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Type 1 IFNs stimulate secretion of IP-10 (CXCL10) which is a critical chemokine to recruit effector T cells to the tumor microenvironment and IP-10 knockout mice exhibit a phenotype with compromised effector T cell generation and trafficking. Type 1 IFNs also induce MHC class 1 upregulation on tumor cells which can enhance anti-tumor CD8 T cell effector response in the tumor microenvironment. Although type 1 IFNs show great promise in potentiating anti-tumor immune response, systemic delivery of type 1 IFNs is associated with toxicity thereby limiting clinical application. In this study, we fused tumor targeting antibodies with IFN-α and showed that the fusion proteins can be produced with high yields and purity. IFN fusions selectively induced IP-10 secretion from antigen positive tumor cells, which was critical in recruiting the effector T cells to the tumor microenvironment. Further, we found that treatment with the anti-PDL1-IFN- α fusion at concentrations as low as 1 pM exhibited potent activity in mediating OT1 CD8+ T cell killing against OVA expressing tumor cells, while control IFN fusion did not exhibit any activity at the same concentration. Furthermore, the IFN-α fusion antibody was well tolerated in vivo and demonstrated anti-tumor efficacy in an anti-PD-L1 resistant syngeneic mouse tumor model. One of the potential mechanisms for the enhanced CD8 T cell killing by anti-PD-L1 IFN fusion was up-regulation of MHC class I/tumor antigen complex. Our data supports the hypothesis of targeting type 1 IFN to the tumor microenvironment may enhance effector T cell functions for anti-tumor immune response., Competing Interests: JG, YX, RI, XL, ML, UL, JH, TS, GB, AS, and TH are employees of AbbVie. MT & CD were AbbVie employees at the time of the study. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

The Bcl-2 family of proteins plays a major role in the regulation of apoptosis, or programmed cell death. Overexpression of the anti-apoptotic members of this family (Bcl-2, Bcl-x(L), and Mcl-1) can render cancer cells resistant to chemotherapeutic agents and therefore these proteins are important targets for the development of new anti-cancer agents. Here we describe the discovery of a potent, highly selective, Bcl-2 inhibitor using SAR by NMR and structure-based drug design which could serve as a starting point for the development of a Bcl-2 selective anti-cancer agent. Such an agent would potentially overcome the Bcl-x(L) mediated thrombocytopenia observed with ABT-263., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Bcl-B protein is an anti-apoptotic member of the Bcl-2 family protein that contains all the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal transmembrane domain. Our previous results showed that Bcl-B binds Bax and suppresses apoptosis induced by over-expression of Bax; however, Bcl-B does not bind or suppress Bak. To explore the molecular basis for the differential binding and suppression of Bax and Bak, we studied the BH3 dimerization domains of Bax and Bak. Chimeric mutants of Bax and Bak were generated that swapped the BH3 domains of these pro-apoptotic proteins. Bcl-B associated with and blocked apoptosis induced by mutant Bak containing the BH3 domain of Bax, but not mutant Bax containing the BH3 domain of Bak. In contrast, Bcl-X(L) protein bound and suppressed apoptosis induction by Bax, Bak and both BH3-domain chimeras. A strong correlation between binding and apoptosis suppression was also obtained using a series of alanine substitutions spanning the length of the Bax BH3 domain to identify critical residues for Bcl-B binding. Conversely, using structure-based modelling to design mutations in the BH3-binding pocket of Bcl-B, we produced two Bcl-B mutants (Leu86-->Ala and Arg96-->Gln) that failed to bind Bax and that also were unable to suppress apoptosis induced by Bax over-expression. In contrast, other Bcl-B mutants that still bound Bax retained protective activity against Bax-induced cell death, thus serving as a control. We conclude that, in contrast with some other anti-apoptotic Bcl-2-family proteins, a strong correlation exists for Bcl-B between binding to pro-apoptotic multidomain Bcl-2 family proteins and functional apoptosis suppression.

We describe a new approach to affinity selection based on the application of centrifugal force to macromolecules in solution. The method relies on the well known macromolecular hydrodynamic principles of centrifugation. It can be automated and operated in a centralized fashion, or it can be decentralized and used by single researchers or networks of researchers with a minimal additional capital investment. In this method, a centrifugal driving force is used to establish a differential and selective concentration gradient between a therapeutic target and potential ligands in compound libraries. This concentration gradient, in turn, drives the binding of ligands. Once formed, the differential concentration gradient of target macromolecules and ligands is fractionated to capture the self-sorting binding events. Ligand binding is defined by the individual ligand binding constants, so tight binding ligands will essentially distribute identically with the protein target, and weaker binding ligands will not. The level of affinity needed to operationally define tight binding can be adjusted by selecting the initial concentration conditions or centrifugal force. A variety of rapid, commonly available, detection methods can be used to assess binding in the fractionated samples. The method can be broadly applied in drug discovery efforts to examine most types of cell-cell, protein-protein, and protein-small molecule interactions. We describe the application of this method to systems of small molecule interactions with several macromolecules of therapeutic interest.

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.

The pyrophosphate-dependent phosphofructokinase (PPi-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5' half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PPi-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PPi-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PPi-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the alpha- and beta-subunits of plant PPi-PFKs. The third group ("X") containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group ("Y") comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PPi-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers.

The prevalent mechanism of bacterial resistance to erythromycin and other antibiotics of the macrolide-lincosamide-streptogramin B group (MLS) is methylation of the 23S rRNA component of the 50S subunit in bacterial ribosomes. This sequence-specific methylation is catalyzed by the Erm group of methyltransferases (MTases). They are found in several strains of pathogenic bacteria, and ErmC is the most studied member of this class. The crystal structure of ErmC' (a naturally occurring variant of ErmC) from Bacillus subtilis has been determined at 3.0 A resolution by multiple anomalous diffraction phasing methods. The structure consists of a conserved alpha/beta amino-terminal domain which binds the cofactor S-adenosyl-l-methionine (SAM), followed by a smaller, alpha-helical RNA-recognition domain. The beta-sheet structure of the SAM-binding domain is well-conserved between the DNA, RNA, and small-molecule MTases. However, the C-terminal nucleic acid binding domain differs from the DNA-binding domains of other MTases and is unlike any previously reported RNA-recognition fold. A large, positively charged, concave surface is found at the interface of the N- and C-terminal domains and is proposed to form part of the protein-RNA interaction surface. ErmC' exhibits the conserved structural motifs previously found in the SAM-binding domain of other methyltransferases. A model of SAM bound to ErmC' is presented which is consistent with the motif conservation among MTases.

An acid protease activity from human brain was found to cleave a fluorogenic peptide substrate encompassing the amino terminus of Alzheimer's amyloid-beta peptide (A beta). The protease was isolated and determined to be cathepsin D based on chromatographic, immunological, and enzymatic data. Analysis of the cleavage sites indicated that cathepsin D hydrolyzed the methionine--aspartate bond generating the in vivo amino terminus of A beta. These data suggested that cathepsin D could be involved in amyloidogenic processing of the amyloid precursor protein. Consequently, cathepsin D from both Alzheimer's-diseased and control brains was compared to determine whether there were any differences which could account for an increase in A beta production in Alzheimer's disease. No differences were detected in isoform composition or tissue content of cathepsin D as measured by 2-D IEF-SDS-PAGE. Enzymological characterization of brain cathepsin D demonstrated that it could undergo a previously undescribed pH-dependent reversible activation. However, that activation appeared identical for both AD and normal brain enzymes. These data demonstrate that concentration, isoform distribution, and several enzymological characteristics of cathepsin D are not distinguishable between AD and normal brain. The pH dependence of cathepsin D activity suggests, however, that its intracellular localization may be important in considering the potential role of cathepsin D in Alzheimer's disease.

The effect of the Kunitz proteinase inhibitor (KPI) on potential beta-amyloid precursor protein (beta PP)-processing activities from control and Alzheimer's disease (AD) brains was examined using fluorogenic substrates designed to mimic the secretory and amyloidogenic cleavages in beta PP. In addition, the level of secretion of KPI-containing beta PP751 and KPI-lacking beta PP695 from transfected cells was examined to assess the effect of the KPI on beta PP secretion. beta PP751 and beta PP695, obtained from conditioned media of transfected cells, had no effect on proteinase activities against the secretory and amyloidogenic substrates in extracts from control and AD brains. At similar concentrations beta PP751, but not beta PP695, completely inhibited the activity of trypsin against these substrates. Serine proteinase inhibitors had only modest effects on activities from brain, whereas cysteine modification completely inhibited them, indicating that these proteinase activities were not of the serine type. Thus, the results do not support a role for the KPI in the secretion of beta PP or in the amyloidogenic cleavage of beta PP. The amounts of beta PP695 and beta PP751 collected from the media of transfected cells after 48 h of growth were similar, indicating an equal rate of secretion. This result suggests that the KPI domain in beta PP751 did not inhibit the secretory cleavage in transfected cells.

The proteases that cleave amyloid precursor protein (APP) leading to generation of amyloid A beta peptide are potential targets for therapeutical intervention of Alzheimer disease. We have been pursuing the identification and characterization of these proteases using as probes the fluorogenic substrates encompassing the cleavage sites of APP that we described recently (Wang, G. T., Krafft, G. A. [1992] Bioorg. Med. Chem. Lett. 2, 1665). This article describes results of experiments designed to examine the effect of Ca(2+) on the cleavage of these substrates by human brain extracts. Fluorogenic substrates encompassing either the N-terminal amyloidogenic cleavage site or the secretory cleavage site were synthesized in five formats with various peripheral residues. Incubation with extracts from normal brain tissue revealed that more negatively charged amyloidogenic substrates were less reactive and exhibited larger rate enhancement in the presence of Ca(2+). The results imply that Ca(2+) stimulation of substrate cleavage by brain proteases occurs primarily as a result of Ca(2+)-substrate interactions, and caution against interpretations that invoke the involvement of Ca(2+)-stimulated proteases in A beta formation.

One of the clinical manifestations of Alzheimer's disease is the deposition of the 39-43 residue amyloid-beta (A beta) peptide in aggregated fibrils in senile plaques. Characterization of the aggregation behavior of A beta is one of the critical issues in understanding the role of A beta in the disease process. Using solution hydrodynamics, A beta was observed to form three types of species in phosphate-buffered saline: insoluble aggregates with sedimentation coefficients of approximately 50,000 S and molecular masses of approximately 10(9) Da, "soluble aggregates" with sedimentation coefficients of approximately 30 S and masses of approximately 10(6) Da, and monomer. When starting from monomer, the aggregation kinetics of A beta 1-40 (A beta 40) and A beta 1-42 (A beta 42), alone and in combination, reveal large differences in the tendency of these peptides to aggregate as a function of pH and other solution conditions. At pH 4.1 and 7.0-7.4, aggregation is significantly slower than at pH 5 and 6. Under all conditions, aggregation of the longer A beta 42 was more rapid than A beta 40. Oxidation of Met-35 to the sulfoxide in A beta 40 enhances the aggregation rate over that of the nonoxidized peptide. Aggregation was found to be dependent upon temperature and to be strongly dependent on peptide concentration and ionic strength, indicating that aggregation is driven by a hydrophobic effect. When A beta 40 and A beta 42 are mixed together, A beta 40 retards the aggregation of A beta 42 in a concentration-dependent manner. Shorter fragments have a decreasing ability to interfere with A beta 42 aggregation. Conversely, the rate of aggregation of A beta 40 can be significantly enhanced by seeding slow aggregating solutions with preformed aggregates of A beta 42. Taken together, the inhibition of A beta 42 aggregation by A beta 40, the seeding of A beta 40 aggregation by A beta 42 aggregates, and the chemical oxidation of A beta 40 suggest that the relative abundance and rates of production of different-length A beta and its exposure to radical damage may be factors in the accumulation of A beta in plaques in vivo.

Amyloid beta (A beta) is a 39-43-residue protein that originates from proteolysis of the beta-protein precursor (beta PP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Mutant beta PP, which incorporates an AD-causing double mutation at positions 687-688, has been shown to enhance A beta production in transfected cells. In this work we investigate the susceptibility of the mutant beta PP sequence to proteolytic cleavage by proteinases from human brain. Internally quenched fluorogenic substrates were used that encompass the NH2-terminal sequence of A beta from wild-type beta PP, the double mutant, and the two single substitutions. Proteinase activity in brain extract cleaved the mutant substrate 100-fold faster than the wild-type substrate and the partial mutants 25-fold faster. The major cleavage site in all substrates was at the amyloidogenic Asp1 site. The brain activity appeared to be cathepsin D (CD), as indicated by similarities to purified CD in 1) the rate and site of substrates cleavage, 2) the pH optima, and 3) the sensitivity to pepstatin A. The increased activity against the mutant substrate was not shared by cathepsins B and C, pepsin, HIV proteinase, and Candida albicans Asp-proteinase. Furthermore, CD cleaved a substrate that incorporates the COOH terminus of A beta at positions equivalent to Thr43 and Ala42, at ratios of 68% and 32%, respectively. CD degraded A beta 1-40 into six fragments but A beta 1-42 was completely resistant to digestion, probably because of its aggregation characteristics. These results indicate that CD is capable of producing the cleavages resulting in A beta production and that it may prove to be a suitable therapeutic target.

The tendency of both labeled and unlabeled beta-amyloid to bind in solution to C1q, the recognition species in the complement cascade, was examined using both hydrodynamic and spectroscopic methods. Potential binding interactions were evaluated using a purified synthetic beta-amyloid 1-40 sequence, alone, and selectively labeled at the amino terminus with spectroscopic probes. The probes permitted both absorbance and fluorescence analyses of beta-amyloid binding interactions. Under conditions used for the analyses beta-amyloid exists exclusively as a monomer in solution, and C1q retains an intact quaternary structure and is capable of binding to IgM. When mixed together the monomeric beta-amyloid does not bind to, or interact with, the complement C1q at concentrations below approximately 100 microM. The data suggest that if beta-amyloid toxicity is associated with complement activation in Alzheimer's disease then monomeric beta-amyloid is likely not responsible for activation through the classical complement pathway.

Fluorogenic peptide substrates designed to encompass the reported alpha-secretory and amyloidogenic cleavage sites of the amyloid-beta precursor protein (beta PP) were used to analyze proteinase activities in brain extracts from control patients and those with Alzheimer's disease (AD). Activity against the secretory substrate at pH 7.5 in control and AD brains produced a major endopeptidase cleavage at the Lys687-Leu688 bond (beta PP770 numbering), consistent with the beta PP secretase cleavage. Activity in control brains against the amyloidogenic substrate at pH 7.5 produced one cleavage at the Ala673-Glu674 bond, two residues C-terminal to the amyloidogenic Met-Asp site. However, in three of four AD brains, the major cleavage was at the Asp-Ala bond, one residue from the amyloidogenic site. Both endopeptidase and carboxypeptidase activities in AD brains were lower than in control brains. Proteinase activities against the secretory substrate had a major optimum at pH 3.0-4.0 and another at pH 6.0-7.5. Proteinase activities against the amyloidogenic substrate had a major optimum at or below pH 3.0 and another at pH 6.0. Using both substrates, activities at low pH were higher in AD-brains than in controls, while at pH above 6.5, activities in control brains were higher than in AD. These results indicate that the levels of proteolytic enzymes in AD brains are altered relative to controls.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii is a non-allosteric enzyme with properties dissimilar to those of other described phosphofructokinases. The enzyme was cloned into pBluescript, sequenced, and expressed in Escherichia coli at levels 15 times higher than those observed in Propionibacterium. The gene consists of 1215 bases which code for a protein of 404 amino acids and a mass of 43,243 daltons. High G + C in the codon usage (66%) of the gene is consistent with the classification of Propionibacterium in the High-G + C subdivision of the Gram-positive bacteria. While showing no sequence identity to the non-allosteric ATP-dependent phosphofructokinase of E. coli, alignments of the amino acid sequence with other PFKs reveal degrees of identities among the amino halves of the proteins, from 26% between the Propionibacterium and potato PPi-PFKs, and 29% between Propionibacterium and E. coli ATP-PFKs. These levels of identities indicate that the amino halves of these proteins are homologous. Identities between the carboxyl half of Propionibacterium PFK and carboxyl halves of other sequences are below 20%, suggesting that the carboxyl half is not homologous. Despite the poor conservation, most of the residues that take part in the binding of fructose-6-P or Mg-PPi could be readily identified by analogy to the structure of the E. coli PFK. Both the fructose-6-P and ATP-binding sites are conserved, indicating that PPi binds to the homologous site of the E. coli ATP-binding site.

Two macros, to calculate percentage identity between protein or DNA sequences using the Microsoft Word word processor, are described. The user prepares an alignment file of multiple sequences which is used by the macros to calculate number of matches, number of mismatches, total number of compared positions, and the percent identity. The macros are especially useful when alignment of multiple sequences is possible only by eye.

Cytosolic fructose-1,6-bisphosphatase from spinach leaves was purified to homogeneity and characterized. The pure enzyme has a subunit mass of 38 kDa, its Km values for fructose 1,6-bisphosphate and Mg2+ are 1.5 microM and 260 mM, respectively, and its Vmax is 110-120 units/mg. It is inhibited by fructose 2,6-bisphosphate and AMP with Ki values of 0.07 microM and 120 microM, respectively. About 90% of the primary structure of the spinach cytosolic fructose-1,6-bisphosphatase has been determined by amino-acid sequencing. The sequence data demonstrate that the cytosolic enzyme lacks the sequence insert characteristic of chloroplast fructose-1,6-bisphosphatase. The data include also the sequences of peptides containing all seven cysteine residues. Only two of the seven cysteines are conserved between the two isozymes, none of which is believed to be involved with the light regulation of the chloroplast enzyme. Sequence comparisons between the spinach cytosolic enzyme and gluconeogenic fructose-1,6-bisphosphatases from other species reveal similarity ranging over 47-54%, which is higher than the 40-45% similarity between the chloroplast enzyme and gluconeogenic fructose-1,6-bisphosphatases. However, similarity between these isozymes and Escherichia coli fructose-1,6-bisphosphatase are 44% and 47% for the cytosolic and chloroplast enzymes, respectively. Similarity between the cytosolic and chloroplast counterparts is 52%, indicating wide divergence between these two fructose-1,6-bisphosphatases.

The effects of calmodulin (CaM) on ATPase activity and ATP-dependent formation of a proton gradient (DeltapH) were studied in tonoplast membrane vesicles from corn (Zea mays L.) roots. At 0.6 micromolar, CaM stimulated ATPase activity by about 20% in the absence of an uncoupler, but by only 4% in its presence. Thus, the uncoupler-dependent increment of activity was decreased 30 to 45% by CaM. The formation of a proton gradient across the membrane vesicle, measured by quinacrine fluorescence quench, was inhibited about 20% by CaM. Its effect was additive to the effect of Ca(2+) and was completely abolished by EGTA. These effects of CaM could be due to stimulation of H(+) efflux or due to inhibition of the H(+)-ATPase. To distinguish between these possibilities, we examined the effect of CaM on dissipation of preformed DeltapH after the ATPase was inhibited. CaM stimulated the dissipation of a preformed DeltapH by 40% after the H(+)-ATPase was inhibited with NO(3) (-). This indicates that CaM facilitates the recycling of protons across the tonoplast membranes and does not regulate the H(+)-ATPase by direct inhibition.

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H(+)-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a K(m) value for MgATP(2-) of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg(2+) and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca(2+), but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H(+)-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.