Localization of carbon monoxide dehydrogenase in acetate-adapted Methanosarcina barkeri

1993 ◽  
Vol 39 (2) ◽  
pp. 223-226 ◽  
Author(s):  
J. U. Gokhale ◽  
H. C. Aldrich ◽  
L. Bhatnagar ◽  
J. G. Zeikus
Keyword(s):  
Carbon Monoxide ◽  
Protein A ◽  
Carbon Monoxide Dehydrogenase ◽  
Methanosarcina Barkeri ◽  
Immunogold Labeling ◽  
Rabbit Antibody ◽  
Methanogenic Bacterium ◽  
Enzyme Localization ◽  
Low Levels ◽  
Lowicryl K4m

Immunogold labeling techniques were utilized to determine the cellular location of carbon monoxide dehydrogenase in the methanogenic bacterium Methanosarcina barkeri grown on acetate to enhance levels of the enzyme. Formaldehyde fixation followed by dehydration in methanol and embedment in Lowicryl K4M resin preserved the antigens. Sections were labelled with primary polyclonal rabbit antibody followed by protein A - gold. Low levels of labeling were improved by use of an antibody to protein A followed by a second incubation on protein A - gold. The enzyme is primarily located in the cytoplasm, which agrees with biochemical results showing that it resides in the soluble cellular fraction.Key words: Methanosarcina barkeri, methanogen, methanogenesis, enzyme localization, immunogold.

scholarly journals Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.

1982 ◽  
Vol 93 (1) ◽  
pp. 223-229 ◽  
Author(s):  
J Roth ◽  
E G Berger
Keyword(s):  
Golgi Apparatus ◽  
Hela Cells ◽  
Protein A ◽  
Rabbit Antibody ◽  
Concerted Action ◽  
Electron Microscope Level ◽  
Thin Sections ◽  
Thiamine Pyrophosphatase ◽  
In Trans ◽  
Lowicryl K4m

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.

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