Degradation of the Large Subunit of Ribulose-1, 5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves

The degradation of the large subunit (LSU) of ribulose- 1, 5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) in wheat (Triticum aestivum L. cv. Yangmai 158) leaves was investigated. A 50 kDa fragment, a portion of the LSU of Rubisco, was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with antibody against tobacco Rubisco in crude enzyme extract of young wheat leaves. The appearance of the 50 kDa fragment was most obvious at 30-35 ℃ and pH 5.5. The LSU and its 50 kDa fragment both existed when the crude enzyme extract was incubated for 60 min. The amount of LSU decreased with incubation time from 0 to 3 h in crude enzyme extract. However, the 50 kDa fragment could not be found any pH from 4.5 to 8.5 in chloroplast lysates of young wheat leaves. In addition,through treatment with various inhibitors, reactions were inhibited by cysteine proteinase inhibitor E-64 or leupeptin.     

Degradation of Rubulose-1．5-Bisphosphate Carboxylase／Oxygenase in Wheat Leaves During Dark-induced Senescence

The degradation of Ribulose-1.5-bisphosphate carboxylase／oxygenase(Rubisco,EC4.1.1.39)in wheat(TnticumaestivumL．CV．Yan6mai 158)leaves dunng dark—induced senescence was studied．An／in vivo degradation product of Rubisco large subunit(LSU)with molecular weiht of 50kD was detected by SDS—PAGE and immunobloted with antibody against tobacco Rubisco．This fragment could also be detected in natural senescence．The result also suggested that the Rubisco holoenzyme had not dissociated when LSU hydrolyzed from 53 kD to 50kD．And．LSUcould be fragmented to 50kD at 30-35℃and at DH7．5 in crude enzyme extracts of wheat leaves dark—induced for 48h．which suggested that maybe LSU was degraded to 50 kD by anunknown protease in chloroplast．     

Cloning of Rat Vitamin K-Dependent γ-Glutamyl Carboxylase and Developmentally Regulated Gene Expression in Postimplantation Embryos

Vitamin K-dependent carboxylase catalyzes the posttranslational modification of glutamate to γ-carboxyglutamate (Gla) in its substrates, the vitamin K-dependent proteins (VKDPs). This modification is required for the activities of the VKDPs. Recent evidence demonstrates previously unrecognized roles for VKDPs as signaling molecules important in the regulation of cell growth, adhesion, and apoptosis, suggesting developmental functions for VKDPs and hence the carboxylase. The tissue distribution and functions of carboxylase in development are unknown. In this study, we isolated and characterized the full-length cDNA encoding the rat carboxylase and analyzed, at the cellular level, the expression of this gene in rat embryos byin situhybridization. We demonstrate that the expression of this gene is highly regulated in a developmental and tissue-specific manner. Hepatocytes, the major site of synthesis of VKDPs of blood coagulation, express carboxylase mRNA late in gestation, in contrast to the central nervous system, mesenchymal, and skeletal tissues which express carboxylase mRNA early during rat embryogenesis. The tissue-specific temporal expression of the carboxylase gene during embryogenesis indicates that vitamin K-dependent carboxylation and the formation of Gla is developmentally regulated. These studies suggest that vitamin K-dependent carboxylation is an important modulator of embryonic VKDP function.     

Evolution of C4 Phosphoenolpyruvate Carboxylase： Enhanced Feedback Inhibitor Tolerance Is Determined by a Single Residue

Dear Editor, Phosphoenolpyruvate carboxylase （PEPC） is the essential enzyme for initial carbon fixation in C4 photosynthesis. The C4-PEPC evolved from a non-photosynthetic C3 ancestor by subtle sequence changes resulting in distinctly different kinetic and regulatory characteristics （Svensson et al., 1997）. Malate and aspartate, which are formed from the carboxyla- tion product of PEPC, serve as feedback inhibitors of the PEPC （Wedding et al., 1990）. Higher malate or aspartate concen- trations during C4 photosynthesis require a reduced sensitiv- ity towards the feedback inhibitors of the C4-PEPC （Jacobs et al., 2008）.     

Effect of Vitamin K-dependent Protein Precursor Propeptide,Vitamin K Hydroquinone,and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO₃, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.     

Diurnal Changes in the Regulatory Properties of Phosphoenolpyruvate Carboxylase in CAM Plants: Are Alterations in the Quaternary Structure Involved?

Day — and night-forms of phosphoenolpyruvate carboxylase (EC 4.1.1.31) (PEP-C) were extracted from leaves of the CAM plant Kalanchoë daigremontiana. During storage after extraction, the day-form spontaneously lost its sensitivity towards the allosteric inhibitor l -malate. This effect was accelerated by inorganic phosphate, PEP, 3-PGA and G-6-P. l -Malate however stabilized the malate sensitivity of the day-form of PEP-C. Crude desalted extracts of the day-form and of the night-form of PEP-C were subjected to ultracentrifugation on a continuous sucrose gradient in the presence of malate and of marker enzymes. Both forms of PEP-C were found to have relative molecular masses of about 200,000. This suggests that both forms represent the dimers of the enzyme protein. The result also suggests that reversible dissociation and association of enzyme subunits is not the mechanism which brings about the interconversion of the two PEP-C forms during the diurnal CAM cycle in vivo.     

Structure and Identification of a Pterin Dehydratase-like Protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) Assembly Factor in the α-Carboxysome

Carboxysomes are proteinaceous bacterial microcompartments that increase the efficiency of the rate-limiting step in carbon fixation by sequestering reaction substrates. Typically, α-carboxysomes are genetically encoded as a single operon expressing the structural proteins and the encapsulated enzymes of the microcompartment. In addition, depending on phylogeny, as many as 13 other genes are found to co-occur near or within α-carboxysome operons. One of these genes codes for a protein with distant homology to pterin-4α-carbinolamine dehydratase (PCD) enzymes. It is present in all α-carboxysome containing bacteria and has homologs in algae and higher plants. Canonical PCDs play an important role in amino acid hydroxylation, a reaction not associated with carbon fixation. We determined the crystal structure of an α-carboxysome PCD-like protein from the chemoautotrophic bacterium Thiomonas intermedia K12, at 1.3-Å resolution. The protein retains a three-dimensional fold similar to canonical PCDs, although the prominent active site cleft present in PCD enzymes is disrupted in the α-carboxysome PCD-like protein. Using a cell-based complementation assay, we tested the PCD-like proteins from T. intermedia and two additional bacteria, and found no evidence for PCD enzymatic activity. However, we discovered that heterologous co-expression of the PCD-like protein from Halothiobacillus neapolitanus with RuBisCO and GroELS in Escherichia coli increased the amount of soluble, assembled RuBisCO recovered from cell lysates compared with co-expression of RuBisCO with GroELS alone. We conclude that this conserved PCD-like protein, renamed here α-carboxysome RuBisCO assembly factor (or acRAF), is a novel RuBisCO chaperone integral to α-carboxysome function.     

Does Decrease in Ribulose-1,5-Bisphosphate Carboxylase by Antisense RbcS Lead to a Higher N-Use Efficiency of Photosynthesis under Conditions of Saturating CO2 and Light in Rice Plants?

Rice (Oryza sativa L.) plants with decreased ribulose-1,5-bisphosphate carboxylase (Rubisco) were obtained by transformation with the rice rbcS antisense gene under the control of the rice rbcS promoter. The primary transformants were screened for the Rubisco to leaf N ratio, and the transformant with 65% wild-type Rubisco was selected as a plant set with optimal Rubisco content at saturating CO2 partial pressures for photosynthesis under conditions of high irradiance and 25[deg]C. This optimal Rubisco content was estimated from the amounts and kinetic constants of Rubisco and the gas-exchange data. The R1 selfed progeny of the selected transformant were grown hydroponically with different N concentrations. Rubisco content in the R1 population was distributed into two groups: 56 plants had about 65% wild-type Rubisco, whereas 23 plants were very similar to the wild type. Although the plants with decreased Rubisco showed 20% lower rates of light-saturated photosynthesis in normal air (36 Pa CO2), they had 5 to 15% higher rates of photosynthesis in elevated partial pressures of CO2, (100-115 Pa CO2) than the wild-type plants for a given leaf N content. We conclude that the rice plants with 65% wild-type Rubisco show a higher N-use efficiency of photosynthesis under conditions of saturating CO2 and high irradiance.     

Isolation and Characterization of cbbL and cbbS Genes Encoding Form I Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Large and Small Subunits in Nitrosomonas sp.…

The cbbL and cbbS genes encoding form I ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large and small subunits in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 were cloned and sequenced. The deduced gene products, CbbL and CbbS, had 93 and 87% identity with Thiobacillus intermedius CbbL and Nitrobacter winogradskyi CbbS, respectively. Expression of cbbL and cbbS in Escherichia coli led to the detection of RubisCO activity in the presence of 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). To our knowledge, this is the first paper to report the genes involved in the carbon fixation reaction in chemolithotrophic ammonia-oxidizing bacteria.     

Effects on the Activity of the Enzyme Phosphoribosyl-Aminoimidazole Carboxylase,Involved in the Biosynthesis of Purine Nucleotides De Novo by Bi-Valent Metal Complexes of the Natural Substrate 5-Amino-1-β-D-Ribofuranosylimidazole-4-Carboxylic Acid 5′-Phosphate

Abstract

A series of bi-valent metal complexes of 5-amino-l-β-D-ribofuranosylimida-zole-4-carboxylic acid and its 5′-phosphate derivative (CAIR), a central intermediate in de novo biosynthesis of purine nucleotides have been synthesised. The nucleotide complexes were found to affect the activity of the enzyme phosphoribosylaminoimidazole carboxylase (EC. 4.1.1.21).     

ATP Hydrolysis Activity and Polymerization State of Ribulose-1,5-Bisphosphate Carboxylase Oxygenase Activase (Do the Effects of Mg2+, K+, and Activase Concentrations Indicate a Functional Similarity to Actin?)

The ATPase activity and fluoresence of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activase were determined over a range of MgCl2, KCl, and activase concentrations. Both salts promoted ADP release from ATP and intrinsic fluorescence enhancement by adenosine 5[prime]-[[gamma]-thio] triphosphate, but Mg2+ was about 10 times more effective than K+. ATPase and fluorescence enhancement both increased from zero to saturation within the same Mg2+ and K+ concentration ranges. At saturating concentrations (5 mM Mg2+ and 22 mM K+), the specific activity of ATPase (turnover time, about 1 s) and specific intrinsic fluorescence enhancement were maximal and unaffected by activase concentration above 1 [mu]M activase; below 1 [mu]M activase, both decreased sharply. These responses are remarkably similar to the behavior of actin. Intrinsic fluorescence enhancement of Rubisco activase reflects the extent of polymerization, showing that the smaller oligomer or monomer present in low-salt and activase concentrations is inactive in ATP hydrolysis. However, quenching of 1-anilinonapthaline-8-sulfonate fluorescence revealed that ADP and adenosine 5[prime]-[[gamma]-thio] triphosphate bind equally well to activase at low- and high-salt concentrations. This is consistent with an actin-like mechanism requiring a dynamic equilibrium between monomer and oligomers for ATP hydrolysis. The specific activation rate of substrate-bound decarbamylated Rubisco decreased at activase concentrations below 1 [mu]M. This suggests that a large oligomeric form of activase, rather than a monomer, interacts with Rubisco when performing the release of bound ribulose-1,5-bisphosphate from the inactive enzyme.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.     

Mechanisms of action of the α-amylase of Micromonospora melanosporea

The -amylase of Micromonospora melanosporea was produced extracellularly during batch fermentation in a 5.0-1 fermentor. The absence of an organic nitrogen source in its growth medium facilitated subsequent purification of the enzyme by ammonium sulphate fractionation and two consecutive Superose-12 gel-filtration steps. The enzyme exhibited maxima for activity at pH 7.0 and 55° C and was 72% stable at pH 6.0–12.0 for 30 min at 40° C. It had a relative molecular mass of 45 000 and an isoelectric point at pH 7.6. The enzyme catalyses the conversion of starch to maltose (53%, w/w) as the predominant final end-product. Initial hydrolysis of this substrate, however, gave rise to the formation of maltooligosaccharides in the range maltotriose to maltohexaose. Maximum yields of these intermediate sugars accumulated to between 31 and 42% (w/w) as the reaction proceeded. The action of the M. melanosporea amylase on high concentrations of saccharides larger than maltotriose resulted in the formation of mainly maltose and maltotriose without concomitant glucose production. A combination of hydrolytic and transfer events is postulated to be responsible for this phenomenon and for the high maltose levels achieved. Correspondence to: C. T. Kelly     

Selection of optimal variants of Gō-like models of proteins through studies of stretching

The Gō-like models of proteins are constructed based on the knowledge of the native conformation. However, there are many possible choices of a Hamiltonian for which the ground state coincides with the native state. Here, we propose to use experimental data on protein stretching to determine what choices are most adequate physically. This criterion is motivated by the fact that stretching processes usually start with the native structure, in the vicinity of which the Gō-like models should work the best. Our selection procedure is applied to 62 different versions of the Gō model and is based on 28 proteins. We consider different potentials, contact maps, local stiffness energies, and energy scales—uniform and nonuniform. In the latter case, the strength of the nonuniformity was governed either by specificity or by properties related to positioning of the side groups. Among them is the simplest variant: uniform couplings with no i, i + 2 contacts. This choice also leads to good folding properties in most cases. We elucidate relationship between the local stiffness described by a potential which involves local chirality and the one which involves dihedral and bond angles. The latter stiffness improves folding but there is little difference between them when it comes to stretching.     

Mechanism of action of α-galactosidase

The effect ofpH on K_m and V_max values of coconut α-galactosidase indicates the involvement of two ionizing groups with pK_a values of 3.5 and 6.5 in catalysis. Chemical modification has indicated the presence of two carboxyl groups, a tryptophan and a tyrosine, at or near the active site of α-galactosidase. Based on these facts a new mechanism of action for α-galactosidase is proposed in which the ionizing group with a pK_a of 3.5 is a carboxyl group involved in stabilizing a carbonium ion intermediate and the ionizing group with a pK_a of 6.5 is a carboxyl group perturbed due to the presence of a hydrophobic residues in its vicinity which donates a H⁺ ion in catalysis.     

Chemistry of the “molecular trap” of protease-catalyzed splicing reaction of complementary segments of α-subunit of hemoglobin A

The complementary fragments of human Hb α, α_1–30, and α_31–141 are spliced together by V8 protease in the presence of 30%n-propanol to generate the full-length molecule (Hb α-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb α is facilitated by the organic cosolvent induced α-helical conformation of product acting as the “molecular trap” of the splicing reaction. The segments α_24–30 and α_31–40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) α_24–40 has been manipulated by engineering the amino acid replacements to the positions α²⁷ and α³¹ to delineate the structural basis of the molecular trap. The location of Glu²⁷ and Arg³¹ residues in the contiguous segment α_24–40 (as well as in other larger segments) is ideal to generate (i, i+4) side-chain carboxylate-guanidino interaction in its α-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at α²⁷ and α³¹ facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The γ-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an α-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu³⁰-Arg³¹ peptide bond. The second structural aspect is a site-specific event, ani, i+4 side-chain interaction in the α-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven “molecular traps” of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.