Calreticulin associates with stress proteins: Implications for chaperone function during heat stress

Acute heat stress leads to the glycosylation of a “prompt” stress glycoprotein, P-SG67/64, identified as calreticulin. In the present study, we used immunoprecipitation to investigate the interactions of P-SG/calreticulin with other proteins during cellular recovery from heat stress. In heat-stressed CHO and M21 cells, both glycosylated and unglycosylated P-SGs interact with HSP90, GRP94, GRP78, and the other prompt stress glycoprotein, P-SG50, in an ATP-independent manner. Specificity of HSP-P-SG interactions was determined by chemical cross-linking with the homo-bifunctional agent DSP (3,3′-dithiobis[succinimidyl propionate]). Characterization of the cross-linked complexes involving calreticulin and heat shock proteins (HSPs) showed an average mass of 400–600 kDa by gel filtration chromatography. Overall, the consistent association of glycosylated and unglycosylated calreticulin with P-SG50 and unglycosylated HSPs suggests that P-SG/calreticulin is an active member of the cast of glycone/aglycone chaperones that cooperate to achieve cellular recovery from acute heat stress. J. Cell. Biochem. 69:30–43, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of a extreme thermostable fructose-1,6-bisphosphate aldolase from hyperthermophilic bacterium Aquifex aeolicus

Fructose-1,6-bisphosphate (FBP) aldolase, is a glycolytic enzyme that catalyzes the reversible condensation reaction of FBP to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). The aldolase gene from Aquifex aeolicus was subcloned, overexpressed in E. coli and purified to 95% homogeneity. The purified enzyme was activated by high concentrations of NH₄⁺ and low concentrations of Co²⁺. The native molecular weight of the purified FBP aldolase was identified as 67 kDa (dimer) by gel filtration chromatography. The enzyme exhibits optimum pH at 6.5 and temperature at 90 °C. Based on the kinetic characterizations, the apparent K_m was calculated to be 4.4 ± 0.07 mM, while V_max was found to be 100 ± 0.02 μM min⁻¹ mg protein⁻¹. The recombinant protein showed extreme heat stability; no activity loss was observed even at 100 °C for 2 h. In addition, the thermophilic enzyme also showed higher stability against several organic solvents viz. acetonitrile, 1,4-dioxane, and methanol. With higher stability against both heat and organic solvents than any other class II aldolase, the A. aeolicus FBP aldolase is an attractive enzyme for use as a biocatalyst for industrial applications.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

The role of glycosylation in storage-protein synthesis in developing pea seeds

Intact pea (Pisum sativum L.) cotyledons were incubated with [¹⁴C]glucosamine at several stages of seed development and the resultant radioactive proteins were analysed by gel electrophoresis combined with immunoaffinity chromatography and sucrose gradient fractionation. Glucosamine was incorporated into at least five vicilin polypeptides (approx. molecular weight 70,000; 50,000, two components; 14,000, two components). No incorporation was detected into the subunits of legumin. Tunicamycin at 50 g/ml largely inhibited glucosamine incorporation but had little effect on the incorporation of ¹⁴C-labelled amino acids into cotyledon proteins, including vicilin. The assembly of vicilin polypeptides into full-sized protein oligomers (7–9 S) was also unaffected by tunicamycin. Chromatography on concanavalin A confirmed that glycosylation of cotyledon proteins was inhibited by tunicamycin. It is concluded that glycosylation of most cotyledonary proteins involves lipid-linked sugar intermediates, but that glycosylation itself is not an essential step in the synthesis of vicilin polypeptides nor in their assembly into oligomers.Abbreviations IgG immunoglobulin G - M W_t approximate molecular weight based on electrophoretic mobility relative to that of protein standards - SDS-PAGE Na-dodecyl sulfate-polyacrylamide gel electrophoresis     

Rate of DNA synthesis as a function of temperature in cultured hamster fibroblasts (V-79) and HeLa-S3 cells

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10⁻¹⁴ g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Intracellular distribution of heat-induced stress glycoproteins

Cellular heat stress results in elevated heat-shock protein (HSP) synthesis and in thermotolerance development. Recently, we demonstrated that protein glycosylation is also an integral part of the stress response with the identification of two major stress glycoproteins, GP50, associated with thermotolerance, and P-SG67, the “prompt” stress glycoprotein induced immediately during acute heat stress. In the present study, we characterized the subcellular location and redistribution of these proteins during the cellular injury and recovery phase. In unheated and heated CHO cells, both stress glycoproteins were present in each subcellular fraction isolated by differential centrifugation. However, the subcellular redistribution in the course of cellular recovery after heat stress was specific for each stress glycoprotein. GP50 was present in all subcellular fractions before heat stress, but showed relatively little redistribution after heat stress. By 24 h of recovery following stress, GP50 showed partial depletion from lysosomes and microsomes, and was mainly present in the mitochondria. Glycosylated P-SG67 was redistributed in a more complex fashion. It was seen predominantly in the lysosomes and microsomes immediately following heat-stress, but after 6 h of recovery following heat stress, it largely disappeared from the microsomes and was present mainly in the cytosol. By 24 h of recovery following heat stress, it was found predominantly in the nucleus-rich fraction and mitochondria. The localization of GP50 and P-SG67 by subcellular fractionation is consistent with immunolocalization studies and contrasts with the translocation of HSP70 after heat stress from cytosol to nuclei and nucleoli. These results reflect a characteristic distribution for each stress glycoprotein; their presence in virtually all subcellular fractions suggests multifunctional roles for the various stress glycoproteins in the cellular heat stress response. J. Cell. Biochem. 66:98–111, 1997. © 1997 Wiley-Liss, Inc.     

Heat Shock Glycoprotein GP50: Product of the Retinoic Acid-Inducible J6 Gene

High intracellular levels of heat shock proteins and enhanced protein glycosylation are two phenomena closely associated with the cellular stress response. GP50 is the major heat-induced glycoprotein in Chinese hamster ovary (CHO) cells; however, GP50 is not well characterized, and its function is unknown. J6 is a gene originally identified in F9 murine teratocarcinoma cells after exposure to retinoic acid. In this study we show that J6 is heat-inducible and codes for a protein that shares characteristics with GP50. Western blotting of CHO cell homogenates, using a J6 polyclonal antibody, showed a single band with a molecular weight identical to that of GP50. Thermotolerant cells showed increased levels of the J6/GP50 protein. Heat-shocked CHO cells also accumulated transiently high levels of J6 mRNA between 2 and 7 h following 10 rain at 45°C. These induction kinetics are similar to those for GP50 labeling with D-[³H]mannose and to the activation of major heat shock genes, e.g., hsp70. Hybrid selection of J6 mRNA from CHO cells, followed by in vitro translation, produced a single band on SDS-PAGE with a molecular weight identical to that of deglycosylated GP50. Neither cellular proliferation (exponential growth versus plateau phase) nor the specific heat shock temperature (41.5°C versus 45°C) had significant effects on J6 induction by heat stress. Stress conditions other than hyperthermia, including ethanol, arsenite, and hypoxia, increased J6 mRNA levels. Conversely, J6 mRNA was reduced by quercetin, brefeldin A, okadaic acid, uv, and hydrogen peroxide. Our data support the hypothesis that J6 is a heat shock gene with a gene product identical to the polypeptide moiety of GP50.     

Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Thermostability of lactate dehydrogenase LDH-A4 isoenzyme: Effect of heat shock protein DnaK on the enzyme activity

Cells exposed to temperature a few degrees higher than their growth temperature synthesize heat shock proteins (hsp) which may then compose even 20% of total protein content. This paper examined the in vitro protective effect of heat shock protein DnaK (70 kDa) from Escherichia coli against the heat inactivation of lactate dehydrogenase isoenzyme LDH-A₄. The LDH-A₄ isoenzyme was purified from fish skeletal muscle using the affinity chromatography on Oxamate-agarose. The enzyme was then heated in the absence and the presence of DnaK protein in a water bath at either 51 or 55°C. The LDH activity was determined by measuring the change in absorbency at 340 nm min⁻¹ at 30°C. The addition of DnaK protein to the LDH-A₄ isoenzyme before heat treatment can protect enzyme activity against mild thermal inactivation. Incubation of the LDH-A₄ isoenzyme at 51°C in the presence of DnaK protein stimulates its activity by about 30%. The presence of 2 mM ATP can raise LDH activity by another 10%. No significant recovery was observed when DnaK protein was added to LDH at 25°C following earlier inactivation. The maximal activities (V_max) in the presence of DnaK protein are almost twice those without DnaK protein in the case of heat-treated LDH-A₄ isoenzyme at 51°C. The observed protection of LDH-A₄ activity increased with the increasing DnaK protein concentration in the incubation medium. Results suggested that the presence of DnaK protein can protect LDH-A₄ from heat inactivation. This action may be important as a part of cellular chaperone machinery capable of repairing heat-induced protein damage. It may have a fundamental role in the acquisition of the thermotolerance to stress temperatures.     

Dynamics of proteolysis and its influence on the accumulation of intracellular recombinant proteins

A method to quantify the impact of proteolysis on accumulation of recombinant proteins in E. coli is described. A much smaller intracellular concentration of staphylococcal protein A (SpA) (14.7 mg · g⁻¹) compared to the fusion protein SpA-βgalactosidase (138 mg · g⁻¹) is explained by a very high proteolysis rate constant of SpA. The SpA synthesis rate reached a maximum one hour after induction and gradually decreased to half of this value at the end of the cultivation. The decrease of the synthesis rate and the 1st order kinetics of proteolysis lead to an equilibrium between synthesis and degradation of SpA from 2 h after induction. This resulted in no further SpA accumulation in cells, though synthesis continued for at least 10 h. Similar experiments with recombinant protein ZZT2 also revealed that most of the synthesized product was degraded. The order of proteolysis kinetics depended on the concentration of the recombinant protein: at low concentrations both SpA and ZZT2 were degraded according to first order kinetics, while at high concentrations ZZT2 was degraded according to zero order kinetics. In a protease Clp mutant the degradation rate decreased and intracellular concentration of ZZT2 increased from 50 mg · g⁻¹ to 120 mg · g⁻¹. The measurements of proteolysis rate throughout the cultivation enabled calculation of a hypothetical accumulation of the product assuming complete stabilization. In this case the concentration would have increased from 50 to 280 mg · g⁻¹ in 11 h. Thus, this method reveals the potential to increase the productivity by eliminating proteolysis.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

Alterations in amounts and covalent modifications of low-molecular-weight chromosomal proteins in Chinese hamster ovary cells during polyamine depletion

The effect of polyamine depletion on phosphorylation and ADP-ribosylation of low-M_r chromosomal proteins was studied in intact, mutant Chinese hamster ovary cells (CHO-P22) devoid of ornithine decarboxylase activity. When starved of polyamines for 6 days, severe polyamine deficiency develops and the cells gradually stop growing. The rate of DNA synthesis was retarded to 16% of the control value and to 29% in density-inhibited cells. The synthesis of high-mobility-group (HMG) proteins was decreased by 65% in polyamine-depleted cells and by 40% in density-inhibited cells. The synthesis of core histones was decreased by 40% both in polyamine-depleted and density-inhibited cells. In polyamine-depleted cells the molar ratio of the higher-M_r HMG proteins (HMG 1 + 2) to the lower-M_r HMG proteins (HMG 14 + P) was about one-half of that found in cells grown in the presence of putrescine or in density-inhibited cells. In contrast to HMG proteins, no major differences were found in the content of core histones in these cell populations. In the perchloric acid-soluble fraction of nuclear proteins, ³²P was incorporated mainly into histone H1, HMG P and a protein migrating more slowly than HMG 1 (protein P1). Specific changes in the ³²P-labeling and migration of a number of protein bands, including histone H1, was observed in polyamine-depleted cells as compared to cells grown in the presence of putrescine or to density-inhibited cells. ADP-ribosylation experiments using [³H]adenosine showed a different pattern of label distribution; the higher-M_r HMG proteins from polyamine-depleted cells contained about one-half the amount of label found in the proteins from control cells. The lower-M_r HMG proteins and histone H1 were the preferentially labeled proteins in polyamine-depleted cells. Labeling of core histones with [³²P]orthophosphate or [³H]adenosine did not differ markedly in the two cell populations. The results obtained using intact polyamine auxotrophic cells indicated that polyamine depletion is connected with more severe alterations in amounts and covalent modifications (phosphorylation and ADP-ribosylation) of HMG chromosomal proteins and histone H1 than core histones.     

Stress protein response in two sibling species of Marenzelleria (Polychaeta: Spionidae): is there an influence of acclimation salinity?

The induction and synthesis of stress proteins in the polychaete sibling species Marenzelleria viridis and M. neglecta was investigated at two different acclimation salinities (10 and 25 ppt). By in vitro labeling of dissected metameres with ³⁵S-methionine/cysteine and electrophoretic separation, four size classes of heat shock proteins (Hsps) were detected corresponding to 86, 78, 75 and 27 kDa. All Hsps, with the exception of Hsp86, represent a complex of multiple isoforms. The sibling species differed in three aspects of their heat shock response: (1) the induction temperature for Hsp75 synthesis was 25 °C and 30 °C in M. viridis and M. neglecta, respectively; (2) the relative level of synthesis of Hsp75 was higher in M. viridis; (3) the heat shock response was inactivated at a higher temperature in M. neglecta compared to M. viridis. The results showed that acclimation salinity had no explicit effect on Hsp synthesis in either species and that M. viridis was thermally more sensitive than its sibling species. We proposed that temperature, alone or in combination with other abiotic factors, plays a far greater role in the biogeographic distribution in Marenzelleria spp. than has been estimated so far.     

Soluble Integrin Ligands and Growth Factors Independently Rescue Neuroblastoma Cells from Apoptosis under Nonadherent Conditions

We have investigated the role of extracellular matrix (ECM) and growth factors in the survival of nonadherent human neuroblastoma cells (line SK-N-BE). Cells cultured in serum-free medium under nonadherent conditions died with apoptotic-like features (cromatin condensation and nuclear fragmentation). SK-N-BE cells underwent neuronal differentiation in response to retinoic acid (RA). While RA itself did not induce apoptosis, differentiation increased the susceptibility of SK-N-BE cells to detachment-induced apoptosis. The appearance of the apoptotic-like phenotype required the maintenance in suspension of SK-N-BE cells for at least 16 h (12.43 ± 1.40% of cells undergoing apoptosis) and the percentage increased up to 46.84 ± 3.15% after 24 h. Suspension-induced apoptosis did not depend on increased intracellular Ca²⁺levels nor onde novoprotein synthesis and was not associated with extensive DNA degradation. Stimulation by soluble collagen I rescued suspended cells from apoptosis, even in the absence of cell adhesion and spreading. The survival promoting effect of ECM was mediated by the integrin receptors, since ([1]) the protective effect of soluble collagen I was blocked by anti-integrin antibodies to β₁and α₁subunits and ([2]) the antibody-induced clustering of α₁, α₃, α_v, β₁, and β₃integrins rescued SK-N-BE cells cultured in suspension from apoptosis. As expected, adhesion on immobilized ECM proteins, collagen I, or laminin (0.1 to 10 μg/ml) also rescued SK-N-BE cells from apoptosis in a dose-dependent manner. Thede novoprotein synthesis was required to promote the survival effect of ECM, since cycloheximide completely abolished the protective effect of collagen I and protection from apoptosis by ECM or by anti-β₁antibody was associated with the increased expression ofbcl-2.In addition to integrin stimulation, serum, insulin, and nerve growth factor inhibited suspension-induced apoptosis of SK-N-BE cells. The survival effect of serum and growth factors did not require the synthesis of new proteins, unlike the ECM effect. These data show that matrix proteins can promote cell survival in neuronal cells via integrin receptors. This effect does not require cell adhesion and the subsequent changes in cell shape as it can be mediated by soluble integrin ligands in suspended cells and involves a signaling pathway different from that triggered by growth factors.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Purification and Characterization of the GroESLx Chaperonins from the Symbiotic X-Bacteria in Amoeba proteus

GroELx and GroESx proteins of symbiotic X-bacteria from Amoeba proteus were overproduced in Escherichia coli transformed with pAJX91 and pUXGPRM, respectively, and their chaperonin functions were assayed. We utilized σ⁷⁰-dependent specific promoters of groEx in the expression vectors and grew recombinant cells at 37°C to minimize coexpression of host groE of E. coli. For purifying the proteins, we applied the principle of heat stability for GroELx and pI difference for GroESx to minimize copurification with the hosts GroEL and GroES, respectively. After ultracentrifugation in a sucrose density gradient, the yield and purity of GroELx were 56 and 89%, respectively. The yield and purity of GroESx after anion-exchange chromatography were 62 and 91%, respectively. Purified GroELx had an ATPase activity of 53.2 nmol Pi released/min/mg protein at 37°C. The GroESx protein inhibited ATPase activity of GroELx to 60% of the control at a ratio of 1 for GroESx-7mer/GroELx-14mer. GroESLx helped refolding of urea-unfolded rhodanese up to 80% of the native activity at 37°C. By chemical cross-linking analysis, oligomeric properties of GroESx and GroELx were confirmed as GroESx₇ and GroELx₁₄ in two stacks of GroELx₇. In this study, we developed a method for the purification of GroESLx and demonstrated that their chaperonin function is homologous to GroESL of E. coli.