Exogenous Protein Substrates Research Articles

Pharmacokinetics mechanism of ABC efflux proteins-mediated seven features of compatibility

ABC efflux proteins are a kind of transporters mediating diversified endogenous and exogenous efflux protein substrates across the plasma membrane by depending on the chemical energy released by ATP hydrolysis. As a vitally important functional membrane, it is widely found in various tissues and organs. The drug changes the expressions and/or functions of the transport proteins, which will affect the disposal process of substrate drugs corresponding to transporters in vivo, and finally lead to the pharmacokinetic interactions. The efflux proteins take part in the absorption, distribution, metabolism and excretion of drugs, and mainly consist of P-glycoprotein(P-gp), multidrug resistance associated protein(MRP) and breast cancer resistance protein(BCRP). The induction effect or inhibition effect of drugs on efflux protein plays a greatly significant role in the drug interaction produced by the compatibility of traditional Chinese medicine, which may be one of the important mechanisms of the theory of seven features of compatibility. In this article, the effects of seven features of compatibility on the ABC efflux transporters were reviewed, in order to reveal the roles of efflux protein in the herb-pairs compatibility, and provide new ideas for the mechanism and rationality of herb compatibility.

Implications of Sortilin in Lipid Metabolism and Lipid Disorder Diseases.

The present review provides a summary of recent evidence of sortilin expression, function, and regulation and its implications in lipid metabolism and development of lipid disorder diseases. As a member of the vacuolar protein sorting 10 protein (Vps10p) receptor family, sortilin mediates intracellular trafficking of diverse endogenous or exogenous protein substrates between the trans-Golgi network (TGN) and plasma membrane compartments. Recent studies reveal that sortilin regulates the expression of lipid genes, plasma lipid level, and the development of lipid disorder diseases. Sortilin promotes atherogenesis by regulating hepatic very low density lipoprotein (VLDL) secretion and plasma lipid level and subsequently macrophage lipid accumulation. Sortilin deficiency is caused by accelerated proteasome degradation under insulin resistance conditions and is thereby implicated in the hyperlipidemia of type 2 diabetes mellitus (T2DM). Sortilin facilitates hepatic cholesterol accumulation by inhibiting hepatic cholesterol catabolism, which promotes the development of nonalcoholic fatty liver disease (NAFLD). Sortilin plays an important role in lipid metabolism and represents a promising therapeutic target for lipid disorder diseases.

Activation, Isolation, and Analysis of the Death-Inducing Signaling Complex.

This protocol describes activation, isolation, and analysis of the CD95 (APO-1/Fas) death-inducing signaling complex (DISC) using affinity purification. Activation is achieved using a biotin-labeled anti-CD95 antibody and the native DISC complex is captured using streptavidin beads. This approach minimizes both the number of steps involved and any potential nonspecific interactions or cross-reactivity of antibodies commonly seen in immunoprecipitations using unlabeled antibodies and protein A/G beads. Composition of the isolated complex is analyzed via western blot to identify known DISC components, and dimerization-induced autocatalytic processing of procaspase-8 at the DISC can be confirmed by detection of caspase-8 cleavage products. The potential for DISC-associated caspase-8 to activate the caspase cascade can be determined by measuring caspase-8-dependent cleavage of the fluorigenic substrate Ac-IETD.AFC, or by performing a bioassay using exogenous protein substrates.

Identification in Lupin Seed of a Serine-Endopeptidase Activity Cleaving between Twin Arginine Pairs and Causing Limited Proteolysis of Seed Storage Proteins

The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.

Plasmodium falciparum possesses a unique dual-specificity serine/threonine and tyrosine kinase, Pfnek3.

Despite the absence of classical tyrosine kinases encrypted in the kinome of Plasmodium falciparum, biochemical analyses have detected significant tyrosine phosphorylation in its cell lysates. Supporting such phosphorylation is critical for parasite development. These observations have thus raised queries regarding the plasmodial enzymes accountable for tyrosine kinase activities in vivo. In the current investigation, immunoblot analysis intriguingly demonstrated that Pfnek3, a plasmodial mitogen-activated protein kinase kinase (MAPKK), displayed both serine/threonine and tyrosine kinase activities in autophosphorylation reactions as well as in phosphorylation of the exogenous myelin basic protein substrate. The results obtained strongly support Pfnek3 as a novel dual-specificity kinase of the malarial parasite, even though it displays a HGDLKSTN motif in the catalytic loop that resembles the consensus HRDLKxxN signature found in the serine/threonine kinases. Notably, its serine/threonine and tyrosine kinase activities were found to be distinctly influenced by Mg(2+) and Mn(2+) cofactors. Further probing into the regulatory mechanism of Pfnek3 also revealed tyrosine phosphorylation to be a crucial factor that stimulates its kinase activity. Through biocomputational analyses and functional assays, tyrosine residues Y117, Y122, Y172, and Y238 were proposed as phosphorylation sites essential for mediating the catalytic activities of Pfnek3. The discovery of Pfnek3's dual role in phosphorylation marks its importance in closing the loop for cellular regulation in P. falciparum, which remains elusive to date.

CoPK32 is a novel stress-responsive protein kinase in the mushroom Coprinopsis cinerea

BackgroundIn a previous study, we conducted an expression cloning screen of a cDNA library prepared from Coprinopsis cinerea mycelia using Multi-PK antibodies and detected a wide variety of Ser/Thr protein kinases. One of the isolated clones, CMZ032, was found to encode a putative Ser/Thr protein kinase designated CoPK32. In the present study, we investigated the biochemical properties and physiological significance of CoPK32. MethodsCoPK32 was expressed in Escherichia coli, and its biochemical properties were examined. The effects of high osmotic stresses on the growth of C. cinerea and on the endogenous CoPK32 activity in mycelia were also examined. ResultsCoPK32 showed autophosphorylation activity and effectively phosphorylated exogenous protein substrates. CoPK32S, a splice variant that was 18 amino acids shorter than CoPK32, showed much lower protein kinase activity than CoPK32. The catalytic properties of CoPK32 deletion mutants suggested that the C-terminal region of CoPK32 was important for the kinase activity and recognition of substrates. CoPK32 was highly expressed in the actively growing region of the mycelial colony. When mycelia were stimulated by high osmotic stresses, endogenous CoPK32 was markedly activated and the mycelial growth was severely inhibited. The activation of CoPK32 activity by high osmotic stresses was abrogated by SB202190 or SB239063 as well-known inhibitors of p38 mitogen-activated protein kinase. ConclusionsCoPK32 is involved in the stress response pathway in mycelia of C. cinerea in response to environmental stresses. General significanceIn C. cinerea, protein kinases such as CoPK32 play important roles in signal transduction pathways involved in stress responses.

A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylated exogenous protein substrates. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Kinetic analysis indicated that the K(m) and V(max) values for SynPTP towards p-nitrophenyl phosphate are similar to those of other known bacterial low molecular weight PTPs. Mutagenic alteration of the predicted catalytic cysteine of PTP, Cys(7), to serine abolished enzyme activity. Using a combination of immunodetection, mass spectrometric analysis and mutagenically altered Cys(7)SerAsp(125)Ala-SynPTP, we identified PsaD (photosystem I subunit II), CpcD (phycocyanin rod linker protein) and phycocyanin-α and -β subunits as possible endogenous substrates of SynPTP in this cyanobacterium. These results indicate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803.

Collaborator of Stat6 (CoaSt6)-associated Poly(ADP-ribose) Polymerase Activity Modulates Stat6-dependent Gene Transcription

The transcription factor Stat6 plays a critical role in interleukin-4-dependent gene activation. To mediate this function, Stat6 recruits canonical transcriptional co-activators including the histone acetyl transferases CREB-binding protein and NCoA-1 and other proteins such as a p100 co-factor. However, much remains unknown regarding the constituents of Stat6 enhancer complexes, and the exact molecular events that modulate Stat6-dependent gene activation are not fully understood. Recently, we identified a novel co-factor, CoaSt6 (collaborator of Stat6), which associates with Stat6 and enhances its transcriptional activity. Sequence homologies place CoaSt6 in a superfamily of poly(ADP-ribosyl)polymerase (PARP)-like proteins. We have demonstrated here that PARP enzymatic activity is associated with CoaSt6, and this function of CoaSt6 can append ADP-ribose to itself and p100. Further, we show that a catalytically inactive mutant of CoaSt6 was unable to enhance Stat6-mediated transcription of a test promoter. Consistent with these findings, chemical inhibition of PARP activity blocked interleukin-4-dependent transcription from target promoters in vivo. Taken together, we have identified a CoaSt6-associated PARP activity and provided evidence for a role of poly(ADP ribosyl)ation in Stat-mediated transcriptional responses involving a novel PARP.

Identification of goat sperm ecto-cyclic AMP independent protein kinase substrate localized on sperm outer surface.

We have demonstrated the location of a cyclic AMP independent serine/threonine protein kinase (ecto-CIK) on the outer surface of mature goat spermatozoa. We purified and characterized the major physiological protein substrate (MPS) of ecto-CIK. 32P-labeled membrane proteins phosphorylated by endogenous ecto-CIK of intact cauda-epididymal spermatozoa was solubilized with 1% Triton X-100 and then fractionated by following several chromatographic techniques like Sephacryl S-300 molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography and chromatofocussing. The MPS of ecto-CIK has been purified to apparent homogeneity and it was found to be a monomeric protein of 100 kDa. Three isoforms of MPS have been found with pI of 6.37, 6.05, and 5.14 and all these isoforms served as the specific substrate of ecto-CIK. The ecto-kinase has nearly 30 times greater affinity for MPS as compared to casein the most potent exogenous protein substrate. Addition of MPS (pI 5.14) antibody caused head-to-head sperm agglutination. The Fv/Fab fragment of anti-MPS caused significant inhibition of sperm motility. The data show that MPS is an ecto-protein localized on the sperm head. MPS may thus play an important role for the regulation of sperm-egg interaction.

Specific Phosphorylation of Exogenous Protein and Peptide Substrates by the Human Cytomegalovirus UL97 Protein Kinase: IMPORTANCE OF THE P+5 POSITION

Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathione S-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, maribavir. Sequencing and mass spectrometric analyses of purified (32)P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.

Mammalian Cell Morphology and Endocytic Membrane Homeostasis Require Enzymatically Active Phosphoinositide 5-Kinase PIKfyve

The dual specificity mammalian enzyme PIKfyve phosphorylates in vitro position d-5 in phosphatidylinositol (PtdIns) and PtdIns 3-P, itself or exogenous protein substrates. Here we have addressed the crucial questions for the identity of the lipid products and the role of PIKfyve enzymatic activity in mammalian cells. CHO, HEK293, and COS cells expressing PIKfyve(WT) at high levels and >90% efficiencies increased selectively the intracellular PtdIns 3,5-P(2) production by 30--55%. In these cell types PtdIns 5-P was undetectable. A kinase-deficient point mutant, PIKfyve(K1831E), transiently transfected into these or other cells elicited a dramatic dominant phenotype. Subsequent to a dilation of the PIKfyve-containing vesicles, PIKfyve(K1831E)-expressing cells progressively accumulated multiple swollen lucent vacuoles of endosomal origin, first in the perinuclear cytoplasm and then toward the cell periphery. Despite their drastically altered morphology, the PIKfyve(K1831E)-expressing cells were viable and functionally active, evidenced by several criteria. This phenotype was completely reversed by introducing PIKfyve(WT) into the PIKfyve(K1831E)-transfected cells. Disruptions of the localization signal in the PIKfyve kinase-deficient mutant yielded a PIKfyve(K1831E Delta fyve) protein, incompetent to vacuolate cells, implying that an active PIKfyve enzyme at distinct late endocytic membranes is crucial for normal cell morphology. This was further substantiated by examining the vacuolation-induced potency of several pharmacological stimuli in cells expressing high PIKfyve(WT) levels. Together, the results indicate that PIKfyve enzymatic activity, possibly through the generation of PtdIns 3,5-P(2), and/or yet to be identified endogenous phosphoproteins, is critical for cell morphology and endomembrane homeostasis.

The Requirement of Tyrosines 579 and 581 for Maximal Ligand-Dependent Activation of the βPDGFR Is Influenced by Noncytoplasmic Regions of the Receptor

Mutating tyrosines 579 and 581 of the β platelet-derived growth factor receptor (βPDGFR) tyrosine kinase to phenylalanines (the F2 mutation) impair activation of the receptor in response to ligand, but mutation of the analogous tyrosines in the αPDGFR has no effect on ligand-dependent receptor activation. We have found that the F2 mutation has only a modest effect on ligand-dependent activation of a chimeric PDGFR composed of the extracellular and transmembrane domains of the αPDGFR and the cytoplasmic domain of the βPDGFR by three measures: (1) the ability to phosphorylate endogenous and exogenous protein substrates in vitro, (2) phosphorylation of tyrosine 857, and (3) binding of the effector proteins PLCγ, RasGAP, and SHP-2. Conversely, the F2 mutation substantially impairs ligand-dependent activation of chimeric PDGFRs that consist of either the extracellular domain alone or the extracellular and transmembrane domains of the βPDGFR and all remaining sequence from the αPDGFR by two measures: (1) phosphorylation of endogenous protein substrates in vitro and (2) binding of PLCγ and SHP-2. Our results indicate that the requirement of tyrosines 579 and 581 for maximal activation of the βPDGFR in response to ligand is primarily determined by noncytoplasmic regions of the receptor.

Chronic contractile activity upregulates the proteasome system in rabbit skeletal muscle.

Remodeling of skeletal muscle in response to altered patterns of contractile activity is achieved, in part, by the regulated degradation of cellular proteins. The ubiquitin-proteasome system is a dominant pathway for protein degradation in eukaryotic cells. To test the role of this pathway in contraction-induced remodeling of skeletal muscle, we used a well-established model of continuous motor nerve stimulation to activate tibialis anterior (TA) muscles of New Zealand White rabbits for periods up to 28 days. Western blot analysis revealed marked and coordinated increases in protein levels of the 20S proteasome and two of its regulatory proteins, PA700 and PA28. mRNA of a representative proteasome subunit also increased coordinately in contracting muscles. Chronic contractile activity of TA also increased total proteasome activity in extracts, as measured by the hydrolysis of a proteasome-specific peptide substrate, and the total capacity of the ubiquitin-proteasome pathway, as measured by the ATP-dependent hydrolysis of an exogenous protein substrate. These results support the potential role of the ubiquitin-proteasome pathway of protein degradation in the contraction-induced remodeling of skeletal muscle.

Deregulation of mitogen-activated protein kinase at low pH due to a structural rearrangement of activation segment

Autophosphorylation of recombinant mitogen-activated protein kinase (MAPK) on Tyr was found to be several-fold stimulated at weakly acidic pH (5.5–6.0), whereas the phosphorylation of a protein substrate, myelin basic protein, was greatly inhibited at pH below 6.0. In contrast to phosphorylation at pH 8.0, both MAPK autophosphorylation and MAPK phosphorylation with upstream MAPK kinase at low pH failed to stimulate essentially its kinase activity towards the exogenous protein substrate. Immunoprecipitation and ELISA with an activation segment-specific antibody, kinetic analysis, and reversible phosphorylation assay revealed a difference in the folding of MAPK activation segment at pH 5.5 and 8.0. The data suggest that a rearrangement of the activation segment at low pH promotes a stable low-activity conformation of the enzyme which is favorable for intramolecular autophosphorylation. In this conformation, the phosphorylation of the exogenous protein substrate is inhibited due to persistent blocking of the enzyme catalytic center by the activation segment.

Substrate affinity of the protein tyrosine kinase pp60c-src is increased on thrombin stimulation of human platelets.

Human blood platelets contain high levels of non-receptor protein tyrosine kinases of the Src family, particularly pp60c-src, suggesting an important role for these enzymes in platelet physiology. Indeed, in response to various agonists of platelet function, a number of proteins become phosphorylated at tyrosine residues. However, no enzymic activation of an Src-related tyrosine kinase has yet been shown in platelets. In searching for the kinase(s) responsible, we found that all agonists tested that directly or indirectly activate protein kinase C in platelets (phorbol 12-myristate, 13-acetate, thrombin, vasopressin, collagen, calcium ionophore A23187) increased the overall activity of pp60c-src determined by IgG phosphorylation in an immunocomplex assay in the presence of low ATP concentrations. On the other hand, elevation of cyclic AMP directly by forskolin or indirectly by prostaglandin E1, or elevation of cyclic GMP by sodium nitroprusside did not significantly affect the activity of the enzyme. To substantiate the differences in enzyme activity, we determined Km and Vmax, values of pp60c-src from resting and thrombin-stimulated platelets. Thrombin treatment increased substrate affinity of pp60c-src as indicated by a 2- to 3-fold decrease in the Km values for ATP and the exogenous protein substrate casein. Vmax. values were only slightly altered under the assay conditions used. To further rule out modifications of pp60c-src in phosphorylation as a probable cause of the changed substrate affinity, we analysed tryptic phosphopeptides of immunoprecipitated, 32P-labelled pp60c-src of unstimulated and stimulated platelets. The platelet agonists listed above induced an increase in pp60c-src phosphorylation at Ser-12, which is the amino acid phosphorylated by protein kinase C. Surprisingly, we found that elevation of cyclic AMP did not affect 32P labelling of pp60c-src. On the basis of our data, we suggest that phosphorylation at Ser-12 might be one of the signal-triggering events that cause the increase in substrate affinity of pp60c-src.

Protein phosphorylation in Rhodnius prolixus oocytes: Identification of a type II casein kinase

A protein kinase activity in chorionated oocytes of Rhodnius prolixus phosphorylates in vitro vitellin (VT), the major yolk protein. Phosphatase inhibitors including NaF, sodium vanadate, β-glycerophosphate and okadaic acid did not alter the protein phosphorylation profile to a visible extent. Among the exogenous protein substrates tested, casein was readily phosphorylated, but histones were not. Several different protein kinase activators, including cAMP, Ca 2+ plus calmodulin, Ca 2+ plus diolein and phosphatidylserine, were added to the reaction media but spermidine was the only effective one, inducing a 2-fold increase in the phosphorylation of VT. A strong inhibition was obtained with nanomolar levels of heparin. The enzyme could also accept GTP as the phosphate donor instead of ATP. These properties identify the major protein kinase activity as a type II casein kinase (CK II). The pH dependence and the effects of mono- and divalent cations on VT phosphorylation were also studied. Gel filtration revealed only one peak of protein kinase activity, with a molecular mass of 170 K, similar to values previously reported in the literature for CK IIs from other organisms.

Ecto-protein kinase and surface protein phosphorylation in PC12 cells: interactions with nerve growth factor.

The phosphorylation of surface proteins by ecto-protein kinase has been proposed to play a role in mechanisms underlying neuronal differentiation and their responsiveness to nerve growth factor (NGF). PC12 clones represent an optimal model for investigating the mode of action of NGF in a homogeneous cell population. In the present study we obtained evidence that PC12 cells possess ecto-protein kinase and characterized the endogenous phosphorylation of its surface protein substrates. PC12 cells maintained in a chemically defined medium exhibited phosphorylation of proteins by [gamma-32P]ATP added to the medium at time points preceding the intracellular phosphorylation of proteins in cells labeled with 32Pi. This activity was abolished by adding apyrase or trypsin to the medium but was not sensitive to addition of an excess of unlabeled Pi. As also expected from ecto-protein kinase activity, PC12 cells catalyzed the phosphorylation of an exogenous protein substrate added to the medium, dephospho-alpha-casein, and this activity competed with the endogenous phosphorylation for extracellular ATP. Based on these criteria, three protein components migrating in sodium dodecyl sulfate gels with apparent molecular weights of 105K, 39K, and 20K were identified as exclusive substrates of ecto-protein kinase in PC12 cells. Of the phosphate incorporated into these proteins from extracellular ATP, 75-87% was found in phosphothreonine. The phosphorylation of the 39K protein by ecto-protein kinase did not require Mg2+, implicating this activity in the previously demonstrated regulation of Ca(2+)-dependent, high-affinity norepinephrine uptake in PC12 cells by extracellular ATP. The protein kinase inhibitor K-252a inhibited both intra- and extracellular protein phosphorylation in intact PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of the purified plasma membrane H+‐ATPase from red beet (Beta vulgaris)

The plasma membrane H+‐ATPase (EC 3.6.1.35) was purified by washing red beet (Beta vulgaris L.) plasma membranes with sodium deoxycholate and separating the ATPase, solubilized with lysophosphatidylcholine, by centrifugation in a glycerol gradient. The purified H+‐ATPase had a sedimentation coefficient of about 8S. In the absence of exogenous protein substrates, the purified ATPase preparation did not present protein kinase activity. Compared with the H+‐ATPase in the plasma membrane, the purified ATPase presented a higher affinity for adenosine 5′‐triphosphate (ATP) and a lower sensitivity to the inhibitors vanadate and inorganic phosphate. These changes in the kinetics of the ATPase could also be observed by treating the membranes with lysophosphatidylcholine, without purifying the enzyme. These results can be explained assuming that lysophosphatidylcholine interacts with the ATPase altering its kinetics probably by stimulating the transformation from the inhibitor‐binding conformation E2 into the ATP‐binding conformation E1.

Phosphatase activity against neurofilament proteins from bovine spinal cord: Effect of aluminium and neuropsychoactive drugs

Protein phosphatase activity associated with neurofilament (NF) rich (Triton X-100 insoluble) fraction was extracted and partially characterised by using known inhibitors of protein phosphatases such as vanadate and fluoride. Protein phosphatase activity was demonstrated with reference to the dephosphorylation of endogenous substrate. NF protein and exogenous protein substrates, casein and phosvitin. Phosphoamino acids and β-glycerophosphate were found to be poor substrates. Further, new observations have been made regarding the in vitro inhibitory effect of aluminium and the differential effects of some of the neuropsychoactive drugs. The findings could possibly lead to studies explaining the biochemical basis of aluminium induced neurotoxicity as well as the side effects associated with the long term medication of neuropsychoactive drugs.

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.