Cotton (Gossypium hirsutum var. Coker 312) seeds were collected at the mid-maturity stage, approximately 25 - 30 days post anthesis. The seed coat was removed before preparation of enzyme extracts.

All operations were carried out at 4˚C. The seeds free of seed coat weighing 91 g were homogenized in 100 mL extraction buffer (50 mM NaOAc, pH 5.0, 150 mM NaCl and 10 mM MgCl₂) using polytron tissuemizer (Tekmar Tissumizer MarkII, Cincinnati, OH) at low, medium and high speed for 30 s each. The homogenate was cooled down on ice for 1 min between bursts. The homogenate was centrifuged at 10,000 g for 15 min and the resulting supernatant was centrifuged at 20,000 g for 30 min at 4˚C (Sorvall RC2B, Miami, FL). This supernatant was ultracentrifuged at 105,000 g for 45 min at 4˚C (Sorvall Discovery 100SE, Hitachi Ltd, Tokyo, Japan) and the resulting supernatant (cytosol) and the pellet (microsomal membranes) were collected. The 105,000 g supernatant was dialyzed to remove Pi from the seed extract against imidazole buffer (25 mM imidazole buffer, pH 6.5, 1 mM MgCl₂) with three 500 mL buffer changes. The dialyzed supernatant became cloudy after dialysis, which was removed by centrifugation at 20,000 g for 30 min at 4˚C. The final supernatant as cytosol and 105,000 g pellet as microsomal membranes were used for determining subcellular distribution of PAP activity. The protein content of the supernatant was determined by Bicinchoninic acid (BCA) method (Thermo Scientific, Rockford, IL).

PAP activity was determined by P_i-release assay. This activity assay was described in details for bitter melon PAP [12] . The Pi-release assay followed the ammonium molybdate-acetone-acid (AMA) method [14] . For standard assay except otherwise noted below, a 50 µL of enzyme extract was added to 900 µL of 50 mM imidazole buffer, pH 5 in a 55˚C water bath. The enzymatic reaction was initiated by the addition of 50 µL of PtdOH/DPA (dioleoyl-phosphatidic acid or 1,2-dioleoyl-sn-glycero-3-phosphate, sodium salt, Avanti Polar Lipids, Inc., Alabaster, Alabama), incubated for 5 min and terminated by 2 mL of AMA reagent. Citric acid (0.1 mL, 1.0 M) was added to each tube 30 s later to fix the color followed by centrifugation at 13,000 g for 7 min (Eppendorf 5415C, Westbury, NY). The absorbance at 355 nm was measured after blanking the spectrophotometer with the appropriate control, which was stopped at zero time. The PAP activity was expressed as nanokatals per milliliter (nkat/mL, nmoles orthophosphate released per sec). One International Unit (IU) is equivalent to 16.67 nkat.

To determine the pH optimum of cottonseed PAP, the reaction mixtures were incubated at 55˚C with different buffer pH using 25 mM glycine-HCl (pH 2 - 3), 50 mM sodium acetate (pH 4 - 5), and 25 mM imidazole (pH 6 - 7). To measure the optimum temperature, the samples were incubated with substrate between 20˚C and 80˚C in 25 mM imidazole, pH 5. The Mg²⁺ dependency was determined by the phosphatase assay with or without 1 mM MgCl₂ in the assay mixtures. The general procedures for PAP characterization were similar to those used for soluble starch synthases [15] .

The K_m and V_max values for cottonseed PAP using the P_i-release assay were determined at 55˚C and pH 5 as mentioned above. The concentration of DPA ranged from 0 to 500 μM. WindowChem’s software Enzyme Kinetics version 1.1 (Fairfield, CA) was used to compute the K_m and V_max values.

PAP family enzymes were classified as either soluble PAP or membrane-bound PAP [9] . Differential centrifugation was used to separate the cytosolic and microsomal membrane fractions for determining the subcellular localization of PAP activity in cottonseed. The seed extract free of seed coat was successively centrifuged at 10,000 g, 20,000 g and 105,000 g. The final pellet and supernatant after ultracentrifugation are generally regarded as the microsomal membranes and the cytosol, respectively [16] . PAP activity in the 10,000 g, 20,000 g and 105,000 g pellet was 6.6%, 12.5% and 9.3% of the total PAP activity (Figure 2). PAP activity in the 105,000 g pellet represented the PAP activity associated with the microsomal membranes, whereas those activities in the 10,000 g pellet and 20,000 g pellet might be due to some seeds not completely homogenized and/or the pellet being contaminated with the cytosol. Following dialysis and centrifugation of the supernatant, the final supernatant contained 69% of the total activity (Figure 2). These subcellular distributions of PAP activity clearly demonstrated that the great majority of PAP activity in cottonseed was soluble and localized in the cytosol. We recognized that this conclusion is only based on cottonseed collected 25 - 30 days post anthesis. It is possible that the ratio of PAP activity distribution could be different in different stages of cottonseeds. Most likely, more membrane-bound PAP could be detected in older seeds.

The linearity between the incubation time and PAP activity was observed in 50 mM imidazole buffer containing 0.3 mM MgCl₂, pH 5 (Figure 3). The linearity between PAP activity and reaction time was much better when the enzymatic assays were performed after incubation within 6 min with R² = 0.9663 (Figure 3(a)) than that incubation extended to 10 min with R² = 0.8466 (Figure 3(b)). Similar linearity of enzymatic reaction was observed in responding to the amount of enzymes used in the assay with R² = 0.9828 (Figure 4). These assay results suggest that the established assays are suitable for characterization of PAP from cottonseed.

To determine the temperature optimum, PAP activity was assayed under a series of reaction temperature ranged from 22˚C to 82˚C with 5˚C interval. Curve-fitting of PAP activity vs. reaction temperature showed that the temperature optimum was at 55˚C (Figure 5). Similarly, to determine the buffer pH optimum, PAP activity was assayed using a series of buffer pH ranged from pH 2 to pH 7 with an interval of 1 pH unit (Figure 6). The pH optimum of PAP was observed at pH 5 under the assay conditions using 5 µL of the enzyme and 500 µM dioleoyl-phosphatidic acid (DPA) in 50 mM

imidazole buffer containing 0.3 mM MgCl₂ (Figure 6). These optimal pH and temperature values are similar to those of bitter melon extract [12] .

Based on the requirement of Mg²⁺ for activity, PAP family enzymes including lipins and lipid phosphate phosphatases (LPPs) are divided into 2 classes: Mg²⁺-dependent PAP and Mg²⁺-independent PAP [11] . It is known that yeast and invertebrates have a single lipin ortholog, but plants have two PAP/lipin genes and mammals have three lipin genes [6] . The lipin family members of PAP are soluble enzymes and require Mg²⁺ for their activity. LPPs also exhibit PAP activity but they are structurally unrelated to lipin proteins. LPPs are localized to the plasma membrane and do not require Mg²⁺ for their activity. Our results showed that PAP activity was not affected or minimally affected by 1 mM MgCl₂ in the assay mixtures (Figure 6). The effects of ion chelaters EDTA and EGTA on PAP activity were measured. However, both chelators did not have significant effects on PAP activity (data not shown). This result further confirmed that the soluble PAP activity was Mg²⁺-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 Mg²⁺-independent enzyme [12] [17] . The overall results suggest that a new class of PAP exists in cottonseed which is a soluble and Mg²⁺-independent enzyme.

The kinetic parameters of PAP were determined using DPA as the substrate under the optimized assay conditions (pH 5, 55˚C and 0.3 mM MgCl₂). The enzyme gave a typical sigmoidal curve for the substrate (Figure 7). The K_m and V_max values of PAP for DPA were calculated to be 539 µM and 2.8 nkat/mg of protein, respectively (Table 1). Triton X-100 did not have significantly effects on PAP activity (Table 1).