Measurement of in situ halophilic glyceraldehyde-3-phosphate dehydrogenase activity from the permeabilized cells of archaebacterium Haloarcula vallismortis.
The cells of Haloarcula vallismortis, an extreme halophilic archaebacterium, were permeabilized by various chemical, physical, and biological treatments. Biological permeabilization by lysozyme and papain showed effective results as observed by studying the in situ activity of halophilic glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) as the model enzyme. Detergents N-cetyl-N, N, N-trimethyl ammonium bromide (CTAB) and digitonin also showed significant results. Other strains of halobacteria could also be permeabilized by lysozyme. The cell morphology did not show any significant change after permeabilization as observed by phase contrast microscopy. The enzyme characteristics of hGAPDH were studied in situ using permeabilized H. vallismortis cells. The properties, like optimum pH, Km for GAP and NAD(+), inhibition by heavy metals, sulphydryl reagents, and other compounds, showed remarkable similarity with those studied in vitro.
Patil
Anita R.
AR
Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai-400085, India.
D'Souza
Stanislaus F.
SF