Diversification of the C-TERMINALLY ENCODED PEPTIDE (CEP) gene family in angiosperms,and evolution of plant-family specific CEP genes

Background

Small, secreted signaling peptides work in parallel with phytohormones to control important aspects of plant growth and development. Genes from the C-TERMINALLY ENCODED PEPTIDE (CEP) family produce such peptides which negatively regulate plant growth, especially under stress, and affect other important developmental processes. To illuminate how the CEP gene family has evolved within the plant kingdom, including its emergence, diversification and variation between lineages, a comprehensive survey was undertaken to identify and characterize CEP genes in 106 plant genomes.

Results

Using a motif-based system developed for this study to identify canonical CEP peptide domains, a total of 916 CEP genes and 1,223 CEP domains were found in angiosperms and for the first time in gymnosperms. This defines a narrow band for the emergence of CEP genes in plants, from the divergence of lycophytes to the angiosperm/gymnosperm split. Both CEP genes and domains were found to have diversified in angiosperms, particularly in the Poaceae and Solanaceae plant families. Multispecies orthologous relationships were determined for 22% of identified CEP genes, and further analysis of those groups found selective constraints upon residues within the CEP peptide and within the previously little-characterized variable region. An examination of public Oryza sativa RNA-Seq datasets revealed an expression pattern that links OsCEP5 and OsCEP6 to panicle development and flowering, and CEP gene trees reveal these emerged from a duplication event associated with the Poaceae plant family.

Conclusions

The characterization of the plant-family specific CEP genes OsCEP5 and OsCEP6, the association of CEP genes with angiosperm-specific development processes like panicle development, and the diversification of CEP genes in angiosperms provides further support for the hypothesis that CEP genes have been integral to the evolution of novel traits within the angiosperm lineage. Beyond these findings, the comprehensive set of CEP genes and their properties reported here will be a resource for future research on CEP genes and peptides.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-870) contains supplementary material, which is available to authorized users.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

Conversion of Methionine to Thiols by Lactococci,Lactobacilli, and Brevibacteria

Methanethiol has been strongly associated with desirable Cheddar cheese flavor and can be formed from the degradation of methionine (Met) via a number of microbial enzymes. Methionine γ-lyase is thought to play a major role in the catabolism of Met and generation of methanethiol in several species of bacteria. Other enzymes that have been reported to be capable of producing methanethiol from Met in lactic acid bacteria include cystathionine β-lyase and cystathionine γ-lyase. The objective of this study was to determine the production, stability, and activities of the enzymes involved in methanethiol generation in bacteria associated with cheese making. Lactococci and lactobacilli were observed to contain high levels of enzymes that acted primarily on cystathionine. Enzyme activity was dependent on the concentration of sulfur amino acids in the growth medium. Met aminotransferase activity was detected in all of the lactic acid bacteria tested and α-ketoglutarate was used as the amino group acceptor. In Lactococcus lactis subsp. cremoris S2, Met aminotransferase was repressed with increasing concentrations of Met in the growth medium. While no Met aminotransferase activity was detected in Brevibacterium linens BL2, it possessed high levels of l-methionine γ-lyase that was induced by addition of Met to the growth medium. Met demethiolation activity at pH 5.2 with 4% NaCl was not detected in cell extracts but was detected in whole cells. These data suggest that Met degradation in Cheddar cheese will depend on the organism used in production, the amount of enzyme released during aging, and the amount of Met in the matrix.The primary classes of compounds that contribute to cheese flavor include amino acids and their degradation products, peptides, carbonyl compounds, and fatty acids. These partition primarily into the aqueous fraction of cheese (3). The volatile fraction of cheese has sulfur-containing compounds such as methanethiol, methional, dimethyl sulfide, dimethyl tetrasulfide, carbonyl sulfide, and hydrogen sulfide (28), and they contribute to the aroma of cheese (7). Methanethiol has been associated with desirable Cheddar-type sulfur notes in good-quality Cheddar cheese (2), and it is also implicated as an influential aroma and flavor compound in many foods, including surface-ripened cheeses that use brevibacteria (17). However, methanethiol, when present alone, does not contribute to typical Cheddar-like flavor notes in cheese (17).Production of methanethiol is important in cheese, but the Met biosynthetic and catabolic pathways vary among bacteria (23). The mechanisms involved and amounts of methanethiol produced during cheese ripening also vary. In an effort to increase and accelerate the development of typical Cheddar cheese flavor, adjunct bacteria have been used during the manufacture of low- and full-fat cheese. Initial selection of flavor adjunct cultures focused on those bacteria used to accelerate flavor development in full-fat cheese, which are typically lactobacilli because they dominate (10⁷ to 10⁹ CFU/g of cheese during storage at 8°C) the microflora during aging (15). The Lactobacillus genus is considered to be a member of the nonstarter lactic acid bacteria subgroup because it is not added with the starter culture for Cheddar cheese. In addition to lactobacilli, micrococci and pediococci have been used as adjunct bacteria to aid in flavor development (20). Brevibacteria, which are normally found on the surfaces of Limburger and other Trappist-type cheeses, are not traditionally used as flavor adjuncts in Cheddar cheese. One advantage these organisms have over other adjuncts is their profuse production of methanethiol (8). Weimer et al. (30) successfully used Brevibacterium linens as an adjunct to improve the flavor of low-fat Cheddar cheese.The mechanism for the production of methanethiol in cheese by bacteria can be a result of the direct catabolism of Met or it can arise from inadvertent catalysis by other enzymes (1, 6, 17). The most direct route to methanethiol is the conversion of Met to methanethiol, ammonia, and α-ketobutyrate (Fig. ​(Fig.1).1). This transformation is catalyzed by inducible Met γ-lyase, a pyridoxal phosphate (PLP)-dependent enzyme (24) which has been purified to homogeneity from Pseudomonas putida (14, 26), Aeromonas spp. (27), and Clostridium sporogenes (16) and partially purified from B. linens (6). Open in a separate windowFIG. 1Metabolic pathways for Met interconversion. The primary intermediates and enzymes are listed. Enzyme 1 is cystathionine γ-lyase, enzyme 2 is cystathionine β-lyase, enzyme 3 is cystathionine β-synthase, enzyme 4 is homocysteine methyltransferase, enzyme 5 is aromatic aminotransferase (tyrB) or transaminase B (ilvE), enzyme 6 is amino acid oxidase, enzyme 7 is Met adenosyltransferase, and enzyme 8 is Met γ-lyase (adapted from reference 18 and 23).Pathways leading away from Met are important to consider because this amino acid is central to many other critical metabolic functions (Fig. ​(Fig.1).1). Utilization of Met for other metabolic functions would lower the pool of Met available for conversion to methanethiol. Methionine adenosyltransferase (S-adenosylmethionine [SAM] synthetase) converts Met into SAM at the expense of one ATP. SAM, one of the major methylating agents in a cell, is also important in the regulation of several of the Met-biosynthetic enzymes (22). Reduced SAM synthetase activity leads to low intracellular levels of SAM, resulting in the induction of the Met-biosynthetic pathway (32).Another mechanism that directs Met away from methanethiol is the deamination reaction to form α-keto γ-methyl thiobutyrate (KMTB). This conversion can be catalyzed by various aminotransferases (33) or amino acid oxidases (21). These enzymes are common in bacteria and are usually the last step in amino acid synthetic pathways (13). Amino acid oxidase activity is a possible route for KMTB production, and it is a possible route for subsequent methanethiol production in cheese, but this is unlikely because cheese tends to be anaerobic. Evidence for the conversion of KMTB to methanethiol is lacking for bacteria; however, this reaction has been shown to take place enzymatically in fungal species (23).When the catabolic pathways for Met are considered, the enzymes involved in the biosynthesis of Met must also be included. Although the principal reactions that these enzymes catalyze are involved in the synthesis of Met, they also coincidentally catalyze catabolic reactions that lead to the production of methanethiol and possibly other cheese flavor compounds. For example, cystathionine β-lyase, which primarily catalyzes the conversion of cystathionine to homocysteine, a reaction involved in the synthesis of Met (29), also catalyzes the conversion of Met to methanethiol, ammonia, and α-ketobutyrate but with 100 times less efficiency than that of its conversion to homocysteine in Lactococcus lactis subsp. cremoris S2 (1). This enzyme was purified from lactococci and has been implicated in the generation of methanethiol in Cheddar cheese (1). Cystathionine γ-lyase catalyzes the α,γ elimination of cystathionine to produce cysteine (Cys), α-ketobutyrate, and ammonia (19). A cystathionine γ-lyase purified from L. lactis subsp. cremoris is capable of catalyzing the α,γ elimination of Met to produce methanethiol at an efficiency much lower than that of the primary reaction it catalyzes (4). These enzymes may be present in the cells and liberated when the cells die and lyse during cheese storage, as occurs in Cheddar cheese ripening (10). With these observations in mind, the objective of this study was to determine the conversion pathways of Met to free thiols under laboratory and cheese-like conditions in bacteria used as starter cultures and flavor adjuncts in Cheddar cheese.     

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor

Summary 80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR ⁺ strain but not by corresponding preparations from an argR ^- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on -galactosidase synthesis.     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

The regulatory effect of phosphates on bone metabolism in vitro

Summary One of the most important indicators in vitro of the bone-cell phenotype is the synthesis of mineralized bone-like tissue. This has been achieved by supplementing isolated bone-cell and tissue cultures with organic phosphates, in particular, -glycerophosphate. To analyze the effects of -glycerophosphate on bone-cell metabolism and osteogenesis in vitro, both biochemical analyses and computer-assisted morphometry were used. Simultaneous autoradiographic and histochemical analyses of proliferating and alkaline phosphatase-positive cells were used to measure osteogenic events at the cellular level. Morphometric data showed that -glycerophosphate-treated cultures mineralized, but exhibited significantly less bone matrix (P < 0.05) than non-mineralizing controls. Cultures treated with inorganic phosphate failed to mineralize. Cellular proliferation was unaffected by -glycerophosphate; however, there was a decrease in the amount of ³H-thymidine incorporation into the DNA of -glycerophosphate-treated cells as detected by autoradiography. The percentage of alkaline phosphatase-positive cells was identical in -glycerophosphate-treated or control cultures. In agreement with previous biochemical results, there was a decrease in the amount of alkaline phosphatase enzyme activity per cell. The kinetics of alkaline phosphatase enzymes were measured on individual cells by microdensitometry. -Glycerophosphate-treated cultures exhibited more rapid reaction rates than control cultures (p < 0.05). Taken together, the results suggest that -glycerophosphate has global effects on bone-cell metabolism in vitro including its importance in mineralization.     

DISCO is key to successful centriole maturation

Centriole maturation is essential for ciliogenesis, but which proteins and how they regulate ciliary assembly is unclear. In this issue, Kumar et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202011133) shed light on this process by identifying a ciliopathy complex at the distal mother centriole that restrains centriole length and supports the formation of distal appendages.

The primary cilium plays a crucial role in embryonic development by allowing the integration of a variety of inputs, including chemical and mechanical signals. Primary cilia are found on most cell types; thereby, mutations in genes encoding cilia components may perturb many cellular functions, including airway mucus clearance, mechanosensation, and cell signaling, which are central regulators of organ function and homeostasis. Numerous mutations leading to ciliary dysfunction have been identified in recent years and thus linked to human cilia-related diseases, called ciliopathies (1, 2). Some of these mutations affect components of the centrioles, which are cytoplasmic cylindrical structures composed by triplets of microtubules arranged in a ninefold symmetry.Cilia originate from centrioles and are anchored to the cell surface. In most mammalian cells, centrioles are present within the centrosome, the main organizing center of microtubules. During G1 phase, cells have one centrosome containing two centrioles of different ages. The older mother centriole is distinguished from the younger daughter centriole by the presence of two sets of appendages organized around its circumference. The centrosome duplicates in S phase and, as a result, a new centriole is formed orthogonally to each parent centriole. The new centrioles subsequently elongate during S and G2 phases, and each daughter cell inherits a parent and a newly formed centriole after mitosis. During this transition, new centrioles become daughter centrioles, and the daughter centriole from the previous cycle acquires appendages to mature into a mother centriole. Distal appendages (DAs) are essential for anchoring the mother centriole to the plasma membrane and for the formation of a cilium (2). The formation of a mature centriole competent for ciliogenesis is therefore a complex process taking place over three successive cell cycles.Different molecular factors required for the progressive maturation of centrioles and the assembly of DAs have been identified in the past, and perturbation of their function has been linked to ciliopathies (2, 3). However, the precise mechanism by which DAs are assembled onto centrioles remains elusive. In this issue, Kumar et al. focused their attention on CEP90, a poorly characterized protein whose mutations have been implicated in several ciliopathies (4). CEP90 is a component of centriolar satellites, which are proteinaceous granules located at the periphery of the centrosome (5, 6). Using a combination of expansion microscopy and structured illumination super-resolution microscopy techniques, the authors found that CEP90 also localized to centrioles, where it formed a discontinuous ring with a ninefold symmetry. CEP90 localized near a well-characterized DA component, CEP164, which was consistent with CEP90 being present at the base of these appendages. Then, they searched for CEP90 interactors. For that, the researchers first had to circumvent the shortcoming of discriminating between interactions that may take place at the centrosome from those occurring within centriolar satellites. To get around this, Kumar et al. used a cell line in which satellite assembly is inhibited. Among the candidates they found interacting with CEP90 at the centrosome were OFD1 and Moonraker (MNR), which are two proteins previously associated with multiple ciliopathies. OFD1 is a centriole component required to restrict centriole elongation and assemble DAs (7). MNR, also called OFIP or KIAA073, is a satellite component necessary for cilia formation (8). Making again use of super-resolution microscopy, the authors showed that all three proteins colocalized at the centriole distal end, with the MNR protein being the closest to the centriole wall, so they named this newly identified complex after DISCO (distal centriole complex).Next, Kumar et al. elegantly demonstrated that, as previously shown for OFD1 (7), inactivating either CEP90 or MNR led to the absence of cilia in cells. In mice, deficiency of any of these proteins resulted in Hedgehog signaling inhibition and early arrest of embryonic development. As reported for OFD1-deficient cells, loss of MNR in human cells resulted in overly long centrioles. However, centriole length was normal in CEP90-deficient cells, suggesting partially distinct functions between members of the DISCO complex. The authors noted that ciliogenesis was blocked at an early stage in CEP90^−/− and MNR^−/− cells and, given that DAs are essential for centriole anchoring and ciliogenesis, they decided to examine DA organization in these cells (4). Indeed, they found that DA components, such as CEP83, were not recruited during centriole maturation in MNR^−/− or CEP90^−/− cells, and DAs were not detected by electron microscopy. These findings pointed out that CEP90 and MNR, like OFD1, were required for the assembly of DAs.Since CEP90 is required for satellite accumulation around the centrosome, and satellites are, in turn, essential for ciliogenesis (6), one possible explanation to their results is that CEP90 might affect DA assembly indirectly via its role in satellite localization. To answer this question, the authors again used cells lacking centriolar satellites. CEP90 was correctly localized at centriole distal ends in these cells, and DAs were formed, supporting a direct requirement for the centriolar pool of CEP90 in DA assembly. Putting all their data together, Kumar et al. proposed the following model: First, MNR is recruited to elongating centrioles, which, in turn, triggers the recruitment of OFD1 to arrest elongation at the end of the first cell cycle. MNR and OFD1 then recruit CEP90, which initiates the recruitment of DA components, including CEP83, at the end of the following cell cycle (Fig. 1). Thus, the DISCO complex allows for coupling the arrest of centriole elongation to centriole maturation across successive cell cycles.Open in a separate windowFigure 1.The DISCO complex restrains centriole elongation and initiates DA assembly. (1) The DISCO complex member MNR is recruited first at the distal end of assembling centrioles. MNR then recruits other members of the complex, including OFD1, which inhibits centriole elongation at the end of the first cell cycle, i.e., when newly formed centrioles become daughter centrioles (DCs). Other members of the complex include CEP90 and possibly also FOPNL. (2) At the end of the following cell cycle, as the daughter centriole matures into a mother centriole (MC), CEP90 initiates the recruitment of CEP83, the most upstream component in DA assembly. A previously identified interaction between OFD1 and another DA component, CEP89, might also contribute to DA organization (10). Proteins are drawn in contact with each other when an interaction or hierarchical recruitment was described (3, 4, 8, 11).Besides OFD1 and MNR proteins, Kumar et al. also identified a protein called FGFR1OP N-Terminal Like (FOPNL or FOR20) as a potential CEP90 interactor (4). Interestingly, this interaction was confirmed in a recent study describing that a complex containing CEP90, OFD1, and FOPNL localizes at the distal end of Paramecium centrioles and is necessary for the recruitment of DA components and centriole docking in Paramecium and human cells (9). FOPNL was previously found in complex with MNR and OFD1 and shown to facilitate their interaction (8). Together, these data suggest that the DISCO complex could also include FOPNL. The functional similarities of some of the components of the DISCO complex between Paramecium and humans strongly suggest that the role of DISCO in centriole maturation and ciliogenesis is broadly conserved across species.Previous studies in different organisms have underpinned the relevance of ciliopathy-associated proteins to ensure normal organism development and tissue function (1, 2). Overall, the findings by Kumar et al. highlight the critical role of a ciliopathy-associated protein complex at distal centrioles in building distal appendages, thus supporting centriole maturation and ciliogenesis in rodents and human cells (4).     

A low molecular weight alkaline proteinase fromConidiobolus coronatus

Summary An extracellular, low molecular weight alkaline proteinase (alkaline proteinase B) has been purified to homogeneity from the culture filtrate ofConidiobolus coronatus (NCIM 1238). A 12-fold purification was achieved with a specific activity of 29,760 u/mg. The enzyme had an optimum pH and temperature of 9.7 and 45°C respectively. It was most active towards casein and had a molecular weight of 6,800, the lowest reported so far. It was stable between pH 6.5–7.5. Alkaline proteinase B is a serine proteinase. It showed an esterolytic activity on N-benzoyl-L-tyrosine ethyl ester (BTEE) and was successfully used to resolve the racemic mixture of D, L-phenylalanine and D,L-phenylglycine and can thus potentially replace subtilisin Carlsberg in resolving the racemic mixture of amino acids.     

Cloning of phoM, a gene involved in regulation of the synthesis of phosphate limitation inducible proteins in Escherichia coli K12

Summary Like the synthesis of alkaline phosphatase, the synthesis of outer membrane PhoE protein is shown to be dependent on the phoM gene product in phoR mutants of E. coli K12. This phoM gene has been cloned into the multicopy vector pACYC184 using selection for alkaline phosphatase constitutive synthesis in a phoR background. The gene was localized on the hybrid plasmids by analysis of deletion plasmids constructed in vitro and of mutant plasmids generated by insertions.Interestingly, two of the selected hybrid plasmids contained the entire phoA-phoB-phoR region of the chromosome, as a multiple copy state of these genes results in the constitutive synthesis of alkaline phosphatase. The presence of multiple copies of the phoM gene hardly influences the level of expression of alkaline phosphatase and PhoE protein in a pho ⁺ strain, but significantly increases the levels of these proteins in aphoR mutant strain.     

Sulfur metabolism in bacteria associated with cheese

Metabolism of sulfur in bacteria associated with cheese has long been a topic of interest. Volatile sulfur compounds, specifically methanethiol, are correlated to desirable flavor in Cheddar cheese, but their definitive role remains elusive. Only recently have enzymes been found that produce this compound in bacteria associated with cheese making. Cystathionine - and -lyase are found in lactic acid bacteria and are capable of producing methanethiol from methionine. Their primary function is in the metabolism of cysteine. Methionine -lyase produces methanethiol from methionine at a higher efficiency than the cystathionine enzymes. This enzyme is found in brevibacteria, bacilli, and pseudomonads. Addition of brevibacteria containing this enzyme improves Cheddar cheese flavor. Despite recent progress in sulfur metabolism more information is needed before cheese flavor associated with sulfur can be predicted or controlled.     

Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences

Summary A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced. The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane. The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site. These features have analogous counterparts in the thylakoid-transfer domain of lumenal polypeptides from chloroplasts of higher plants. These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides. In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts.     

Changes in survival rate enzyme activities and in Escherichia coli with ozone

Summary With ozone, it is important to know the decomposition of cell components in order to explain the bactericidal effects. We chose Escherichia coli as a bactericidal model with ozone and observed the changes in activities of alkaline phosphatase and -galactosidase, located in the periplasm and cytoplasm, respectively. The activity of -galactosidase more rapidly than that of alkaline phosphatase in cells. In addition, we present an equation to describe our results. Correspondence to: Y. Takamoto     

Enhancement of Glucose transport in clone 9 cells by exposure to alkaline pH: Studies on potential mechanisms

Summary Incubation of a nontransformed rat liver cell line. Clone 9, at pH 8.5 resulted in an 16-fold stimulation of cytochalasin B-inhibitable 3-O-methylglucose (3-OMG) transport, an effect that was independent of the presence of serum. Exposure to 100 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated 3-OMG uptake, and the enhancement was not additive to that produced by incubation at pH 8.5. In cells depleted of protein kinase C activity by a 20-hr exposure to TPA, however, the stimulation of 3-OMG transport in response to incubation at alkaline pH was still fully demonstrable. In control and alkaline pH-exposed cells, the inhibition of 3-OMG uptake by cytochalasin B was consistent with a single-site ligand binding model (K ₁10^–7 m). Northern blot analysis demonstrated the presence of only the human erythrocyte/rat brain/HepG2 cell glucose transporter-mRNA isoform (EGT), and the abundance of this mRNA was unchanged following exposure to alkaline pH. Immunoblot analysis, using polyclonal antibodies directed against the carboxy-terminal dodecapeptide of EGT, demonstrated and 2.0-fold increase in the abundance of transporters in partially purified plasma membrane fractions following incubation at pH 8.5, while EGT abundance was unchanged in whole-cell extracts. It is concluded that the stimulation of glucose transport in response to incubation of Clone 9 cells at alkaline pH does not require the presence of serum or activation of protein kinase C, and that the response is at least in part mediated by an increase in the number of glucose transporters in the plasma membrane.     

Cellulolytic complex of Aspergillus niger under conditions for citric acid production. Isolation and characterization of two β-(1→4)-glucan hydrolases

Summary Two -(14)-endoglucanases have been purified from industrial waste broth of Aspergillus niger grown under conditions which produce citric acid. Molecular weights for endoglucanase A were 43,000 and 25,000 for endoglucanase B. Both enzymes exhibited very similar properties: a rather broad pH optimum between pH 2 and 7 for CM-cellulose hydrolysis and an inability to degrade crystalline cellulose. The endoglucanases have a higher thermal stability at acid pH (up to 60°C) than at alkaline pH. They are inhibited by iodine, HgCl₂ and N-bromosuccinimide.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

The 40s Ω-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1–38 heme-containing N-fragment with the residues 57–104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega ()-loop region. The fragment complex shows compact structure, native-like -helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1–38/57–104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (pK_a1=6.72) and a high pK_a-associated state transition (pK_a2=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (pK_a=8.9). The data indicate that the 40s -loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.