6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei.
Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37 degrees C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37 degrees C in comparison with those maintained at 0 degrees C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37 degrees C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37 degrees C.
Martelli
A M
AM
Dipartimento di Morfologia Umana Normale, Trieste, Italy.
Neri
L M
LM
Zamai
L
L
Bareggi
R
R
Manzoli
L
L
Cocco
L
L
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry*; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008855:Microscopy, Electron, Scanning; D015530:Nuclear Matrix / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry*; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature; D014407:Tumor Cells, Cultured
D005452:Fluoresceins; D009687:Nuclear Proteins; D013438:Sulfhydryl Compounds; C050046:6-iodoacetamidofluorescein; D003545:Cysteine