Calcium can function as a second messenger through stimulation of calcium-dependent protein kinases. A protein kinase that requires calcium but not calmodulin or phospholipids for activity has been purified from soybean. The kinase itself binds calcium with high affinity. A complementary DNA clone for this kinase has been identified; it encodes a protein with a predicted molecular mass of 57,175 daltons. This protein contains a catalytic domain similar to that of calmodulin-dependent kinases and a calmodulin-like region with four calcium binding domains (EF hands). The predicted structure of this kinase explains its direct regulation via calcium binding and establishes it as a prototype for a new family of calcium-regulated protein kinases.
A protein kinase that is activated by calcium and lipid has been partially purified from the plasma membrane of oat roots. This protein kinase cross-reacts with four monoclonal antibodies directed against a soluble calcium-dependent protein kinase from soybean described previously [Putman-Evans, C. L., Harmon, A. C., & Cormier, M. J. (1990) Biochemistry 29, 2488-2495; Harper, J. F., Sussman, M. R., Schaller, G. E., Putnam-Evans, C., Charbonneau, H., & Harmon, A. C. (1991) Science 252, 951-954], indicating that the oat enzyme is a member of this calcium-dependent protein kinase family. Immunoblots demonstrate that the membrane-derived protein kinase is slightly larger than that observed in the cytosolic fraction of oat. Limited digestion of the membrane-derived kinase with trypsin generates a smaller water-soluble kinase that is still activated by calcium but is no longer activated by lipid. When posthomogenization proteolysis is minimized, the bulk of the immunoreactive kinase material is localized in the membrane. These results suggest that a calcium-dependent protein kinase observed in the supernatant fraction of oat extracts may originate in situ from a calcium- and lipid-dependent protein kinase which is associated with the oat plasma membrane. They further indicate that, in contrast to animal cells, the predominant calcium- and lipid-dependent protein kinase associated with the plasma membrane of plant cells has biochemical properties and amino acid sequence unlike protein kinase C.
In crude extracts of plant tissue, the Mr = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4°C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cationtransporting ATPases.The plasma membrane of higher plants and fungi contains an electrogenic, proton-pumping ATPase (H+-ATPase). The H+-ATPase uses the chemical energy of ATP to extrude protons into the external medium. This generates a proton electrochemical gradient, also termed a protonmotive force, which is used to drive solute uptake systems. In addition to providing a driving force for nutrient uptake, the H+-ATPase helps to establish the turgor, pH, and ionic composition necessary for growth of the cell (4, 18).The H+-ATPase belongs to a class of cation-transporting ATPases with similar enzymatic and structural properties. Other members include the Na+, K+-ATPase, the H+, K+-ATPase, and the Ca2+-ATPase of animal membranes, and the K+-ATPase of Escherichia coli. All of these enzymes are inhibited by van-adate and have a catalytic subunit of M, = 100,000 that is phosphorylated during the reaction cycle. The complete amino acid sequences for many of the cation-transporting ATPases (6,10,16) MATERIALS AND METHODSPlasma Membrane Isolation. Oat root plasma membranes were isolated and assayed for ATPase activity as described by Surowy and Sussman (20). Briefly, oat roots were homogenized, strained through cheesecloth, and centrifuged at 8,000g for 15 min to remove mitochondria and cellular debris. Microsomes were pelleted from the supernatant by centrifugation at 48,000g for 1.5 h, and then applied to a discontinuous sucrose gradient. After centrifugation at 200,000g for 1.5 h, plasma membranes were collected at the 33%146% (w/w) sucrose interface. ATPase activity was assayed colorimetrically by measuring the release of inorganic phosphate from ATP. Plasma membrane protein was assayed by the method of Lowry (9), using BSA as standard. For the experiments described here, 200 mg of oat plasma membrane protein obtained from 3.6 kg of oat roots were used. Specific activity of the H+-ATPase in these membranes was 1.3 ,umol inorganic phosphate released x (mg protein)-I x min-. ...
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