Gideon M. Polya

Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin.

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6+/-0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma-thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC(50) 2 µM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor.

Inhibition of signal-regulated protein kinases by plant-derived hydrolysable tannins

A variety of hydrolysable tannins purified from Phyllanthus amarus are potent inhibitors of rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK) with IC50 values (concentrations for 50% inhibition) in the range 0.2-1.7 microM. The three most effective compounds of this series of hydrolysable tannins have five phenolic substituents. These three compounds are also the most effective inhibitors of wheat embryo Ca(2+)-dependent protein kinase (CDPK), rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC) and Ca(2+)-calmodulin-dependent myosin light chain kinase (MLCK). The order of sensitivity for protein kinase inhibition by the hydrolysable tannins studied is cAK > CDPK > PKC > MLCK. Thus the IC50 values for protein kinase inhibition by the most potent compound are 0.2 microM (for cAK), 1.8 microM (for CDPK), 26 microM (for PKC) and 56 microM (for MLCK) when protein kinase affinity is measured using synthetic peptide substrates. These hydrolysable tannin inhibitors found are the most specific and potent plant-derived inhibitors of cAK yet found.

Selective inhibition of cyclic AMP-dependent protein kinase by amphiphilic triterpenoids and related compounds.

A set of plant- and animal-derived amphiphilic triterpenoids have been shown to be potent and selective inhibitors of the catalytic subunit of rat liver cyclic AMP-dependent protein kinase (cAK). Thus plant-derived 18 alpha- and 18 beta-glycyrrhetinic acid, ursolic acid, oleanolic acid and betulin and animal-derived lithocholic acid, 5-cholenic acid and lithocholic acid methyl ester are inhibitors of cAK with IC50 values (concentrations for 50% inhibition) in the range 4-20 microM. These compounds are ineffective or relatively ineffective as inhibitors of various other eukaryote signal-regulated protein kinases namely wheat embryo Ca(2+)-dependent protein kinase (CDPK), avian calmodulin-dependent myosin light chain kinase (MLCK) and rat brain Ca(2+)- and phospholipid-dependent protein kinase C (PKC). These naturally occurring triterpenoids have a common structural motif involving polar residues located at opposite ends of an otherwise non-polar triterpenoid nucleus. A variety of triterpenoids not possessing this structural motif are relatively inactive as inhibitors of cAK and of CDPK, PKC and MLCK. The terpenoid amphiphilic compound crocetin is also a potent and relatively selective inhibitor of cAK (IC50 value for cAK 3.0 microM). 12-Hydroxystearic acid and 10-hydroxydecanoic acid do not inhibit CDPK, PKC or MLCK but are selective inhibitors of cAK (IC50 values 127 and 138 microM, respectively), consistent with a simple model for amphiphile inhibition of cAK involving two polar groups separated by a non-polar region. However, laurylgallate and 15-pentadecanolide are also potent and selective inhibitors of cAK (IC50 values 1.5 and 20 microM, respectively) although the structures of both of these compounds involve a large non-polar portion associated with only one polar region. Crocetin and the plant-derived amphiphilic triterpenoids described here are the most potent non-aromatic plant-derived inhibitors of cAK yet found.

Resolution of Ca2+—calmodulin-activated protein kinase from wheat germ

A soluble Ca2+‐ and Ca2+—calmodulin‐activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca2+‐dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3‐fold and up to 16‐fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca2+‐dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca2+‐dependent fashion. This type of Ca2+‐activated protein kinase may be involved in stimulus—response coupling in plants.

Properties of a high-affinity cytokinin-binding protein from wheat germ

A soluble protein that interacts with a range of cytokinins was extensively purified from wheat (Triticum aestivum L.) germ. This protein has a Kd for kinetin of 2×10-7 M. The binding of kinetin to the protein is inhibited by low concentrations of synthetic and naturally-occurring cytokinins including N6-benzyladenine, N6-benzyladenosine, kinetin riboside, N6-dimethylallyladenine, N6-dimethylallyladenosine, zeatin, zeatin riboside, N6-dimethyladenine and N6-dimethyladenosine. Adenine, adenosine and several non-N6-substituted adenine derivatives were ineffective as inhibitors of kinetin binding. While N6-butyryl-3′,5′-cyclic AMP, N6,2-O′-dibutyryl-3′,5′-cyclic AMP and 2′,3′-cyclic AMP inhibited binding of kinetin to the protein, 3′,5′-cyclic AMP was ineffective. The kinetin-binding protein is heat-labile and pronase-sensitive. Kinetin-binding activity exactly co-chromatographs with a single peak of carbohydrate and protein on gel-filtration and is displaced from concanavalin A-Sepharose 4B by α-methylglucoside. On gel filtration, the kinetin-binding protein behaves as a soluble protein with an apparent molecular weight of 180,000 daltons.

Activation of wheat embryo calcium-regulated protein kinase by unsaturated fatty acids in the presence and absence of calcium

Unsaturated fatty acids (oleic, linoleic and arachidonic acids) markedly activate extensively purified wheat embryo Ca2+‐regulated protein kinase in the absence of Ca2+ or at very low free Ca2+ levels (<0.4 μM). While oleic and linoleic acids also activate in the presence of Ca2+ (10−6−10−4 M), arachidonic acid inhibits at free Ca2+ greater than 10−5 M. Phosphatidylserine does not substantially activate in the absence of Ca2+ or the presence of high Ca2+. Stearic and arachidic acids are less effective than the unsaturated fatty acids as activators in the absence of Ca2+. This type of plant protein kinase may be regulated in vivo independently of Ca2+ through release of unsaturated fatty acids.

Phosphorylation of style S-RNases by Ca2+-dependent protein kinases from pollen tubes

Solanaceous plants with gametophytic self-incompatibility produce ribonucleases in the transmitting tract of the style that interact with self-pollen and inhibit its growth. These ribonucleases are a series of allelic products of the S-locus, which controls self-incompatibility. Little is known about the pollen components involved in this interaction or whether a signal transduction pathway is activated during the self-incompatibility response. We have partially purified a soluble protein kinase from pollen tubes of Nicotiana alata that phosphorylates the self-incompatibility RNases (S-RNases) from N. alata but not Lycopersicon peruvianum. The soluble protein kinase (Nak-1) has several features shared by the calcium-dependent protein kinase (CDPK) class of plant protein kinases, including substrate specificity, calcium dependence, inhibition by the calmodulin antagonist calmidazolium, and cross-reaction with monoclonal antibodies raised to a CDPK from soybean. Phosphorylation of S2-RNase by Nak-1 is restricted to serine residues, but the site(s) of phosphorylation has not been determined and there is no evidence for allele-specific phosphorylation. The microsomal fraction from pollen tubes also phosphorylates S-RNases and this activity may be associated with proteins of Mr∼60 K and 69 K that cross-react with the monoclonal antibody to the soybean CDPK. These results are discussed in the context of the involvement of phosphorylation in other self-incompatibility systems.

Purification and Sequencing of Radish Seed Calmodulin Antagonists Phosphorylated by Calcium-Dependent Protein Kinase

A family of radish (Raphanus sativus) calmodulin antagonists (RCAs) was purified from seeds by extraction, centrifugation, batch-wise elution from carboxymethyl-cellulose, and high performance liquid chromatography (HPLC) on an SP5PW cation-exchange column. This RCA fraction was further resolved into three calmodulin antagonist polypeptides (RCA1, RCA2, and RCA3) by denaturation in the presence of guanidinium HCI and mercaptoethanol and subsequent reverse-phase HPLC on a C8 column eluted with an acetonitrile gradient in the presence of 0.1% trifluoroacetic acid. The RCA preparation, RCA1, RCA2, RCA3, and other radish seed proteins are phosphorylated by wheat embryo Ca2+-dependent protein kinase (CDPK). The RCA preparation contains other CDPK substrates in addition to RCA1, RCA2, and RCA3. The RCA preparation, RCA1, RCA2, and RCA3 inhibit chicken gizzard calmodulin-dependent myosin light chain kinase assayed with a myosin-light chain-based synthetic peptide substrate (fifty percent inhibitory concentrations of RCA2 and RCA3 are about 7 and 2 [mu]M, respectively). N-terminal sequencing by sequential Edman degradation of RCA1, RCA2, and RCA3 revealed sequences having a high homology with the small subunit of the storage protein napin from Brassica napus and with related proteins. The deduced amino acid sequences of RCA1, RCA2, RCA3, and RCA3[prime] (a subform of RCA3) have agreement with average molecular masses from electrospray mass spectrometry of 4537, 4543, 4532, and 4560 kD, respectively. The only sites for serine phosphorylation are near or at the C termini and hence adjacent to the sites of proteolytic precursor cleavage.

Partial purification and characterization of a second Calmodulin-activated Ca2+-dependent protein kinase from wheat germ

Abstract A soluble Ca 2+ -dependent protein kinase was partially purified wheat germ by a procedure involving adsorption to DEAE-cellulose, Ca 2+ -dependent binding to phenyl-Sepharose, (NH 4 ) 2 SO 4 precipitation and gel filtration. The protein kinase ( M r 86 000) catalyzes the phosphorylation of histones, casein and phosvitin. Calmodulin activates the phosphorylation of histones catalyzed by the protein kinase. The protein is largely dependent upon Ca 2+ for activity. The rate of casein phosphorylation is half-maximal at 0.3 μM free Ca 2+ and maximal at 3 μM free Ca 2+ ; much higher free Ca 2+ concentrations are required for half-maximal (60 μM) and maximal (500 μM) rates of histone phosphorylation. Millimolar Mg 2+ is required in addition to Ca 2+ for maximal activity of the enzyme and millimolar Mn 2+ can substitute for the (Ca 2+ + Mg 2+ ) requirement. The protein kinase is inhibited by the calmodulin antagonists, chlorpromaxine and fluphenazine. The K m for ATP is 16 μM and the enzyme phosphorylates serine and threonine residues of casein. The enzyme is very similar to a Ca 2+ - and calmodulin-activated protein kinase isolated from wheat-germ chromatin, but differs from this enzyme in various properties, notably apparent molecular size and insensitivity to nucleotides that inhibit the chromatin-derived enzyme.

Inhibition of eukaryote signal-regulated protein kinases by plant-derived catechin-related compounds

The cladodes of Phyllocladus trichomanoides, the bark of Pseudotsuga menziesii and the heartwood of Acacia melanoxylon contain catechin derivatives that are potent inhibitors of rat brain protein kinase C. Most of these compounds are also inhibitors of bovine heart cyclic AMP-dependent protein kinase catalytic subunit and wheat Ca(2+)-dependent protein kinase. However, these compounds are either not inhibitors or are relatively poor inhibitors of avian myosin light chain kinase. The most potent protein kinase inhibitors are the procyanidin dimer, trimer and tetramer from the bark of Pseudotsuga menziesii. These compounds are among the most potent plant-derived protein kinase inhibitors yet found.