Soluble Cytoplasmic Enzyme Research Articles

Unique Structural Fold of LonBA Protease from Bacillus subtilis, a Member of a Newly Identified Subfamily of Lon Proteases.

ATP-dependent Lon proteases are key participants in the quality control system that supports the homeostasis of the cellular proteome. Based on their unique structural and biochemical properties, Lon proteases have been assigned in the MEROPS database to three subfamilies (A, B, and C). All Lons are single-chain, multidomain proteins containing an ATPase and protease domains, with different additional elements present in each subfamily. LonA and LonC proteases are soluble cytoplasmic enzymes, whereas LonBs are membrane-bound. Based on an analysis of the available sequences of Lon proteases, we identified a number of enzymes currently assigned to the LonB subfamily that, although presumably membrane-bound, include structural features more similar to their counterparts in the LonA subfamily. This observation was confirmed by the crystal structure of the proteolytic domain of the enzyme previously assigned as Bacillus subtilis LonB, combined with the modeled structure of its ATPase domain. Several structural features present in both domains differ from their counterparts in either LonA or LonB subfamilies. We thus postulate that this enzyme is the founding member of a newly identified LonBA subfamily, so far found only in the gene sequences of firmicutes.

Subcellular coordination of plant cell wall synthesis.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis.

Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the new millennium.

English

Glutathione S-transferases are multifunctional proteins involved in diverse intracellular events such as primary and secondary metabolisms, stress metabolism, herbicide detoxification and plant protection against ozone damages, heavy metals and xenobiotics. The plant glutathione S-transferase superfamily have been subdivided into eight classes. Phi, tau, zeta, theta, lambda, dehydroascorbate reductase and tetrachlorohydroquinone dehalogenase classes are soluble and one class is microsomal. Glutathione S-transferases are mostly soluble cytoplasmic enzymes. To date, the crystal structures of over 200 soluble glutathione S-transferases, present in plants, animals and bacteria have been resolved. The structures of glutathione S-transferase influence its function. Phylogenetic analysis suggests that all soluble glutathione S-transferases have arisen from an ancient progenitor gene, through both convergent and divergent pathways. Key words: Glutathione S-transferases (GST), classification, structure, evolution, phylogenetic analysis, xenobiotics.

Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.

Allosteric Activation of Human Glucokinase by Free Polyubiquitin Chains and Its Ubiquitin-dependent Cotranslational Proteasomal Degradation

Human glucokinase (hGK) is a monomeric enzyme highly regulated in pancreatic beta-cells (isoform 1) and hepatocytes (isoforms 2 and 3). Although certain cellular proteins are known to either stimulate or inhibit its activity, little is known about post-translational modifications of this enzyme and their possible regulatory functions. In this study, we have identified isoforms 1 and 2 of hGK as novel substrates for the ubiquitin-conjugating enzyme system of the rabbit reticulocyte lysate. Both isoforms were polyubiquitinated on at least two lysine residues, and mutation analysis indicated that multiple lysine residues functioned as redundant acceptor sites. Deletion of its C-terminal alpha-helix, as part of a ubiquitin-interacting motif, affected the polyubiquitination at one of the sites and resulted in a completely inactive enzyme. Evidence is presented that poly/multiubiquitination of hGK in vitro serves as a signal for proteasomal degradation of the newly synthesized protein. Moreover, the recombinant hGK was found to interact with and to be allosterically activated up to approximately 1.4-fold by purified free pentaubiquitin chains at approximately 100 nm (with an apparent EC(50) of 93 nm), and possibly also by unidentified polyubiquitinated proteins assigned to their equilibrium binding to the ubiquitin-interacting motif site. The affinity of pentaubiquitin binding to hGK is regulated by the ligand (d-glucose)-dependent conformational state of the site. Both ubiquitination of hGK and its activation by polyubiquitin chains potentially represent physiological regulatory mechanisms for glucokinase-dependent insulin secretion in pancreatic beta-cells.

A cDNA cloned from Physarum polycephalum encodes new type of family 3 β-glucosidase that is a fusion protein containing a calx-β motif

The microplasmodia of Physarum polycephalum express three types of β-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane β-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane β-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane β-glucosidase 1 sequence and were highly homologous with the primary structures of fungal β-glucosidases. Notably, the C-terminal half of membrane β-glucosidase 1 contains two calx-β motifs, which are known to be Ca 2+ binding domains in the Drosophila Na +/Ca 2+ exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane β-glucosidase 1 differs from all previously identified family 3 β-glucosidases. In addition to cDNA for membrane β-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane β-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other β-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane β-glucosidase is a new type family 3 enzyme fused with the Calx-β domain. We propose that Calx-β domain may modulate the β-glucosidase activity in response to changes in the Ca 2+ concentration.

A novel mechanism for mitogen-activated protein kinase localization.

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.

NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.

Peroxidase involvement in the defense response of red raspberry to Didymella applanata (Niessl/Sacc.)

Peroxidase activity of red raspberry canes was dependent on the cultivar and influenced the subsequent lignification. After inoculation with Didymella applanata, responsible for the spur blight cane disease, the activity of soluble cytoplasmic enzyme increased in the moderately resistant ‘Latham’ and susceptible ‘Malling Promise’, similarly for syringaldazine and guaiacol as hydrogen donors. Systemic induction found in ‘Latham’ was recognized as a symptom of defence mechanism responsible for fungal restriction. Locally enhanced peroxidase activity in the ‘M.Promise’ tissues was related to the local lignification and/or may be associated with the loss of cell integrity caused by pathogen penetration. Pathogen-induced changes of cell wall peroxidases were similar in both cultivars mentioned above. No influence of the infection was found in the high susceptible Zeva cultivar. Using native-PAGE analysis and horizontal starch electrophoresis of soluble fraction five constitutive acidic isoperoxidases were detected in ‘Latham’ and three in ‘M. Promise’. The infection process was accompanied by the appearance of two new anodic isoforms.

Crystal structure of rat biliverdin reductase.

Biliverdin reductase (BVR) is a soluble cytoplasmic enzyme that catalyzes the conversion of biliverdin to bilirubin using NADH or NADPH as electron donor. Bilirubin is a significant biological antioxidant, but it is also neurotoxic and the cause of kernicterus. In this study, we have determined the crystal structure of rat BVR at 1.4 A resolution. The structure contains two domains: an N-terminal domain characteristic of a dinucleotide binding fold (Rossmann fold) and a C-terminal domain that is predominantly an antiparallel six-stranded beta-sheet. Based on this structure, we propose modes of binding for NAD(P)H and biliverdin, and a possible mechanism for the enzyme.

Argininosuccinate synthetase and argininosuccinate lyase are localized around mitochondria: An immunocytochemical study

Argininosuccinate synthetase and argininosuccinate lyase are soluble cytoplasmic enzymes of the urea cycle. Previous biochemical studies using permeabilized hepatocytes showed that these enzymes are organized in situ, and function as if they are located next to the outer membrane of mitochondria. We have now confirmed and extended those observations in intact liver by means of immunocytochemistry at the electron microscope level. Morphometric analysis of the electron micrographs shows that argininosuccinate synthetase and argininosuccinate lyase are located in the immediate vicinity of the mitochondria, predominantly next to the cytoplasmic surface of the outer membrane. Some immuno-specific protein is also observed in the endoplasmic reticulum in the immediate vicinity of the mitochondria. These results support our previous biochemical findings, and additionally suggest that virtually all of the argininosuccinate synthetase and argininosuccinate lyase of the liver parenchymal cell are located just outside the mitochondria. © 1996 Wiley-Liss, Inc.

Transport of glucose and cellobiose by Candida wickerhamii and Clavispora lusitaniae.

The cellular location of beta-1,4-glucosidase activity from, as well as the transport of glucose and cellobiose into, cells of Clavispora lusitaniae NRRL Y-5394 and Candida wickerhamii NRRL Y-2563 was investigated. The beta-glucosidase from Cl. lusitaniae appeared to be a soluble cytoplasmic enzyme. This yeast transported both glucose and cellobiose when grown in medium containing cellobiose as the sole carbon source. Glucose, but not cellobiose, uptake was observed for cells grown on glucose. The Ks and Vmax values for cellobiose transport were different when Cl. lusitaniae was cultured either aerobically (0.11 mM, 6.28 nmol.min-1.mg-1) or anaerobically (0.25 mM, 3.88 nmol-1.min-1.mg-1). The Ks and Vmax values for glucose transport (0.23-1.10 mM and 17.2-33.9 nmol.min-1.mg-1) also differed with the various growth conditions. The beta-glucosidase from C. wickerhamii was extracytoplasmically located. This yeast transported glucose, but not cellobiose, under all growth conditions tested. The Ks for glucose uptake was 0.13-0.28 mM when C. wickerhamii was cultured on cellobiose and 0.25-0.30 mM when cultured on glucose. The Vmax values for glucose uptake were greater for cells cultured on cellobiose (35.0-37.9 nmol.min-1.mg-1) than for cells cultured on glucose (15.6-21.4 nmol.min-1.mg-1). Cellobiose did not inhibit glucose uptake in either yeast. Glucose partially inhibited cellobiose transport in C. lusitaniae, but only if the yeast was grown aerobically. In both yeasts, sugar transport was sensitive to carbonyl cyanide p-trifluoromethoxyphenylhydrazone and 1799, but insensitive to valinomycin.

Soluble and Bound Apoplastic Activity for Peroxidase, β-d-Glucosidase, Malate Dehydrogenase, and Nonspecific Arylesterase, in Barley (Hordeum vulgare L.) and Oat (Avena sativa L.) Primary Leaves

An intercellular washing solution containing about 1% of the soluble protein, 0.3% or less of the glucose-6-phosphate dehydrogenase activity, but up to 20% of the peroxidase and beta-d-glucosidase activity of barley (Hordeum vulgare L.) or oat (Avena sativa L.) primary leaves was obtained by vacuum infiltrating peeled leaves with pH 6.9 buffered 200 millimolar NaCl. After this wash, segments were homogenized in buffer, centrifuged, and the supernatant was assayed for soluble cytoplasmic enzymes. The pellet was washed and resuspended in 1 molar NaCl to solubilize enzymes strongly ionically bound to the cell wall. The final pellet was assayed for enzyme activity covalently bound in the cell wall. Apoplastic (intercellular washing solution, ionically bound, and covalently bound) fractions contained up to 76% of the beta-d-glucosidase activity, 36% of the peroxidase activity, 11% of the nonspecific arylesterase activity, 4% of the malate dehydrogenase activity, but less than 2% of the glucose-6-phosphate dehydrogenase activity of peeled leaf segments. The partitioning and salt-solubility of the enzymes between the apoplast and symplast differed considerably between these two species. Intercellular washing fluid prepared by centrifuging unpeeled leaves had higher activity for glucose-6-phosphate dehydrogenase, less soluble protein, and less peroxidase activity per leaf than intercellular washing solution obtained by our peeling-infiltration-washing technique. The results are discussed in relation to the roles of these enzymes in phenolic metabolism in the cell wall.

Ecto-enzyme activity of human erythrocyte adenosine deaminase.

Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl-, which is precipitated at its locus of formation by added Ag+, and the precipitated AgCl converted into the electron dense Ag0 upon exposure to light. From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane. The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.

Channeling of Urea Cycle Intermediates in Situ in Permeabilized Hepatocytes

Preferential use of endogenously generated intermediates by the enzymes of the urea cycle was observed using isolated rat hepatocytes made permeable to low molecular weight compounds with alpha-toxin. The permeabilized cells synthesized [14C]urea from added NH4Cl, [14C]HCO3-, ornithine, and aspartate, using succinate as a respiratory substrate; with all substrates saturating, about 4 nmol of urea were formed per min/mg dry weight of cells. Urea usually accounted for about 40-50% of the total (NH3 + ornithine)-dependent counts, arginine for less than 10%, and citrulline for about 30%. Very tight channeling of arginine between argininosuccinate lyase and arginase was shown by the fact that the addition of a 200-fold excess of unlabeled arginine to the incubations did not decrease the percentage of counts found in urea or increase that found in arginine, even though a substantial amount of the added arginine was hydrolyzed inside the cells. The channeling of argininosuccinate between its synthetase and lyase was demonstrated by similar observations; unlabeled argininosuccinate added in 200-fold excess decreased the percentage of counts in urea by only 25%. Channeling of citrulline from its site of synthesis by ornithine transcarbamylase in the mitochondrial matrix to argininosuccinate synthetase in the cytoplasmic space was also shown. These results strongly suggest that the three "soluble" cytoplasmic enzymes of the urea cycle are grouped around the mitochondria and are spatially organized within the cell in such a way that intermediates can be efficiently transferred between them.

Immunocytochemical localization of two key enzymes of the 2-hydroxyglutarate pathway of glutamate fermentation in Acidaminococcus fermentans

We have investigated the in situ location of glutaconyl-CoA decarboxylase and 2-htdroxyglutaryl-CoA dehydratase in Acidaminococcus fermentans using the antibody-gold and protein A-gold techniques carried out as a post-embedding immunoelectron microscopic procedure. Polyclonal antisera were raised in rabbits against homogeneous fractions of the enzymes. Anaerobically grown cells of A. fermentans of the late exponential growth phase were fixed with 0.2% glutaraldehyde and 0.3% formaldehyde (final concentrations) in the growth medium. Dehydration of the cells was achieved with methanol. The cells were embedded in the low temperature embedding resin Lowicryl K4M. The markers indicative for antigenic sites of the two enzymes unequivocally demonstrate that the sodium pump glutaconyl-CoA decarboxylase is located at the cell periphery being a membrane-bound enzyme as expected whereas 2-hydroxyglutaryl-CoA dehydratase is a soluble cytoplasmic enzyme.

Effect of Forskolin on Voltage-Gated K + Channels Is Independent of Adenylate Cyclase Activation

Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.

Subcellular localization of a carboxypeptidase activity in maize root homogenates

Maize root homogenates were prepared and centrifuged to sediment the mitochondria. The supernatants (6 KS) and pellets (6 KP) were collected and fractionated on linear sucrose density gradients. The distribution of carboxypeptidase was similar to that of α-mannosidase (a soluble vacuolar enzyme) and glucose-6-phosphate dehydrogenase (a soluble cytoplasmic enzyme). Carboxypeptidase was therefore a soluble in maize root cells and cannot be used as a marker for the tonoplast.

Subcellular localization of high- and low-molecular weight acid phosphatases from chicken liver

1. 1. The subcellular localization of high and low molecular weight acid phosphatases in chicken liver was studied. 2. 2. The high molecular weight acid phosphatase is mainly associated with the particulate fraction, particularly with the mitochondrial-lysosomal fraction, whereas the low molecular weight form seems to be a soluble cytoplasmic enzyme. 3. 3. Biochemical properties including optimal pH, molecular weight determination and the effect of some modifier substance indicate that mitochondria-lysosomes and microsomes contain the same high molecular weight acid phosphatase form.