Purification, characterization, and bioinformatics studies of phosphatidic acid phosphohydrolase from <i>Lagenaria siceraria</i>

Abstract

Phosphatidic acid phosphohydrolase (PAP), EC 3.1.3.4, is the penultimate step in the Kennedy pathway of triacylglycerol (TAG) synthesis leading to the formation of diacylglycerol (DAG), which is a key intermediate in TAG synthesis. We partially purified a soluble PAP from mid maturing seeds of bottle gourd, Lagenaria siceraria. The steps include both anionic and cationic ion exchanger columns. Catalytic characterization of the partially purified PAP revealed that the optimum pH and temperature for activity were at 5.5˚C and 45˚C. Under optimum assay condition using dioleoyl phosphatidic acid (DPA) as the substrate, the V max and K m were 0.36 ηkat/mg of protein and 200 µM, respectively. For the synthetic sub-K m were 33.0 nkat/mg of protein and 140 µM, respectively. The activity was neither inhibited nor enhanced by the presence of Mg 2+ at a concentration range of 0 to 10 mM. Two major protein bands at 42-kDa and 27-kDa were visible in SDS-PAGE after partial purification. Bioinformatics analysis of trypsinized protein fractions containing PAP activity showed peptide sequences with sequence homology to various phosphate metabolizing enzymes including cucumber and castor bean purple acid phosphatase, polyphosphate kinase, fructose biphosphate aldolase, and enolase from various dicotyledonous plants including rice, corn, grape, and Arabidopsis lyrata.

Characterization of a Soluble Phosphatidic Acid Phosphatase in Bitter Melon (Momordica charantia)

“…This result further confirmed that the soluble PAP activity is Mg 2+ -independent in bitter melon extract. These assay results confirm the previous observations from crude extract of bitter melon and partially purified bottle gourd PAP that these PAPs are Mg 2+ -independent enzyme [39] , [41] . The overall results suggest that a new class of PAP exists in bitter melon and bottle gourd which is a soluble and Mg 2+ -independent enzyme.…”

“…These K m and V max values of the purified PAP from bitter melon were approximately 4-fold and 56-fold, respectively, of those obtained from crude bitter melon extract [39]. These K m and V max values of the purified PAP from bitter melon were approximately 3-fold of those obtained from partially purified bottle gourd PAP [41]. The differences in PAP kinetic parameters between the partially purified PAP and crude PAP preparations are in agreement with some observations from previous publications.…”

“…Further purification with Affigel Blue resin did not result in any improvement of purity and actually reduced the total activity of PAP (data not shown). The yield and specific activity of PAP from bitter melon is much higher than those of PAP purified from Lagenaria siceraria (bottle gourd, opo squash or long melon) [41]. Additional purification steps resulted in significant loss of activity and the protein yield was low, probably due to PAP-associated with other proteins (see below).…”

Identification of an Mg<sup>2+</sup>-Independent Soluble Phosphatidate Phosphatase in Cottonseed (<i>Gossypium hirsutum L.</i>)

“…This result further confirmed that the soluble PAP activity was Mg 2+ -independent in cottonseed extract. These assay results confirm the previous observations from crude extract of bitter melon and partially purified bottle gourd PAP that these PAPs are Mg 2+ -independent enzyme [12] [17]. The overall results suggest that a new class of PAP exists in cottonseed which is a soluble and Mg 2+ -independent enzyme.…”

Agronomic evaluation and quality characteristics of three Cucurbitaceae varieties acclimated in Benin

“…In order to achieve this objective, the present study was interested in seeds of some Cucurbitaceous such as C. lanatus, L. siceraria and C. edulis. Although, several studies were carried out on their pharmacological potential (Prajapati et al, 2010) and the various rough levels of nutriments (Nmila et al, 2002;Sabo et al, 2005a, b;Sadou et al, 2007;Ullah et al, 2012), very few works were focused on the biochemical composition of these seeds (Abiodun and Adeleke, 2010). In Benin, previous researches were focused on the agronomic evaluation of some species of Cucurbitaceous used in the foodstuff in Benin (Achigan Dako et al, 2006) in other to develop a model of prediction.…”