A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the “LID” domain. The sequence ¹²⁹Cys-X₅-His-X₁₅-Cys-X₂-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.     

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from <Emphasis Type="Italic">Desulfovibrio gigas</Emphasis>: a novel metal-containing adenylate kinase from Gram-negative bacteria

Adenylate kinases (AK) from Gram-negative bacteria are generally devoid of metal ions in their LID domain. However, three metal ions, zinc, cobalt, and iron, have been found in AK from Gram-negative bacteria. Crystal structures of substrate-free AK from Desulfovibrio gigas with three different metal ions (Zn²⁺, Zn-AK; Co²⁺, Co-AK; and Fe²⁺, Fe-AK) bound in its LID domain have been determined by X-ray crystallography to resolutions 1.8, 2.0, and 3.0 Å, respectively. The zinc and iron forms of the enzyme were crystallized in space group I222, whereas the cobalt-form crystals were C2. The presence of the metals was confirmed by calculation of anomalous difference maps and by X-ray fluorescence scans. The work presented here is the first report of a structure of a metal-containing AK from a Gram-negative bacterium. The native enzyme was crystallized, and only zinc was detected in the LID domain. Co-AK and Fe-AK were obtained by overexpressing the protein in Escherichia coli. Zn-AK and Fe-AK crystallized as monomers in the asymmetric unit, whereas Co-AK crystallized as a dimer. Nevertheless, all three crystal structures are very similar to each other, with the same LID domain topology, the only change being the presence of the different metal atoms. In the absence of any substrate, the LID domain of all holoforms of AK was present in a fully open conformational state. Normal mode analysis was performed to predict fluctuations of the LID domain along the catalytic pathway.     

A novel iron protein from Desulfovibrio gigas

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. Elemental analysis of a crude protein preparation gave 5.3% iron, 2.9% sulfur and 11.9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be dissociated from the protein by treatment with 5% SDS. The iron-free protein was purified by a combination of organic extraction and DEAE-cellulose chromatography. The purified protein showed only one major band, M_r 14 000, by SDS-polyacrylamide gel electrophoresis. The protein could be reconstituted upon treatment with an appropriate mixture of FeS and β-mercaptoethanol. The reconstituted protein had the same physical and chemical properties as the native protein. The amino acid composition was not unusual except for the high isoleucine content.     

Differential steric effects in the axial ligation of pyridine and imidazole to α- and β-alkylcobinamides

One subclass of B₁₂-requiring enzymes is now known to bind their B₁₂ coenzymes “base-off,” with a histidine residue from the protein supplying an imidazole ligand to the cobalt center. Recent results from Sirovatka and Finke (J.M. Sirovatka and R.G. Finke, J.Am. Chem. Soc. 119, (1997) 3057) show that imidazole has an extraordinary trans effect on the mode of carbon–cobalt bond cleavage in coenzyme B₁₂ analogs, compared to pyridine or the natural 5,6-dimethylbenzimidazole ligand, and it was suggested that a differential steric effect could, in part, account for the uniqueness of the imidazole ligand. Such a differential steric effect for imidazole and pyridine is now demonstrated by studies of the thermodynamics of ligation of these ligands to the α and β diastereomers of two alkylcobinamides (RCbi⁺s, derivatives of cobalamins which lack the normal axial nucleotide) based on the known differences in steric crowding of the α (“lower”) and β (“upper”) axial ligand positions of cobalt corrinoids. Imidazole binds more tightly than pyridine to both diastereomers of NCCH₂Cbi⁺ and CF₃Cbi⁺, in all cases due to a more favorable entropy change, which is the result of lowered steric interference with corrin side chain thermal motions.     

Isolation of cobalt hyper-resistant mutants of Saccharomyces cerevisiae by in vivo evolutionary engineering approach

Cobalt is an important element with magnetic properties used in various industrial applications, but is also needed for biological activity. Very little is known about the cellular response of living systems to cobalt stress. Towards investigating this mechanism, we isolated individual Saccharomyces cerevisiae cells resistant to high cobalt concentrations up to 8 mmol l⁻¹, by employing four different ‘in vivo’ evolutionary engineering strategies: selection under constant or gradually increasing stress levels, and selection under continuous or pulse exposure to cobalt stress. Selection under continuous exposure to gradually increasing cobalt stress levels yielded the most resistant cell population to cobalt. However, the resistance was highly heterogeneous within the mutant populations ranging from 3- to 3700-fold survival rate of isolated individuals to 8 mmol l⁻¹ CoCl₂ in the most resistant population. Moreover, cobalt-resistant individual colonies were associated with 2–4-times lower intracellular cobalt contents as compared to wild-type, and with cross-resistance to metals such as nickel, zinc, manganese, but not to copper and chromium ions. Contrary to mutants evolved under continuous exposure to cobalt, those isolated by pulse exposure strategy also exhibited resistance to heat shock and hydrogen peroxide stress. Taken together, this study reinforced the fact that evolutionary engineering is useful in selecting strains with very specific phenotypes, and further illustrated the importance of the strategy chosen to isolate the best evolved strain.     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Australopithecus, Meganthropus and Ramapithecus

The South African members of Australopithecus form a single group, trending from earlier, more gracile or smaller forms, to later, more robust or larger forms, in accordance with the “Law of Cope”. This is supported by the evidence of the lower first deciduous molar: it is only slightly molarized in the earlier, smaller forms and astonishingly heavily molarized in the later, robust forms, such as that of Kromdraai. Hence, Robinson's view that two different genera are represented by the gracile and robust forms is not supported here. A. robustus is seen as a late offshoot of the Australopithecus stem. The resemblance of the Taung dm₁ to that of Sinanthropus, except for the more differentiated talonid of the Taung specimen, suggests that the separation of the Australopithecinae and Homininae must have taken place at an earlier stage than that represented by the oldest South African Australopithecinae. The lower jaw of Meganthropus of Java combines certain characteristics of A. africanus with those of A. robustus: Meganthropus might provisionally be called “Australopithecoid”. The geographically intermediate India has yielded a hominid, Ramapithecus punjabicus, but the author does not consider “Kenyapithecus” to be a hominid. “K. wickeri” is a pongid species of its own and “K. africanus” a Proconsul. Ramapithecus sensu stricto is known only from the Indian Siwaliks and the author suggests that the transition from Ramapithecus to a still unknown Australopithecus took place in the same region prior to their migration into Africa and Southeast Asia.     

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.     

On the reversibility of the B1 2r−B1 2s couple in cobalamins

The cobalt(II)—cobalt(I) interconversion in a number of vitamin B_{1 2} derivatives was investigated by cyclic voltammetry. Particular attention was focused on the factors determining whether the interconversion is reversible. When the lower axial coordination position is occupied by a strong ligand, such as the benzimidazole nucleotide in “base on” cobalamins, the cobalt(II)—cobalt(I) interconversion is irreversible due to a slow reduction of the cobalt(II). However, when the lower axial coordination position is free of a strong ligand, as in most cobinamides or in “base off” cobalamins, the cobalt(II)—cobalt(I) interconversion is nearly perfectly reversible. Possible implications of the observations to B_{1 2}-dependent enzymes are briefly discussed.     

Dinucleoside 5′,5-P,P-Triphosphate Hydrolase from Yellow Lupin (Lupinus luteus) Seeds: Purification to Homogeneity and Hydrolysis of mRNA 5′-Cap Analogs

Three separable forms of diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15MKCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS–polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain ofM_r41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5′-cap structure, have been tested as potential substrates of the lupin “Ap₃A hydrolase.” All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m⁷Gp₃G, et⁷Gp₃G, and bz⁷Gp₃G were hydrolyzed randomly, whereas m⁷Gp₃A, m⁷Gp₃C, and m⁷Gp₃U yielded at least 80% m⁷GMP plus corresponding NDP and 20% or less NMP plus m⁷GDP. Hydrolysis of the guanosine-containing hybrids, Ap₃G, Cp₃G, and Up₃G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m⁷G- moiety were hydrolyzed 2- to 4.5-fold faster than Ap₃A. Thus a general name, “dinucleoside triphosphate hydrolase,” is more appropriate for the enzymatic activity described.     

Differential genotoxic effects of antitumor trans-[PtCl2(E-iminoether)2] and cisplatin in Escherichia coli

In the present work, we measured survival and the platinum on the genome after treatment of repair-proficient or repair-deficient Escherichia coli strains with trans-[PtCl₂(E-iminoether)₂] and compared these results with the effects of “classical” cisplatin. We found that toxicity of antitumor trans-[PtCl₂(E-iminoether)₂] in repair-deficient trains was much less than that of cisplatin. This markedly reduced toxicity was not a consequence of the reduced uptake or low levels of DNA binding in the bacteria cells but rather appeared to reflect DNA binding mode of this trans-platinum drug different from that of cisplatin.     

Expression of a Zinc-Binding Domain of Boar Spermatidal Transition Protein 2 in Escherichia coli

Transition protein 2 (TP2; 137 amino acid residues) from boar late spermatid nuclei has three potential zinc finger motifs in the N-terminal region. Gel shift assays revealed that boar TP2 recognized a CpG island sequence in a zinc-dependent manner. However, there was some nonspecific recognition of the oligonucleotide. Then, we constructed the expression system of zinc-binding domain of TP2 (TP2Z) (residues 1–103) in Escherichia coli. Double-stranded DNA fragments encoding TP2Z were synthesized as 18 fragments with 103 residues, annealed, and cloned into the expression plasmid pET11d. TP2Z was expressed upon induction with 1 mM isopropylthiogalactoside and extracted with acid including 0.71 M 2-mercaptoethanol. TP2Z was purified by ion-exchange chromatography on Fractogel EMD SO⁻₃ and HPLC on Nucleosil 300 7C18 and on Diol-120. Atomic absorption and CD spectroscopy showed that TP2Z bound three atoms of zinc per molecule of the protein and underwent a zinc-dependent conformational change in a manner similar to that for intact TP2. Gel shift assays indicated that TP2Z recognized a CpG island sequence more specifically than intact TP2 and that the specificity is dependent on zinc.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using C labels detected by nuclear magnetic resonance and C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [¹⁴CH₃]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Zinc-, cobalt- and iron-chelated forms of adenylate kinase from the Gram-negative bacterium Desulfovibrio gigas

Adenylate kinase (AK) from the sulphate-reducing bacterium Desulfovibrio gigas (AK) has been characterized earlier as a Co²⁺/Zn²⁺-containing enzyme, which is an unusual characteristic for adenylate kinases from Gram-negative bacteria, in which these enzymes are normally devoid of metal ions. AK was overexpressed in E. coli and homogeneous Co²⁺-, Zn²⁺- and Fe²⁺-forms of the enzyme were obtained under in vivo conditions. Their structural stability and spectroscopic and kinetic properties were compared. The thermal denaturation of Co²⁺- and Zn²⁺-forms of AK was studied as a cooperative two-state process, sufficiently reversible at pH 10, which can be correctly interpreted in terms of a simple two-state thermodynamic model. In contrast, the thermally induced denaturation of Fe²⁺-AK is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Practically identical contents of secondary-structure elements were found for all the metal-chelated-forms of AK upon analysis of circular dichroism data, while their tertiary structures were significantly different. The peculiar tertiary structure of Fe²⁺-AK, in contrast to Co²⁺- and Zn²⁺-AK, and the consequent changes in the physico-chemical and enzymatic properties of the enzyme are discussed.     

The structure of Escherichia coli membranes studied by fluorescence measurements of lipid phase transitions

Lipid phase transitions in Escherichia coli membranes and in dispersions of the extracted lipids were studied using the negatively charged fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS⁻) and the hydrophobic fluorescence probe N-phenyl-1-naphthylamine (NPN). The fluorescence change, ΔI, at the phase transition approaches a limiting value (ΔI)_lim with increasing dye concentration. A comparison of the limiting values (Δ)_lim^NPN obtained for membranes and the lipid standard allows us to estimate the lipid fraction, ρ, in the membrane that takes part in the phase transition (ρ = 80%). The same procedure carried out with ANS⁻ yields a value of 42.5% for the lipid fraction that is accessible from the aqueous phase. These values, combined with published freeze-etching data for the particle density within the fracture plane of membranes are used to quantify the Davson-Danielli-Robertson-Benson-Singer membrane model which assumes a fluid lipid bilayer with “integral” proteins embedded in the lipid matrix and surface proteins attached to the lipid head groups. It appears that on the average one “integral” membrane protein is surrounded by about 600 lipid molecules and that about 130 of these molecules are closely coupled to the protein molecule, forming an halo in which the chain-chain interaction between the lipids is disturbed. About half of the bilayer surface is covered with proteins; part of these seem to be stacked.     

Second-messenger-induced signalling events in pollen tubes of Papaver rhoeas

A role for cytosolic free Ca²⁺ (Ca²⁺_i) in the regulation of growth of Papaver rhoeas pollen tubes during the self-incompatibility response has recently been demonstrated [Franklin-Tong et al. Plant J. 4:163–177 (1993); Franklin-Tong et al. Plant J. 8:299–307 (1995); Franklin-Tong et al. submitted to Plant J.]. We have investigated the possibility that Ca²⁺_i is more generally involved in the regulation of pollen tube growth using confocal laser scanning microscopy (CLSM). Data obtained using Ca²⁺ imaging, in conjunction with photolytic release of caged inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], point to a central role of the phosphoinositide signal transduction pathway in the control of Ca²⁺ fluxes and control of pollen tube growth. These experiments further revealed that increases in cytosolic levels of Ins(1,4,5)P₃ resulted in the formation of distinct Ca²⁺ waves. Experiments using the pharmacological agents heparin, neomycin and mastoparan further indicated that Ca²⁺ waves are propagated, at least in part, by Ins(1,4,5)P₃-induced Ca²⁺ release rather than by simple diffusion or by “classic” Ca²⁺-induced Ca²⁺ release mechanisms. We also have data which suggest that Ca²⁺ waves and oscillations may be induced by photolytic release of caged Ca²⁺. Ratio-imaging has enabled us to identify an apical oscillating Ca²⁺ gradient in growing pollen tubes, which may regulate normal pollen tube growth. We also present evidence for the involvement of Ca²⁺ waves in mediating the self-incompatibility response. Our data suggest that changes in Ca²⁺_i and alterations in growth rate/patterns are likely to be closely correlated and may be causally linked to events such as Ca²⁺-induced, or Ins(1,4,5)P₃-induced wave formation and apical Ca²⁺ oscillations.Presented at the 1997 SEB Annual Meeting: Interactive MultiMedia Biology - Experimental Biology Online Symposium, Canterbury, 7-11 April     

Interaction between uranium and the cytochrome c 3 of Desulfovibrio desulfuricans strain G20

Cytochrome c₃ of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c₃ in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c₃ (cycA) to lacZ. Instead, cytochrome c₃ protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c₃ with U(IV) was interpreted to be non-specific, since pure cytochrome c₃ adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe₂O₃), and commercially available U(IV) oxide.An erratum to this article can be found at     

Interrelationships between Growth Yield,ATPase and Adenylate Kinase Activities in Zymomonas mobilis

The presence of cytoplasmic and membrane‐bound adenylate kinase (EC 2.7.4.3) as well as inorganic pyrophosphatase (EC 3.6.1.1) was detected in Zymomonas mobilis ATCC 29191. An increase in the molar growth yield (Y_X/S) of Z. mobilis under aerobic growth conditions appeared to be in proportion to a reduction of membrane‐bound adenylate kinase (mAK) and ATPase activities and to an increase in cytoplasmic adenylate kinase (AK) activity. Significant (1 — P < 0.01) multiple regressions were observed between the values of Y_X/S (dependent variable), ATPase and AK or AK and mAK as independent variables, suggesting that a combined operation of these phosphohydrolases and phosphotransferases would be responsible for the biomass yield in Z. mobilis.     

Carbohydrate content and monosaccharide composition of Rapana thomasiana grosse (Gastropoda) hemocyanin and its structural subunits. Comparison with gastropodan hemocyanins

The hemocyanin of Rapana thomasiana grosse (marine snail, gastropod) is a glycoprotein with a carbohydrate content of 8.9% (w/w) and monosaccharide constituents xylose, fucose, 3-OOmethylgalactose, mannose, galactose, N-acetylgalactosamine and N-acetylglucosamine residues. The two structural subunits of this oxygen carrier, RHSS1 and RHSS2, are unevenly glycosylated. On subtracting the carbohydrate contribution from the M_r values of 250 and 450 kDa attributed to the two subunits, values of 2.18 × 10⁵ daltons and 4.30 × 10⁵ daltons were calculated for the polypeptide part of the “light” and “heavy” subunits, respectively. Comparison of the monosaccharide compositions of gastropodan hemocyanins revealed qualitative similarities, as well as relationships between the quantities, of the individual monosaccharides: Man 3MeGal > GlcNAc GalNAc and Fuc Xyl