Question

1. This procedure recommends preparing E coli as spheroplasts to expose the alkaline phosphatase. Why is this a preferred strategy than simply completely lysing the bacteria completely?

2. Explain the unit 'Unit' when attempting to express a quantity of enzyme. Relate your definiton to the reaction taking place in the PNPP reaction.

Answer 1

1. Alkaline phosphatase is one of several hydrolytic enzymes in Escherichia coli that appear to be localized in a periplasmic space that lies exterior to the bacterial cell membrane. Spheroplast is a bacterium bound by its plasma membrane and the whole having a spherical form. Under conditions that permitted continued protein synthesis, spheroplast of Escherichia coli were unable to form active alkaline phosphatase, although they synthesised protein that antigenically related to alkaline phosphatase subunits. This cross reaction protein primarily detected in the medium of spheroplast culture, and properties that closely resembled those of alkaline phosphatase subunits. These result suggest that formation of the active alkaline phosphatase dimer by intact E coli cells proceeds by a pathway in which inactive subunits released from polyribosomes diffuses through the bacterial cell membrane to a periplasmic space where subsequent dimerization to active enzyme occurs. This pathway provides a possible mechanism for the specific localization of this enzyme to the E coli periplasmic space. So, this is preferred strategy than simply completely lysing the bacteria.

2. Most of the chemical reaction that takes place within a cell involve protein catalysts called enzymes. The enzyme unit is a unit for the amount of a particular enzyme. One U is defined as the amount of enzyme that produces a certain amount of enzymatic activity, that is the amount catalyzes the conversion of 1 micromole of substrate per minute under the specific conditions.

p-Nitrophenyl phosphate (pNPP) is a non proteinaceous, non specific substrate used to assay protein, alkaline and acid phosphatases. It can be used for a quick analysis of the protein phosphatase activity under any conditions. Phosphatases catalyze the hydrolysis of pNPP liberating inorganic phosphate and the conjugate base of para-nitrophenol(pNP). PNPP has Km values for protein phosphatase in the range of 0.5-10 mM. One unit of the protein phosphatases activity is defined as the amount of enzyme that hydrolzes 1 nano mole of PNPP in one minute under standard reaction condition. Protein phosphatase, enzyme that control the removal of phosphate group from protein molecules regulate cellular process such as proliferation, cell attachment. PNPP phosphatase assay is optimized to detect the phosphatase activity in biological sample using PNNP as a colorimetric substrate for most phosphatases.