I am working on enzyme assays for PMM and PMI from leucocytes. In order to choose the method for leucocyte disruption I want to know if these enzymes are membrane bound or free. I have been looking up for references but could not get satisfactory answers. please help!
Phosphomannomutases PMM1 and PMM2 are soluble proteins. Here is a paper that describes heterologous expression, purification and x-ray crystal structure of PMM1.
http://www.jbc.org/content/281/21/14918.full
Phosphomannose isomerase is also a soluble protein. This paper shows that its activity is found in the 100,000 g supernatant of tissue extracts.
http://glycob.oxfordjournals.org/content/12/7/435.long
Dear Mihika Dave
Phosphomannomutase(PMM) and Phosphomannose isomerase(PMI) are not are not membrane associated.
Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of fructose 6-phosphate and mannose 6-phosphate
The PMM2 gene provides instructions for making an enzyme called phosphomannomutase 2 (PMM2). This enzyme is involved in a process called glycosylation, which attaches groups of sugar molecules (oligosaccharides) to proteins.
Mutations in the gene PMM2 ( the most common subtype is CDG-Ia (also referred to as PMM2-CDG) where the genetic defect leads to the loss of phosphomannomutase 2, the enzyme responsible for the conversion of mannose-6-phosphate into mannose-1-phosphate) in no way affect the state of the PMI gene , the correlation of the intercellular exchange at the level of the proteins is missing.
If I'm wrong-please arguments to prove the opposite.
Best
Dear Dr. Petrov,
Thank you for the answer.
I am trying to perform these assays from leukocytes. For disruption I am using pottering (manual shearing of cells using pestle) and subsequently the supernatant obtained is used as the cell extract for reactions.
Do I need to use a harsher technique for disruption?
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