An oxygen transporter hemocyanin can act on the late pathway of melanin synthesis

5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) are precursors of eumelanin. The effects of crustacean hemolymph proteins on these eumelanin-related metabolites were investigated. Zymogram analysis indicated that polymers of hemocyanin (Hc) subunits converted DHI into black pigment while no effects were observed using DHICA as a substrate. Spectrum changes for mixtures of purified Hc and DHI showed a profile similar to oxidized DHI by mushroom tyrosinase while Hc had only slight effects on DHICA. Typical inhibitors of tyrosinase and phenoloxidase severely hampered the production of oxidized DHI. Taken together with previous results, these data indicate that Hc plays a crucial role in the conversion of DHI in the hemolymph of crustaceans, which promotes late reactions in the melanin synthetic pathway as well as early reactions (oxidation of tyrosine and DOPA to dopaquinone).     

Subunit compositions of crustacean haemocyanins are species-specific: evidence from non-decapod species

Electrophoretic examination of dissociated haemocyanin subunits from a number of amphipod, decapod and isopod crustaceans supports the hypothesis that subunit composition is species-specific, despite marked within-species variation in many species. General patterns of heterogeneity on native PAGE gels were also evident between groupings within the Amphipoda. Gammarid amphipods could be split into two groups; one characterised by a high degree of heterogeneity and the other by a low degree of heterogeneity. The talitrid amphipods generally displayed a low degree of heterogeneity similar to, although still distinct from, the second gammarid category. Haemocyanin from the Hyalidae, a family allied to the talitrids was highly heterogeneous, similar to the first gammarid group and unlike the talitrids. Isopod haemocyanin banding patterns were more similar to one another than to any of the amphipod or decapod species examined. In general, the molecular weights of the amphipod Hcs tended to be greater than those of the isopods, with the decapods being lowest of all. It is suggested that Hc subunit heterogeneity may be a useful tool for investigating speciation and speciation events, and for reliably separating very closely-related species (e.g. Gammarus spp.), purely on the basis of their Hc subunit compositions.     

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.     

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies.     

The activation of human skin fibroblast procollagenase. Sequence identification of the major conversion products

Human skin collagenase is secreted by cultured fibroblasts in a proenzyme form and can be activated to a catalytically competent enzyme by a number of processes. All modes of activation studied lead to conversion of the proenzyme to a stable 42-kDa active enzyme, concomitant with removal of an 81-amino acid peptide from the amino-terminal end of the molecule. The sequence of events leading to the formation of this enzyme form has been determined by analyzing the primary structure of the conversion intermediates. Trypsin-induced activation of procollagenase occurs as a result of the initial cleavage of the peptide bond between Arg-55 and Asn-56, generating a major intermediate of 46 kDa. Treatment of the proenzyme with organomercurials, which have no intrinsic ability to cleave peptide bonds, initially results in activation of the enzyme without loss of molecular weight. This is followed by conversion to two lower molecular weight species of 44 and 42 kDa, the latter corresponding to the stable active enzyme form. The final cleavage producing this form of collagenase is not restricted to a single polypeptide bond but can occur on the amino-terminal side of any one of three contiguous hydrophobic residues, Phe-100, Val-101, Leu-102. The data suggest that both trypsin and organomercurials activate procollagenase by initiating an intramolecular autoproteolytic reaction resulting in the formation of a stable 42-kDa active enzyme species.     

Binding of YC-1/BAY 41-2272 to soluble guanylate cyclase: A new perspective to the mechanism of activation

Soluble guanylate cyclase (sGC), a heterodimeric heme protein, catalyses the conversion of GTP in to cyclic GMP, which acts as a second messenger in cellular signaling. Nitric oxide activates this enzyme several hundred folds over its basal level. Carbon monoxide, along with some activator molecules like YC-1 and BAY, also synergistically activate sGC. Mechanism of this synergistic activation is a matter of debate. Here we review the existing literature to identify the possible binding site for YC-1 and BAY on bovine lung sGC and its mechanism of activation. These two exogenous compounds bind sGC on α subunit inside a pocket and thus exert allosteric effect via subunit interface, which is relayed to the catalytic site. We used docking studies to further validate this hypothesis. We propose that the binding of YC-1/BAY inside the sensory domain of the α subunit modulates the interactions on the subunit interface resulting in rearrangements in the catalytic site into active conformation and this partly induces the cleavage of Fe-His bond.     

Studies on the irreversible step of pepsinogen activation

The bond cleavage step of pepsinogen activation has been investigated in a kinetic study in which the denatured products of short-term acidifications were separated on SDS-polyacrylamide gels and the peptide products were quantitated by densitometry. Although several peptide products were observed, under the conditions of the experiments (pH values between 2.0 and 2.8, 22 degrees C), the only one that was a product of an initial bond cleavage was the 44-residue peptide, which upon removal from pepsinogen yields pepsin. The rate constant for this bond cleavage is 0.015 s-1 at pH 2.4, which is the same as that at which the alkali-stable potential activity of pepsinogen had been found to convert to the alkali-labile activity of pepsin. When the conversion of zymogen to enzyme was followed by the change in fluorescence of adsorbed 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS), the rate of change in TNS fluorescence was the same as the conversion to alkali lability. However, pepstatin blocked the bond cleavage of pepsinogen to pepsin, but it permitted the fluorescence change to proceed. In fact, it accelerated the apparent rate of change of TNS fluorescence by shifting the pKa of an essential conjugate acid from 1.7 to 2.6. The conversion to alkali lability, therefore, may be considered to be a composite of a relatively slow conformational change (at the measured rate), followed immediately by a relatively fast bond cleavage.     

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.     

Identifying and modulating disulfide formation in the biopharmaceutical production of a recombinant protein vaccine candidate

Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.     

Identification of Two Forms of Qβ Replicase with Different Thermal Stabilities but Identical RNA Replication Activity

The enzyme Qβ replicase is an RNA-dependent RNA polymerase, which plays a central role in infection by the simple single-stranded RNA virus bacteriophage Qβ. This enzyme has been used in a number of applications because of its unique activity in amplifying RNA from an RNA template. Determination of the thermal stability of Qβ replicase is important to gain an understanding of its function and potential applications, but data reported to date have been contradictory. Here, we provide evidence that these previous inconsistencies were due to the heterogeneous forms of the replicase with different stabilities. We purified two forms of replicase expressed in Escherichia coli, which differed in their thermal stability but showed identical RNA replication activity. Furthermore, we found that the replicase undergoes conversion between these forms due to oxidation, and the Cys-533 residue in the catalytic β subunit and Cys-82 residue in the EF-Tu subunit of the replicase are essential prerequisites for this conversion to occur. These results strongly suggest that the thermal stable replicase contains the intersubunit disulfide bond between these cysteines. The established strategies for isolating and purifying a thermally stable replicase should increase the usefulness of Qβ replicase in various applications, and the data regarding thermal stability obtained in this study may yield insight into the precise mechanism of infection by bacteriophage Qβ.     

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.     

The activation of pro-urokinase by plasma kallikrein and its inactivation by thrombin

Plasma kallikrein was found to be a good activator of pro-urokinase, the inactive zymogen form of urokinase. The complete activation of pro-urokinase by plasma kallikrein was obtained in 2 h with an enzyme/substrate weight ratio of 1/30. The rate of activation of pro-urokinase by plasma kallikrein was comparable to that catalyzed by plasmin and trypsin. The rate of activation of pro-urokinase by factor XIIa was approximately one-seventh of that by plasma kallikrein. The activation of the zymogen was due to the cleavage of a single internal peptide bond, resulting in the conversion of a single chain pro-urokinase (Mr = 55,000) into two-chain urokinase (Mr = 33,000 and 22,000), and these two chains were linked by a disulfide bond(s). These results indicate an important role of plasma kallikrein for the activation of pro-urokinase in the factor XII-dependent intrinsic pathway of fibrinolysis. Thrombin also converted pro-urokinase to a two-chain form that was not activatable by plasmin, plasma kallikrein, and factor XIIa. Thrombin specifically cleaved the Arg 156-Phe 157 bond which is located 2 residues prior to the activation site of Lys 158-Ile 159.     

Glucagon stimulation of rat hepatic phenylalanine hydroxylase through phosphorylation in vivo

Phenylalanine hydroxylase activities in extracts of livers from rats pretreated with glucagon are higher than in controls. This time-dependent activation is seen when the hydroxylase is assayed in the presence of tetrahydrobiopterin, but not in the presence of 2-amino-4-hydroxy-6,7-dimethyltetrahydropterin. A maximum 4-fold stimulation of hydroxylase activity was correlated with a conversion of the multiple forms of the enzyme to a single form. This form is characterized by an increased extent of phosphorylation compared to the unactivated enzyme. Incorporation of radioactive inorganic phosphate into phenylalanine hydroxylase following administration of glucagon was determined after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis showed that stimulation of enzyme activity is accompanied by incorporation of 32Pi into the protein to the extent of 0.7 mol/mol of hydroxylase subunit. These results demonstrate the phosphorylation of hepatic phenylalanine hydroxylase in vivo and strongly support the idea that the activity of this enzyme can be hormonally regulated through a phosphorylation mechanism.     

Involvement of sulfhydryl groups in the activation mechanism of the ATPase activity of chloroplast coupling factor 1

Activation of the ATPase activity and the exposition of a new adenine nucleotide binding site of chloroplast coupling factor 1 (CF1) by dithioerythritol at 25 degrees C were reversed by oxidants. The ATPase activity elicited by heat (63 degrees C, 4 min) was slightly inhibited by oxidants and was partially additive with the activity induced by dithioerythritol. Titration of the thiols of CF1 and determination of their subunit distribution before and after activation by dithioerythritol show an increase of the free groups from 8 to 10 with the appearance of the 2 new thiols on the gamma subunit. These thiols were available to reagents in nondenatured enzyme and were reoxidized to a disulfide bond by iodosobenzoate or CuCl2. It is concluded that the mechanisms of CF1 activation by dithioerythritol and by heat are different and that the former involves a net reduction of a disulfide bond of the gamma subunit.     

Cysteine-rich module structure reveals a fulcrum for integrin rearrangement upon activation

Cysteine-rich repeats in the integrin beta subunit stalk region relay activation signals to the ligand-binding headpiece. The NMR solution structure and disulfide bond connectivity of Cys-rich module-3 of the integrin beta2 subunit reveal a nosecone-shaped variant of the EGF fold, termed an integrin-EGF (I-EGF) domain. Interdomain contacts between I-EGF domains 2 and 3 observed by NMR support a model in which the modules are related by an approximate two-fold screw axis in an extended arrangement. Our findings complement a 3.1 A crystal structure of the extracellular portion of integrin alphaVbeta3, which lacks an atomic model for I-EGF2 and a portion of I-EGF3. The disulfide connectivity of I-EGF3 chemically assigned here differs from the pairings suggested in the alphaVbeta3 structure. Epitopes that become exposed upon integrin activation and residues that restrain activation are defined in beta2 I-EGF domains 2 and 3. Superposition on the alphaVbeta3 structure reveals that they are buried. This observation suggests that the highly bent alphaVbeta3 structure represents the inactive conformation and that release of contacts with I-EGF modules 2 and 3 triggers a switchblade-like opening motion extending the integrin into its active conformation.     

An iron-dependent 5'-nucleotide phosphoribohydrolase activity in the venom of Crotalus atrox and its effect upon the determination of mammalian ribonucleotide reductase

There is an iron-dependent activity in the venom of Crotalus atrox which appears to hydrolyze the N-glycosidic sugar-base bond of pyrimidine and purine nucleotides. Maximal activation of this activity occurred at 1.0 mmol/l and 0.8 mmol/l FeCl3 for cytidine diphosphate (CDP) and ADP substrates, respectively. The release of free base affects the background values of control experiments carried out during nucleotide-labelled assays of ribonucleotide reductase which require the conversion of nucleotides to nucleosides by snake venom treatment. When the reductase is examined in intact cells, a situation closely resembling normal physiological conditions for the enzyme, FeCl3 was found to be an inhibitor of ADP reductions and varied from a mild stimulator to a significant inhibitor of CDP reductions depending upon FeCl3 concentration. Possible explanations for previously observed variability in ribonucleotide reductase activity in the presence of iron are suggested.     

Histoplasma capsulatum secreted gamma-glutamyltransferase reduces iron by generating an efficient ferric reductant

The intracellular fungal pathogen Histoplasma capsulatum (Hc) resides in mammalian macrophages and causes respiratory and systemic disease. Iron limitation is an important host antimicrobial defence, and iron acquisition is critical for microbial pathogenesis. Hc displays several iron acquisition mechanisms, including secreted glutathione-dependent ferric reductase activity (GSH-FeR). We purified this enzyme from culture supernatant and identified a novel extracellular iron reduction strategy involving gamma-glutamyltransferase (Ggt1) activity. The 320 kDa complex was composed of glycosylated protein subunits of about 50 and 37 kDa. The purified enzyme exhibited gamma-glutamyl transfer activity as well as iron reduction activity in the presence of glutathione. We cloned and manipulated expression of the encoding gene. Overexpression or RNAi silencing affected both GGT and GSH-FeR activities concurrently. Enzyme inhibition experiments showed that the activity is complex and involves two reactions. First, Ggt1 initiates enzymatic breakdown of GSH by cleavage of the gamma-glutamyl bond and release of cysteinylglycine. Second, the thiol group of the released dipeptide reduces ferric to ferrous iron. A combination of kinetic properties of both reactions resulted in efficient iron reduction over a broad pH range. Our findings provide novel insight into Hc iron acquisition strategies and reveal a unique aspect of Ggt1 function in this dimorphic mycopathogen.     

Structure and function of cholera toxin and hormone receptors

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active ¹²⁵I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside G_MI. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyme to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the “active” subunit of the toxin can specifically adsorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergentsolubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct pertubation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.