Influence of glycosylation on the oligomeric structure of yeast acid phosphatase

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units. Dissociation to dimer was in all cases accompanied with a loss of most of the enzyme activity. The underglycosylated acid phosphatase, with less than eight carbohydrate chains per subunit, secreted from cells treated with moderate tunicamycin concentrations, contained besides octamers a high proportion of the dimers. With decreasing levels of enzyme glycosylation, the proportion of dimers increases and the amount of octamers correspondingly decreases. Furthermore, underglycosylated octamers were found to be significantly less stable than the fully glycosylated ones. This showed that carbohydrate chains play a significant role in the octamer formation in vivo, and in stabilization of the enzyme octameric form.     

Over-expression, purification and characterization of the oligomerization dynamics of an invertebrate mitochondrial creatine kinase

Mitochondrial creatine kinase (MtCK) plays a central role in energy homeostasis within cells that display high and variable rates of ATP turnover. Vertebrate MtCKs exist primarily as octamers but readily dissociate into constituent dimers under a variety of circumstances. MtCK is an ancient protein that is also found in invertebrates including sponges, the most primitive of all multi-cellular animals. We have cloned, expressed, and purified one of these invertebrate MtCKs from a marine polychaete worm, Chaetopterus variopedatus (CVMtCK). Size exclusion chromatography and dynamic light scattering (DLS) were used to characterize oligomeric state in comparison with that of octameric chicken sarcomeric isoform (SarMtCK). At protein concentrations >1 mg/ml, CVMtCK was predominantly octameric (>90%). When diluted to 0.1 mg/ml, CVMtCK dissociated into dimers much more rapidly than SarMtCK when observed under identical conditions. The rate of dissociation for both MtCKs increased as temperature rose from 2 to 28 degrees C, and in CVMtCK, fell at higher incubation temperatures. The fraction of octameric CVMtCK at equilibrium increased with temperature and then fell. Temperature transition studies showed that octamers and dimers were rapidly interconvertible on a similar time scale. Importantly, when CVMtCK was converted to the transition state analog complex (TSAC), both size exclusion chromatography and DLS showed that there was minimal dissociation of octamers into dimers while SarMtCK octamers were highly unstable as the TSAC. These results clearly show distinct differences in octamer stability between CVMtCK and SarMtCK, which could impact function under physiological circumstances. Furthermore, the large yield of recombinant protein and high stability of CVMtCK in the TSAC suggest that this protein might be a good target for crystallization efforts.     

pH-, temperature- and ion-dependent oligomerization of Sulfolobus solfataricus recombinant amidase: a study with site-specific mutants

Recombinant amidase from Sulfolobus solfataricus occurred as a dimer of 110 kDa comprising identical subunits. Only dimers were present at pHs above 7.0, but with decreasing pH, dimers associated into octamers, with complete oligomerization occurring at pH 3.0. Oligomerization showed reversible temperature-dependence, with octamer formation increasing with temperature from 36 °C to between 70 and 80° C. Increasing salt concentrations, favored dissociation of the octamers. Among the three investigated factors affecting the dimer–octamer equilibrium, the most important was pH. Among four mutants obtained by site-specific mutagenesis and selection for pH and temperature sensitivity, the T319I and D487N mutant amidases, like that of the native Sulfolobus solfataricus, responded to changes in pH and temperature with a conformational change affecting the dimer–octamer equilibrium. The Y41C and L34P mutant amidases were unaffected by pH and temperature, remaining always in the dimeric state. The differences among mutants in protein conformation must be related to the position of the introduced mutation. Although the L34P and Y41C mutations are located in the helical region 33–48 (LLKLQLESYERLDSLP), which is close to the amino-terminal segment of the protein, the T319I mutation is located in a strand on the surface of the protein, which is far from, and opposite to, the amino-terminal segment. The D487N mutation is located in the center of the protein, far distant from the 33–48 segment. These observations suggest that the segment of the protein closest to the amino-terminus plays a key role in the association of dimers into octamers.     

Functional studies with the octameric and dimeric form of mitochondrial creatine kinase. Differential pH-dependent association of the two oligomeric forms with the inner mitochondrial membrane

Phosphate extraction of mitochondrial creatine kinase (Mi-CK, EC 2.7.3.2) from freshly isolated intact mitochondria of chicken cardiac muscle, after short swelling in hypotonic medium, yielded more than 90% of octameric and only small amounts of dimeric Mi-CK as judged by fast protein liquid chromatography-gel permeation analysis of the supernatants immediately after extraction of the enzyme. In extraction buffer, octameric Mi-CK displayed a tendency to dissociate, albeit at a slow rate with a half-life of approximately 3-5 days, into stable dimers. Experiments with purified Mi-CK octamers or dimers, or defined mixtures thereof, incubated under identical conditions with Mi-CK-depleted mitoplasts revealed that both oligomeric forms of Mi-CK can rebind to mitoplasts. However, the association of Mi-CK was strongly pH-dependent and, in addition, octameric and dimeric Mi-CK showed different pH dependences of rebinding. Therefore, it was possible under certain pH conditions to rebind either both oligomeric forms or selectively the octamers only. Furthermore, evidence is presented that Mi-CK dimers partially form octamers upon rebinding to the inner membrane. The differential association of the two oligomeric Mi-CK forms with the inner mitochondrial membrane together with the dynamic equilibrium between octameric and dimeric Mi-CK (Schlegel, J., Zurbriggen, B., Wegmann, G., Wyss, M., Eppenberger, H.M., and Wallimann, T. (1988) J. Biol. Chem., 263, 16942-16953) suggest that both oligomeric forms are physiologically relevant. A change in the octamer to dimer ratio may influence the association behavior of Mi-CK in general and thus modulate mitochondrial energy flux as discussed in the phosphoryl creatine circuit model (Wallimann, T., Schnyder, T., Schlegel, J., Wyss, M., Wegmann, G., Rossi, A.-M., Hemmer, W., Eppenberger, H.M., and Quest, A.F.G. (1989) Prog. Clin. Biol. Res. 315, 159-176.     

Dissociation of the Octameric Enolase from S. Pyogenes - One Interface Stabilizes Another

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO₄, a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO₄ dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.     

Native mitochondrial creatine kinase forms octameric structures. I. Isolation of two interconvertible mitochondrial creatine kinase forms, dimeric and octameric mitochondrial creatine kinase: characterization, localization, and structure-function relationships

The mitochondrial isoform of creatine kinase (Mi-CK, EC 2.7.3.2) purified to homogeneity from chicken cardiac muscle by the mild and efficient technique described in this article was greater than or equal to 99.5% pure and consisted of greater than or equal to 95% of a distinct, octameric Mi-CK protein species, with a Mr of 364,000 +/- 30,000 and an apparent subunit Mr of 42,000. The remaining 5% were dimeric Mi-CK with an apparent Mr of 86,000 +/- 8,000. Octamerization was not due to covalent linkages or intermolecular disulfide bonding. Upon dilution into buffers of low ionic strength and alkaline pH, octameric Mi-CK slowly dissociated in a time-dependent manner (weeks-months) into dimeric Mi-CK. However, the time scale of dimerization was reduced to minutes by the addition to diluted Mi-CK octamers of a mixture of Mg2+, ADP, creatine and nitrate known to induce a transition-state analogue complex (Milner-White, E.J., and Watts, D. C. (1971) Biochem. J. 122, 727-740). The conversion was fully reversible, and octamers were reformed by simple concentrations of Mi-CK dimer solutions to greater than or equal to 1 mg/ml at near neutral pH and physiological salt concentrations in the absence of adenine nucleotide. After separation of the two Mi-CK species by gel filtration, electron microscopic analysis revealed uniform square-shaped particles with a central negative-stain-filled cavity in the octamer fractions and "banana-shaped" structures in the dimer fractions. Mi-CK was localized inside the mitochondria by immunogold labeling with polyclonal antibodies. A dynamic model of the octamer-dimer equilibrium of Mi-CK and the preferential association of the octameric Mi-CK form with the inner mitochondrial membrane is discussed in the context of regulation of Mi-CK activity, mitochondrial respiration, and the CP shuttle.     

Native mitochondrial creatine kinase forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurements by scanning transmission electron microscopy, and image analysis of single mitochondrial creatine kinase octamers

Electron micrographs of negatively stained and metal-shadowed mitochondrial creatine kinase (Mi-CK) molecules purified as described by Schlegel et al. (Schlegel, J., Zurbriggen, B., Wegmann, E., Wyss, M., Eppenberger, H. M., and Wallimann, T. (1988) J. Biol Chem. 263, 16942-16953) revealed a homogeneous population (greater than or equal to 95%) of distinctly sized square-shaped, octameric particles with a side length of 10 nm that frequently exhibited a pronounced 4-fold axis of symmetry. The cube-like molecules consist of four dimers that are arranged around a stain-accumulating central cavity of 2.5-3 nm in diameter. This interpretation is supported by single particle averaging including correlation analysis by computer. Upon prolonged storage or high dilution, the cube-like octamers tended to dissociate into "banana-shaped" dimers. Sedimentation velocity and sedimentation equilibrium experiments yielded an s value of 12.8-13.5 S and an Mr of 328,000 +/- 25,000 for the octameric cubes. An s value of 5.0 S and a Mr of 83,000 +/- 8,000 was found under conditions which revealed banana-shaped dimers. These dimers proved to be very stable, as their dissociation into monomers of 45 kDa (s value = 2.0 S) required 6 M guanidine HCl. Thus, the oligomeric structures observed in the electron microscope are identified as Mi-CK dimers (banana-shaped structures) and cubical Mi-CK octamers assembled from four Mi-CK dimers. The octameric nature of native Mi-CK and the formation of Mi-CK dimers were confirmed by direct mass measurements of individual molecules by scanning transmission electron microscopy yielding a molecular mass of 340 +/- 55 kDa for the octamer and 89 +/- 27 kDa for the dimer. A structural model of Mi-CK octamers and the possible interaction with ATP/ADP-translocator molecules as well as with the outer mitochondrial membrane is proposed. The implications with respect to the physiological function of Mi-CK as an energy-channeling molecule at the producing side of the phosphoryl creatine shuttle are discussed.     

Association-dissociation of mammalian brain glutamine synthetase: effects of metal ions and other ligands

Glutamine synthetase from ovine brain has been found to exist in vivo and in vitro as a Mn4E complex, where E is octameric enzyme [F. C. Wedler, R. B. Denman, and W. G. Roby (1982) Biochemistry 24, 6389-6396]. Previously observed anomolous effects of added metal ions and protein concentration on the observed specific activity in vitro can now be explained in terms of association-dissociation of the native octamer. In the absence of glycerol, added to stabilize the enzyme for long-term storage, activity decreases sharply below 4 micrograms/ml (20 nM octamer) in assay mixtures due to dissociation of octamer to tetramer and thence to inactive monomer. No dimeric species were detectable under any conditions. The octameric species Mn4EMn4 could be activated further by Mn(II) to form a species Mn4EMn4Mn8 that has a specific activity of ca. 900 U/mg in the transferase assay. Enzyme with one Mn(II)/subunit, Mn4EMn4, associated to octamers more extensively than Mn4E. At the low concentrations of enzyme at which the tetramer predominates, addition of substrates alone or in pairs caused partial reassociation to octamers, the most effective combinations being ATP and glutamate, ADP and L-glutamine, or ATP and L-methionine sulfoximine. Analysis of the data by the methods of Kurganov or Thomes and co-workers indicate that the tetramer/octamer equilibrium has a Kd value of ca. 2.5 X 10(-6) M, comparable to values calculated for other enzyme systems. The specific activities for octamer and monomer in the Mg(II)-dependent transferase assay were calculated to be 200 +/- 20 and 0 U/mg, respectively. Direct determination of the specific activity of pure tetramer is hampered by its substrate-promoted reassociation to octamer under assay conditions. Tetramers, produced by 2 M urea and then immobilized on CNBr-activated Sepharose 4B, exhibited a specific activity that was 86% of that of the identically treated octamers. This indicates a specific activity of ca. 172 (+/- 20) for tetramers in solution. Light-scattering experiments showed that, with 1.7-2.0 Mn(II) bound per subunit, the octameric enzyme octamers can associate further to an oligomeric species (Mn4EMn4Mn8)n, where n greater than or equal to 5. This oligomerization also was promoted strongly by lanthanide ions. Mg(II), however, caused only the association of tetramer to octamer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallographic analysis reveals octamerization of viroplasm matrix protein P9-1 of Rice black streaked dwarf virus

The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.     

Characterization of the molecular properties of KtrC,a second RCK domain that regulates a Ktr channel in Bacillus subtilis

RCK (regulating conductance of K⁺) domains are common regulatory domains that control the activity of eukaryotic and prokaryotic K⁺ channels and transporters. In bacteria these domains play roles in osmoregulation, regulation of turgor and membrane potential and in pH homeostasis. Whole-genome sequencing unveiled RCK gene redundancy, however the biological role of this redundancy is not well understood. In Bacillus subtilis, there are two closely related RCK domain proteins (KtrA and KtrC) that regulate the activity of the Ktr cation channels. KtrA has been well characterized but little is known about KtrC. We have characterized the structural and biochemical proprieties of KtrC and conclude that KtrC binds ATP and ADP, just like KtrA. However, in solution KtrC exist in a dynamic equilibrium between octamers and non-octameric species that is dependent on the bound ligand, with ATP destabilizing the octameric ring relative to ADP. Accordingly, KtrC-ADP crystal structures reveal closed octameric rings similar to those in KtrA, while KtrC-ATP adopts an open assembly with RCK domains forming a super-helix. In addition, both KtrC-ATP and -ADP octamers are stabilized by the signaling molecule cyclic-di-AMP, which binds to KtrC with high affinity. In contrast, c-di-AMP binds with 100-fold lower affinity to KtrA. Despite these differences we show with an E. coli complementation assay that KtrC and KtrA are interchangeable and able to form functional transporters with both KtrB and KtrD. The distinctive properties of KtrC, in particular ligand-dependent assembly/disassembly, suggest that this protein has a specific physiological role that is distinct from KtrA.     

Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli

The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.     

Determination of the multimerization state of the hepatitis delta virus antigens in vivo

Hepatitis delta virus expresses two essential proteins, the small and large delta antigens, and both are required for viral propagation. Proper function of each protein depends on the presence of a common amino-terminal multimerization domain. A crystal structure, solved using a peptide fragment that contained residues 12 to 60, depicts the formation of an octameric ring composed of antiparallel coiled-coil dimers. Because this crystal structure was solved for only a fragment of the delta antigens, it is unknown whether octamers actually form in vivo at physiological protein concentrations and in the context of either intact delta antigen. To test the relevance of the octameric structure, we developed a new method to probe coiled-coil structures in vivo. We generated a panel of mutants containing cysteine substitutions at strategic locations within the predicted monomer-monomer interface and the dimer-dimer interface. Since the small delta antigen contains no cysteine residues, treatment of cell extracts with a mild oxidizing reagent was expected to induce disulfide bond formation only when the appropriate pairs of cysteine substitution mutants were coexpressed. We indeed found that, in vivo, both the small and large delta antigens assembled as antiparallel coiled-coil dimers. Likewise, we found that both proteins could assume an octameric quaternary structure in vivo. Finally, during the course of these experiments, we found that unprenylated large delta antigen molecules could be disulfide cross-linked via the sole cysteine residue located within the carboxy terminus. Therefore, in vivo, the C terminus likely provides an additional site of protein-protein interaction for the large delta antigen.     

Transcriptional analysis of the nitrile-degrading operon from Rhodococcus sp. ACV2 and high level production of recombinant amidase with an Escherichia coli– T7 expression system

Northern blotting analysis with RNA probes derived from amidase and nitrile hydratase genes from Rhodococcus sp. ACV2 revealed that both genes are part of the same operon. RNase protection mapping and sequence analysis indicated that the operon is probably under the control of a sigma 70-like promoter located upstream from the amidase gene. Plasmids were constructed with the cloned genes under tac and lac promoter control. Expression of amdA was demonstrated in Escherichia coli. In another construction, the amdA gene was inserted under the control of the bacteriophage T7 promoter. Large amounts of recombinant amidase (at least 20% of total proteins) in a soluble and active form were obtained with the E. coli-T7 expression system by lowering the growth temperature to 29 degrees C, without IPTG induction. The ratio of amidase activity of strain ACV2 to E. coli was approximately 1:3. Purification of the recombinant amidase was carried out in one chromatographic step, giving an enzyme preparation that could be used directly in a biotechnological process.     

The role of an absolutely conserved tryptophan residue in octamer formation and stability in mitochondrial creatine kinases

In most organisms, mitochondrial creatine kinase (MtCK) is present as dimers and octamers with the latter predominating under physiological conditions. An absolutely conserved tryptophan residue (Trp-264 in chicken sarcomeric MtCK) appears to play a key role in octamer stability. Recently, it has been shown that the sponge Tethya aurantia, a member of the most ancient group of living multi-cellular animals, expresses an obligate, dimeric MtCK that lacks this absolutely conserved tryptophan residue, instead possessing a tyrosine in this position. In the present study we confirm that the absolutely conserved tryptophan residue is lacking in other sponge MtCKs where it is instead substituted by histidine or asparagine. Site directed mutations of the Trp-264 in expression constructs of chicken sarcomeric MtCK and the octameric MtCK from the marine worm Chaetopterus destabilized the octameric quaternary structure producing only dimers. A Tyr-->Trp mutation in an expression construct of the Tethya MtCK construct failed to produce octamerization; Tyr-->His and Tyr-->Asn mutations also yielded dimers. These results, in conjunction with analysis of homology models of Chaetopterus and Tethya MtCKs, strongly support the view that while the absolutely conserved tryptophan residue is important in octamer stability, octamer formation involves a complex suite of interactions between a variety of residues.     

High-level secretory expression of penicillin amidase from Providencia rettgeri in Saccharomyces cerevisiae: purification and characterization

Heterologous production of the heterodimeric penicillin G amidase (PAC) from Providencia rettgeri was optimized in Saccharomyces cerevisiae. Several factors, including the effect of different growth and induction conditions, were identified to be critical for the enzyme overproduction and secretion. The PAC yield was significantly increased by more than 500-fold compared to that obtained in the native bacterium, and the recombinant enzyme was almost entirely secreted. Electrophoretic characterization of the secreted rPAC(Pr), which was purified over 20-fold by a combination of hydrophobic interaction and ion-exchange chromatography, demonstrated a microheterogeneity of the recombinant enzyme. The recombinant PAC(Pr) was further characterized in terms of specific activity, pH, and temperature profiles and kinetic parameters. The data presented here suggest that by overexpressing rPAC(Pr) in S.cerevisiae and purifying secreted enzyme from culture medium one can readily obtain a large amount of an alternative source of penicillin amidase with properties comparable to that of todays main industrial source of enzyme.     

Cloning, expression, and characterization of polyamidase from Nocardia farcinica and its application to polyamide modification

Polyamidase was able to hydrolyze the amide bond of insoluble polymer. In the present study, a polyamidase from Nocardia farcinica CGMCCC4.1166 was cloned and expressed in E. coli BL21(DE3). The recombinant polyamidase was purified to homogeneity, through a combination of chromatography of anion exchange, and hydrophobic interaction. The purified enzyme was characterized in detail. The optimum temperature of the enzyme was 50°C, and it was stable below 40°C. The enzyme had an optimum pH of 8.0, with pH stability between pH 7.0 and 9.0. The enzyme does not need metal ion as cofactor. In addition, when the enzyme was utilized to hydrolyze polyamide, the monomeric product of adipic acid was verified by HPLC analysis; as well, the wettability and dyeability of polyamide fabric after enzyme treatment were significantly improved, which differed from those of its inactive S173A mutant, and the amidase from Rhodococcus pyridinivorans. Furthermore, the structural features near the active site of polyamidase, different from other amidases, were explored.     

Recombinant hemoglobin betaG83C-F41Y

We have engineered a stable octameric hemoglobin (Hb) of molecular mass 129 kDa, a dimer of recombinant hemoglobin (rHb betaG83C-F41Y) tetramers joined by disulfide bonds at the beta83 position. One of the major problems with oxygen carriers based on acellular hemoglobin solutions is vasoactivity, a limitation which may be overcome by increasing the molecular size of the carrier. The oxygen equilibrium curves showed that the octameric rHb betaG83C-F41Y exhibited an increased oxygen affinity and a decreased cooperativity. The CO rebinding kinetics, auto-oxidation kinetics, and size exclusion chromatography did not show the usual dependence on protein concentration, indicating that this octamer was stable and did not dissociate easily into tetramers or dimers at low concentration. These results were corroborated by the experiments with haptoglobin showing no interaction between octameric rHb betaG83C-F41Y and haptoglobin, a plasma glycoprotein that binds the Hb dimers and permits their elimination from blood circulation. The lack of dimers could be explained if there are two disulfide bridges per octamer, which would be in agreement with the lack of reactivity of the additional cysteine residues. The kinetics of reduction of the disulfide bridge by reduced glutathione showed a rate of 1000 M(-1) x h(-1) (observed time coefficient of 1 h at 1 mM glutathione) at 25 degrees C. Under air, the cysteines are oxidized and the disulfide bridge forms spontaneously; the kinetics of the tetramer to octamer reaction displayed a bimolecular reaction of time coefficient of 2 h at 11 microM Hb and 25 degrees C. In addition, the octameric rHb betaG83C-F41Y was resistant to potential reducing agents present in fresh plasma.     

Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp.

A constitutively expressed aliphatic amidase from a Rhodococcus sp. catalyzing acrylamide deamination was purified to electrophoretic homogeneity. The molecular weight of the native enzyme was estimated to be 360,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation yielded a homogeneous protein band having an apparent molecular weight of about 44,500. The amidase had pH and temperature optima of 8.5 and 40 degrees C, respectively, and its isoelectric point was pH 4.0. The amidase had apparent K(m) values of 1.2, 2.6, 3.0, 2.7, and 5.0 mM for acrylamide, acetamide, butyramide, propionamide, and isobutyramide, respectively. Inductively coupled plasma-atomic emission spectometry analysis indicated that the enzyme contains 8 mol of iron per mol of the native enzyme. No labile sulfide was detected. The amidase activity was enhanced by, but not dependent on Fe(2+), Ba(2+), and Cr(2+). However, the enzyme activity was partially inhibited by Mg(2+) and totally inhibited in the presence of Ni(2+), Hg(2+), Cu(2+), Co(2+), specific iron chelators, and thiol blocking reagents. The NH2-terminal sequence of the first 18 amino acids displayed 88% homology to the aliphatic amidase of Brevibacterium sp. strain R312.     

Structure of the mitochondrial creatine kinase octamer: high-resolution shadowing and image averaging of single molecules and formation of linear filaments under specific staining conditions.

The combination of high-resolution tantalum/tungsten (Ta/W) shadowing at very low specimen temperature (-250 degrees C) under ultrahigh vacuum (less than 2 x 10(-9) mbar) with circular harmonic image averaging revealed details on the surface structure of mitochondrial creatine kinase (Mi-CK) molecules with a resolution less than 2.5 nm. Mi-CK octamers exhibit a cross-like surface depression dividing the square shaped projection of 10 x 10 nm into four equally sized subdomains, which correspond to the four dimers forming the octameric Mi-CK molecule. By a combination of positive staining (with uranyl acetate) and heavy metal shadowing, internal structures as well as the surface relief of Mi-CK were visualized at the same time at high resolution. Computational image analysis revealed only a single projection class of molecules, but the ability of Mi-CK to form linear filaments, as well as geometrical considerations concerning the formation of octamers by four equal, asymmetric dimers, suggest the existence of at least two distinct faces on the molecule. By image processing of Mi-CK filaments a side view of the octamer differing from the top-bottom projections of single molecules became evident showing a funnel-like access each form the top and bottom of the octamer connected by a central channel. The general structure of the Mi-CK octamer described here is relevant to the localization of the molecule at the inner-outer mitochondrial contact sites and to the function of Mi-CK as an "energy channeling" molecule.     

Cloning and over-expression in Escherichia coli of the gene encoding NADPH group III alcohol dehydrogenase from Thermococcus hydrothermalis. Characterization and comparison of the native and the recombinant enzymes.

A NADP-dependent group III alcohol dehydrogenase (ADH) was purified from the hyperthermophilic strictly anaerobic archaeon Thermococcus hydrothermalis, which grows at an optimum temperature of 85 degrees C and an optimum pH of 6. The gene encoding this enzyme was cloned, sequenced, and over-expressed in Escherichia coli. The recombinant enzyme was purified, characterized and compared with the native form of the enzyme. The enzyme structure is pH-dependent, being a 197-kDa tetramer (subunit of 45 kDa) at pH 10.5, the pH optimum for alcohol oxidation, and a 80.5-kDa dimer at pH 7.5, the pH optimum for aldehyde reduction. The kinetic parameters of the enzyme show that the affinity of the enzyme is greater for the aldehyde substrate and NADPH cofactor, suggesting that the dimeric form of the enzyme is probably the active form in vivo. The ADH of T. hydrothermalis oxidizes a series of primary aliphatic and aromatic alcohols preferentially from C2 to C8 but is also active towards methanol and glycerol and stereospecific for monoterpenes. T. hydrothermalis ADH is the first Thermococcale ADH to be cloned and overproduced in a mesophilic heterologous expression system, and the recombinant and the native forms have identical main characteristics.