Purification and Characterization of Actin from Maize Pollen

Abstract

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold. amino acid composition, circular dichroism spectrum, and activation of myosin ATPase activities. MATERIALS AND METHODS ChemicalsDEAE-cellulose and Sephacryl S-200 were obtained from Pharmacia. ATP-Na2 salt, imidazole, SDS, acrylamide, bismethylene-acrylamide, Tris, and monoclonal antibodies against smooth-muscle a-actin were from Sigma. All other reagents used were of analytical grade. Maize PollenMaize (Zea mays L.) plants were grown in the Experimental Station of Beijing Agricultural University in Beijing.The cultivar was Nongda No. 14. Pollen were collected in the middle of July when the plants were in pollination stage. About 1 kg fresh weight of maize pollen was collected and stored in a desiccator placed in a freezer (-700C) for use. It has been well established that the actomyosin system is universally distributed in higher plants (9). The actomyosin system is an important component of cytoskeleton, which plays a pivotal role in the maintenance of cell shape, cell motility, mitosis, and cytokinesis. The presence of actin and myosin in higher plants has been identified in Amaryllis, Haemanthus, tomato, conifer root, pollen of Luffa, pea tendrils, and pollen tube of lily (3,5,13,17,20,21,23) by biochemical and immunological methods. But the concentrations of actin in plant are lower than they are in animal cytoplasmic tissues (14). So the biochemical and physiological studies of plant actomyosin are limited, unlike the cases in other nonmuscle cells such as slime molds and Acanthamoeba castellanii. However, we found that pollen is a rich source of actin for purification. We have developed an efficient method to prepare sufficient quantities of pure actin for biochemical and physiological studies such as the conformational changes during the polymerization of actin and the reassembly of microfilaments in vitro. Here we describe this method, which includes acetone powder preparation, ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephacryl S-200 gel filtration. Furthermore, the pollen actin w...

Actin Purified from Maize Pollen Functions in Living Plant Cells.

“…The average yield of 6 mg of actin from 10 g of pollen is at least threefold higher than the yield obtained using a previous method for isolating actin from maize (Liu and Yen, 1992). Liu and Yen (1992) developed a protocol involving the preparation of a pollen acetone powder, low-ionic-strength extraction, ammonium sulfate precipitation, anion exchange chromatography, polymerization and depolymerization, and size exclusion chromatography. Their procedure provides up to 2 mg of actin from 10 g of pollen at an apparent purity of 92% (Liu and Yen, 1992).…”

“…Liu and Yen (1992) developed a protocol involving the preparation of a pollen acetone powder, low-ionic-strength extraction, ammonium sulfate precipitation, anion exchange chromatography, polymerization and depolymerization, and size exclusion chromatography. Their procedure provides up to 2 mg of actin from 10 g of pollen at an apparent purity of 92% (Liu and Yen, 1992). Our new strategy generates polymerizable actin that is >98% pure in less than half of the time.…”

A Gelsolin-like Protein from Papaver rhoeas Pollen (PrABP80) Stimulates Calcium-regulated Severing and Depolymerization of Actin Filaments

“…In addition to being essential for sexual reproduction, pollen is an ideal choice of material for studies of the cytoskeleton. Actin is one of the most abundant proteins in pollen, representing 5-20% of total cellular protein (1,2). Cytoskeletal genes are among the most abundantly expressed classes of transcripts in Arabidopsis pollen (3), and several classes of actin-binding protein (ABP) 1 have been isolated and characterized biochemically (4 -6).…”

Latrunculin B Has Different Effects on Pollen Germination and Tube Growth

“…Pollen is a rich source of actin, which accounts for 2 to 20% of the total soluble protein in this tissue (Andersland et al, 1992;Liu and Yen, 1992;Ren et al, 1997;Vidali and Hepler, 1997). It also contains several actin binding proteins such as profilin, actin depolymerizing factor, spectrin, 115-and 135-kD actin-bundling proteins, and myosin (reviewed in Asada and Collings, 1997;Staiger et al, 1997;de Ruijter and Emons, 1999).…”