromobimane