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PubMed1978

Theory of proton hyperfine interactions in bacterial photosynthetic systems. Bacteriopheophytin cation and anion.

The electronic structures of the cation and anion of bacteriopheophytin alpha monomer are investigated by the self-consistent charge extended Hückel procedure including both pi and sigma electrons in the molecule. The calculated electron distributions are tested by comparison of the predicted hyperfine fields at proton sites with experimental data in both the ions and most important, by their ability to explain the observed trend in the hyperfine fields in going from the cation bacteriopheophytin+ alpha to the anion, a trend that is similar in many respects to the corresponding observed trend for the bacteriochlorophyll alpha cation and anion. Good agreement is obtained with experiment both for the absolute values of the observed proton hyperfine fields in both bacteriopheophytin a cation and anion as well as the ratio of the corresponding fields for the two systems. In particular, our calculated electron distributions in the two molecules lead, for the cation, to substantially different proton hyperfine fields for the two methyl groups attached to rings I and III, while for the anion, the corresponding fields are much closer to each other, a trend in good agreement with recent data. Also explained are the features of larger methine hyperfine constants in the anion as compared to the cation and the reverse trend for the protons in rings II and IV. Other features of the calculated electron distributions in the cation and anion are discussed and compared with each other. Possible additional measurements in the two systems that could provide further tests of the theoretically obtained electron distribution will be pointed out.

Bacteriochlorophylls↗
PubMed1994

Cationization of a monoclonal antibody to the human immunodeficiency virus REV protein enhances cellular uptake but does not impair antigen binding of the antibody.

Replication of the human immunodeficiency virus (HIV) within cells may be blocked by neutralization of viral-specific proteins that are absolutely required for growth of the virus. One such viral-specific protein is REV, and a monoclonal antibody (mAb) against the REV protein is a potential therapeutic for acquired immune deficiency syndrome (AIDS). However, in order to effect 'intracellular immunization', mAbs must be enabled to target the intracellular compartment. One strategy for transcellular drug delivery of mAb-based therapeutics is cationization, and the present studies describe the cationization of a murine mAb specific to the REV protein of HIV-1. The isoelectric point (pI) of the mAb was raised from 6.6 to more than 9.5. There was virtually no difference in binding to wild-type REV protein between the native or cationized anti-REV mAb, based on studies with a solid-phase immunoradiometric assay. The uptake of the [125I] native anti-REV mAb by human peripheral blood lymphocytes (PBLs) was negligible; however, there was a marked increase in both total cell binding and endocytosis by the human PBLs of the [125I] cationized anti-REV mAb. In conclusion, these studies show that an anti-REV mAb may be cationized to markedly increase endocytosis of the antibody and that this cationization reaction does not significantly alter the affinity of the antibody for its target protein. Cationized anti-REV mAbs may allow for intracellular immunization of the virus and are potential therapeutics for the treatment of HIV.

Antibodies, Monoclonal↗
PubMed1994

CCK-8-evoked cationic currents in substantia nigra dopaminergic neurons are mediated by InsP3-induced Ca2+ release.

Our recent study demonstrated that by activating CCK-A receptors, CCK-8 excites substantia nigra (SN) dopaminergic (DA) neurons via increasing a non-selective cationic conductance. In the present study, we further studied the molecular mechanism by which CCK-8 induces cationic currents in SN DA neurons. CCK-8-evoked inward currents were inhibited by the intracellular perfusion of GDP-beta-S (1 mM). In DA neurons internally perfused with GTP-gamma-S (0.5 mM), the inward currents produced by CCK-8 became irreversible. Pretreating DA neurons with 500 ng/ml pertussis toxin (PTX) did not significantly affect the ability of CCK-8 to induce cationic currents. Intracellular application of heparin (2 mg/ml), an inositol 1,4,5-trisphosphate (InsP3) receptor antagonist, and buffering intracellular calcium with the Ca(2+)-chelator BAPTA (10 mM) suppressed CCK-8-evoked cationic currents. Dialyzing DA neurons with protein kinase C (PKC) inhibitors, staurosporine and PKC(19-31), failed to prevent CCK-8 from generating cationic currents. It is concluded that PTX-insensitive G-proteins mediate CCK-8-induced enhancement of cationic conductance of SN DA neurons. The coupling mechanism via G-proteins is likely to involve the generation of InsP3, and subsequent InsP3-evoked Ca2+ release from the intracellular store results in activating the non-selective cationic conductance.

Alkaloids↗
PubMed2004

Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer.

This investigation is focused on the enhancement of in vitro transfection activity by optimizing cationic lipid and matrix lipid composition of solid lipid nanoparticles (SLN). For this purpose SLN were formulated by using two different matrix lipids and six different cationic detergents. These 12 formulations were tested for physical parameters such as particle size, zeta potential and DNA-binding capacity, and also for their biological properties such as cytotoxicity and in vitro transfection efficiency. The SLN were produced by hot high-pressure homogenization, all formulations were physically stable and showed a highly positive surface charge (+34 to +45 mV). In vitro cytotoxicity measurements on COS-1 cells revealed that cytotoxicity is strongly dependent on the cationic lipid used. SLN made from one-tailed cationic detergents were highly cytotoxic. In contrast the two-tailed cationic lipids were all well tolerated. Transfection activity seems to be determined by both the cationic lipid and the matrix lipid used. Here, the combination of cetylpalmitate and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride led to significantly higher transfection efficiencies than in all other tested combinations. These results indicate that well tolerated and highly efficient in vitro transfection could be achieved with SLN whenever selecting good combinations of two-tailed cationic lipids and matrix lipids.

Animals↗
PubMed2005

Natural vermiculite as an exchanger support for heavy cations in aqueous solution.

The natural highly charged lamellar silicate vermiculite was investigated as an exchanger matrix in doubly distilled water solution to exchange magnesium inside the lamella with the heavy cations copper, nickel, cobalt, and lead at the solid/liquid interface. The extension of each exchange reaction was dependent on time of reaction, pH, and cation concentration. The maximum time presented the following order Pb2+ < Ni2+ < Cu2+ < Co2+, which corresponds to 12, 24, 48, and 72 h, respectively. The best performance was observed for nickel, as represented by the exchange capacity Nf, which gave values 0.59, 0.76, 0.84, and 0.93 mmol g(-1) for Pb2+ < Co2+ < Cu2+ < Ni2+, respectively. This capacity is dependent on pH interval variation from 1 to 9, being significantly increased in alkaline condition. The isotherm data were adjusted to a modified Langmuir equation and from the data the spontaneous Gibbs free energy was calculated. Linear correlations were obtained through Gibbs free energy or the maximum capacity against the cationic radius plot, with the lowest values for the largest cation lead. An exponential correlation was also observed for the maximum capacity versus enthalpy of hydration plot, indicating a difficulty of the less hydrated cation, lead, in exchanging with magnesium inside the lamellar space, as suggested by the proposed mechanism. The saturated matrices with cations presented a decrease in interlayer distance in comparison with the original vermiculite, which can be related to the hydrated phases, characteristic for each cation, with a lowest value for lead.

Journal Article↗
PubMed2000

DOTAP cationic liposomes prefer relaxed over supercoiled plasmids.

Cationic liposomes and DNA interact electrostatically to form complexes called lipoplexes. The amounts of unbound (free) DNA in a mixture of cationic liposomes and DNA at different cationic lipid:DNA molar ratios can be used to describe DNA binding isotherms; these provide a measure of the binding efficiency of DNA to different cationic lipid formulations at various medium conditions. In order to quantify the ratio between the various forms of naked DNA and supercoiled, relaxed and single-stranded DNA, and the ratio between cationic lipid bound and unbound DNA of various forms we developed a simple, sensitive quantitative assay using agarose gel electrophoresis, followed by staining with the fluorescent cyanine DNA dyes SYBR Green I or SYBR Gold. This assay was compared with that based on the use of ethidium bromide (the most commonly used nucleic acid stain). Unlike ethidium bromide, SYBR Green I DNA sensitivity and concentration-dependent fluorescence intensity were identical for supercoiled and nicked-relaxed forms. DNA detection by SYBR Green I in solution is approximately 40-fold more sensitive than by ethidium bromide for double-stranded DNA and approximately 10-fold for single-stranded DNA, and in agarose gel it is 16-fold more sensitive for double-stranded DNA compared with ethidium bromide. SYBR Gold performs similarly to SYBR Green I. This study shows that: (a) there is no significant difference in DNA binding isotherms to the monocationic DOTAP (DOTAP/DOPE) liposomes and to the polycationic DOSPA (DOSPA/DOPE) liposomes, even when four DOSPA positive charges are involved in the electrostatic interaction with DNA; (b) the helper lipids affect DNA binding, as DOTAP/DOPE liposomes bind more DNA than DOTAP/cholesterol; (c) in the process of lipoplex formation, when the DNA is a mixture of two forms, supercoiled and nicked-relaxed (open circular), there is a preference for the binding to the cationic liposomes of plasmid DNA in the nicked-relaxed over the supercoiled form. This preference is much more pronounced when the cationic liposome formulation is based on the monocationic lipid DOTAP than on the polycationic lipid DOSPA. The preference of DOTAP formulations to bind to the relaxed DNA plasmid suggests that the binding of supercoiled DNA is weaker and easier to dissociate from the complex.

Benzothiazoles↗
PubMed1996

The role of helper lipids in cationic liposome-mediated gene transfer.

In the procedure for cationic liposome-mediated transfection, the cationic lipid is usually mixed with a "helper lipid" to increase its transfection potency. The importance of helper lipids, including dioleoylphosphatidylcholine (DOPC) and phosphatidylethanolamine (dioleoyl PE), DO was examined. Freeze-fracture electron microscopy of DNA:cationic complexes containing the pSV-beta-GAL plasmid DNA, the cationic lipid dioleoyl trimethylammonium propane, and these helper lipids showed that the most efficient mixtures were aggregates of ensheathed DNA and fused liposomes. PE-containing complexes aggregated rapidly when added to culture media containing polyanions, whereas PC-containing complexes did not. However, more granules of PC-containing complexes were formed on cell surfaces after the complexes were added to Chinese hamster ovary (CHO) cells in transfection media. Pronase treatment inhibited transfection, whereas dilute poly-L-lysine enhanced transfection, indicating that the attachment of DNA:liposome complexes to cell surfaces was mediated by electrostatic interaction. Fluorescence spectroscopy studies confirmed that more PC-containing complexes than PE-containing complexes were associated with CHO cells, and that more PC-containing complexes were located in a low pH environment (likely to be within endosomes) with time. Cytochalasin-B had a stronger inhibitory effect on PC-containing liposome-mediated than on PE-containing liposome-mediated transfection. Confocal microscopic recording of the fluorescently label lipid and DNA uptake process indicated that many granules of DNA:cationic liposome complexes were internalized as a whole, whereas some DNA aggregates were left out on the cell surfaces after liposomes of the complexes fused with the plasma membranes. For CHO cells, endocytosis seems to be the main uptake pathway of DNA:cationic liposome complexes. More PC-containing granules than PE-containing granules were formed on cell surfaces by cytoskeleton-directed membrane motion, after their respective DNA:liposome complexes attached to cell surfaces by electrostatic means. Formation of granules on the cell surface facilitated and/or triggered endocytosis. Fusion between cationic liposomes and the cell membrane played a secondary role in determining transfection efficiency.

Animals↗
PubMed1997

Extracellular Mg2+ inhibits receptor-mediated Ca(2+)-permeable non-selective cation currents in aortic smooth muscle cells.

The effects of extracellular Mg2+ on receptor-mediated Ca(2+)-permeable non-selective cation currents were investigated in a cultured aortic smooth muscle cell line (A7r5) from rat thoracic aorta, using the whole-cell voltage-clamp technique. Under the Cs(+)-containing internal solution, both vasopressin and endothelin-1 (100 nM) activated a long-lasting inward current with a high noise level. The reversal potential of these agonists-induced current was approximately +0 mV, and was not significantly altered by the replacement of [Cl-]i or [Cl-]o, suggesting that the inward current was a cation-selective channel. La3+ and Cd2+ (1 mM) almost completely abolished the vasopressin or endothelin-induced non-selective cation current; however, nifedipine (10 microM) failed to inhibit it significantly. Extracellular Mg2+ (3-20 mM) also markedly inhibited the vasopressin- or endothelin-induced non-selective cation current in a concentration-dependent manner. When a non-hydrolysable GTP-analogue, GTP gamma S (1 mM), was applied from the patch pipette, the non-selective cation current was gradually activated even in the absence of agonist (vasopressin or endothelin-1), probably due to the direct activation of GTP-binding proteins coupled to the receptors. Extracellular Mg2+ (3-20 mM) also suppressed the activation of non-selective cation current induced by GTP gamma S, suggesting that the inhibitory sites of Mg2+ are not located on the receptors. These results suggest that extracellular Mg2+ inhibits receptor-mediated non-selective cation current, which may contribute to the relaxation effects of Mg2+ in vascular smooth muscle cells.

Animals↗
PubMed2003

Adsorption of cations onto the surfaces of silver nanoparticles.

The effects of cations on the absorption spectra of silver sols have been investigated by the UV-vis spectrometry and TEM. Experiments showed that injection of certain amounts of transition metal cations into silver sols resulted not only in the aggregation of silver nanoparticles but also in the appearance of a new band centered near 510 nm in the absorption spectra of silver sols. However, the new band was not observed in the presence of alkaline earth metal cations or the Mv2+ cations. The peak position of the new band depends on the nature as well as the concentration of metal cations used. Comparing the peak positions of the new bands, it was found that the new band induced by the injection of Cr3+ was red-shifted with respect to those induced by Cu2+, Zn2+, or the Cd2+ cations. It is reasonable that this band near 510 nm should be attributed to the coeffects of the adsorption of metal cations onto the surfaces of silver nanoparticles and the aggregation of silver nanoparticles.

Journal Article↗
PubMed1996

Injection of rat hepatocyte poly(A)+ RNA to Xenopus laevis oocytes leads to expression of a constitutively-active divalent cation channel distinguishable from endogenous receptor-activated channels.

The expression of hepatocyte plasma membrane receptor-activated divalent cation channels in immature (stages V and VI) Xenopus laevis oocytes and the properties which allow these channels to be distinguished from endogenous receptor-activated divalent cation channels were investigated. Divalent cation inflow to oocytes housed in a multiwell plate was measured using the fluorescent dyes Fluo-3 and Fura-2. In control oocytes, ionomycin, cholera toxin, thapsigargin, 3-fluoro-inositol 1,4,5-trisphosphate (InsP3F) and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulated Ca2+ and Mn2+ inflow following addition of these ions to the oocytes. Ionomycin-, cholera-toxin-, thapsigargin- and InsP3F-stimulated Ca2+ inflow was inhibited by Gd3+ (half maximal inhibition at less thari 5 microM Gd3+ for InsP3F-stimulated Ca2+ inflow). GTP gamma S-stimulated Ca2+ inflow was insensitive to 50 microM Gd3+ and to SK&F 96365. These results indicate that at least three types of endogenous receptor-activated Ca2+ channels can be detected in Xenopus oocytes using Ca(2+)-sensitive fluorescent dyes: lanthanide-sensitive divalent cation channels activated by intracellular Ca2+ store depletion, lanthanide-sensitive divalent cation channels activated by cholera toxin, and lanthanide-insensitive divalent cation channels activated by an unknown trimeric G-protein. Oocytes microinjected with rat hepatocyte poly(A)+ RNA exhibited greater rates of Ca2+ and Mn2+ inflow in the basal (no agonist) state, greater rates of Ca2+ inflow in the presence of vasopressin or InsP3F and greater rates of Ba2+ inflow in the presence of InsP3F, when compared with "mock"-injected oocytes. In poly(A)+ RNA-injected oocytes, vasopressin- and InsP3F-stimulated Ca2+ inflow, but not basal Ca2+ inflow, was inhibited by Gd3+. It is concluded that at least one type of hepatocyte plasma membrane divalent cation channel, which admits Mn2+ as well as Ca2+ and is lanthanide-insensitive, can be expressed and detected in Xenopus oocytes.

Animals↗
PubMed1994

Increased serum levels of eosinophil cationic protein and eosinophil-derived neurotoxin (protein X) in vernal keratoconjunctivitis.

PURPOSE: To evaluate whether the eosinophil preformed mediators, eosinophil cationic protein X, and eosinophil-derived neurotoxin/eosinophil protein X (EDN/EPX) are detectable in serum and can be used as markers of eosinophilic inflammation. METHODS: A competitive radioimmunoassay was used to detect serum levels of eosinophil cationic protein and EDN/EPX in 31 patients with vernal keratoconjunctivitis (VKC) and in 15 healthy controls. RESULTS: Mean serum levels of both eosinophil cationic protein and EDN/EPX were significantly higher in patients with VKC than in controls. There was a good correlation between serum levels of eosinophil cationic protein and EDN/EPX. No significant difference in mean serum eosinophil cationic protein levels was observed between patients with positive radioallergosorbent test (RAST) results and those with negative RAST results who have VKC, whereas mean EDN/EPX and total immunoglobulin E serum levels were significantly higher in patients with positive RAST results than in those with negative RAST results who have VKC. Serum eosinophil cationic protein, but not EDN/EPX, levels were significantly related to total immunoglobulin E levels. CONCLUSION: The authors' data indicate that serum levels of eosinophil cationic protein and EDN/EPX are useful markers of eosinophilic ocular inflammation in VKC.

Adolescent↗
PubMed1998

Characterization of cationic amino acid transporter and its gene expression in rat hepatic stellate cells in relation to nitric oxide production.

BACKGROUND/AIMS: Nitric oxide is a potent mediator of hepatic sinusoidal hemodynamics and affects hepatic stellate cells (Ito cells, fat-storing cells). Although nitric oxide production may depend on the induction of inducible nitric oxide synthase and on transport of extracellular L-arginine, the precise mechanisms controlling nitric oxide production in stellate cells have not been well characterized. METHODS: Using stellate cells prepared from the male Wistar rat, kinetic analysis of L-arginine transport and reverse transcription-polymerase chain reaction for cationic amino acid transporter were carried out. The effect of tumor necrosis factor-alpha and interferon-gamma on L-arginine transport, mRNA expression of cationic amino acid transporter and inducible nitric oxide synthase, and nitric oxide production of stellate cells was assessed. RESULTS: The L-arginine transport system functioning in the transformed hepatic stellate cells was system y+, possibly mediated by cationic amino acid transporter-1 and cationic amino acid transporter-2B (Km approximately 50 microM). Tumor necrosis factor-alpha enhanced cationic amino acid transporter-2B mRNA expression and L-arginine transport, whereas cationic amino acid transporter-1 mRNA expression remained unchanged. Interferon-gamma induced the expression of inducible nitric oxide synthase mRNA without obvious changes in L-arginine transport. Interferon-gamma in combination with tumor necrosis factor-alpha induced nitric oxide production with an enhancement in cationic amino acid transporter-2B mRNA expression, inducible nitric oxide synthase mRNA expression, and L-arginine transport, while extracellular L-lysine competitively inhibited this nitric oxide production. CONCLUSIONS: In transformed hepatic stellate cells, tumor necrosis factor-alpha and interferon-gamma have a crucial role in nitric oxide production, and extracellular L-arginine transport and inducible nitric oxide synthase expression are regulated in a differential cytokine-specific manner. As the estimated Km of L-arginine transporter in transformed hepatic stellate cells is very similar to the physiological L-arginine concentration in portal vein, we assume that increased portal L-arginine concentration may easily affect sinusoidal blood flow through enhancement of autocrine nitric oxide production in transformed hepatic stellate cells of diseased liver.

Amino Acid Transport Systems, Basic↗
PubMedDate not supplied

Transport of a reactive tracer in saturated alluvium described using a three-component cation-exchange model.

A weakly sorbing cation, lithium, will be used as a reactive tracer in upcoming field tracer tests in the saturated alluvium south of Yucca Mountain, Nevada. One objective of the field tests is to determine how well field-scale reactive transport can be predicted using transport parameters derived from laboratory experiments. This paper describes several laboratory lithium batch sorption and column transport experiments that were conducted using ground water and alluvium obtained from the site of the planned field tests. In the batch experiments, isotherms were determined over 2.5 orders of magnitude of lithium concentrations, corresponding to the range expected in the field tests. In addition to measuring equilibrium lithium concentrations, concentrations of other cations, namely Na(+), K(+), and Ca(2+), were measured in the batch tests to determine Li(+)-exchangeable equilibria. This information was used in conjunction with alluvium cation exchange capacity measurements to parameterize a three-component cation-exchange model (EQUIL) that describes lithium sorption in the alluvium system. This model was then applied to interpret the transport behavior of lithium ion in saturated alluvium column tests conducted at three different lithium bromide injection concentrations. The concentrations were selected such that lithium ion either dominated, accounted for a little over half, or accounted for only a small fraction of the total cation equivalents in the injection solution. Although tracer breakthrough curves differed significantly under each of these conditions, with highly asymmetric responses occurring at the highest injection concentrations, the three-component cation-exchange model reproduced the observed transport behavior of lithium and the other cations in each case with a similar set of model parameters. In contrast, a linear K(d)-type sorption model could only match the lithium responses at the lowest injection concentration. The three-component model will be used to interpret the field tests, with the expectation that it will help refine estimates of effective flow porosity, particularly if the lithium response curves are asymmetric.

Adsorption↗
PubMed1996

Levels of eosinophil cationic protein and myeloperoxidase from chronic middle ear effusion in patients with allergy and/or acute infection.

BACKGROUND AND OBJECTIVE: Allergy may play a role in the middle ear inflammation that leads to otitis media with effusion. The purpose of this study was to determine whether an elevated mediator correlated with the patient's disease and thus could be used to differentiate allergy vs. infection as the cause of the middle ear inflammation. METHODS: WE evaluated 57 individuals with otitis media with effusion, 32 with persistent effusion but no recent acute infection, 14 with recent infection and purulent otitis media with effusion, and II healthy subjects. The mediator activity of eosinophils and neutrophils in effusion was studied in patients characterized as having allergy by positive intradermal skin test results and positive radioallergosorbent test results. Eosinophils were characterized by measurement of eosinophil cationic protein in the effusion. Neutrophils were characterized by measurement of myeloperoxidase in the effusion. The levels of eosinophil cationic protein and myeloperoxidase in patients with and without allergy were correlated to patient history. RESULTS: Significantly elevated levels of both eosinophil cationic protein and myeloperoxidase indicated that inflammation in the ear of patients with otitis media with effusion was characterized by a pronounced involvement of both eosinophils and neutrophils. Eighty-nine percent of all patients with disease had allergy. A higher ratio of myeloperoxidase to eosinophil cationic protein in patients with purulent otitis media with effusion indicated that in patients with a superimposed acute infection, neutrophil activity was increased even further. The level of eosinophil cationic protein was elevated only during the effusion of patients with allergies as compared with controls (p < 0.01). Among 29 cases of nonpurulent otitis media with effusion, 96.5% had allergic immune-mediated disease proved by skin testing, which was related clinically to their ear disease. Eighty-nine percent (89.6%) of these patients had eosinophil cationic protein levels greater than 10 microgram/L. CONCLUSION: Middle ear eosinophil cationic protein may be used as a marker of related allergy.

Adolescent↗
PubMed2000

NMR, UV-vis and CD study on the interaction of pradimicin BMY-28864 with divalent cations of alkaline earth metal.

In order to clarify the mechanism of the calcium-activated anti-fungal action of pradimicin derivatives, we investigated the compatibility of four divalent metal cations, Ca2+, Mg2+, Sr2+ and Ba2+, when combined with pradimicin BMY-28864 in an aqueous solution. The 1H NMR studies suggest that all the tested cations can induce a chemical exchange between two types of aggregation of BMY-28864 in an aqueous solution. The exchange rate, however, varies according to the cation species. In descending order they are: Ba2+ > Sr2+ > Ca2+ > Mg2+. The differences in the binding ability of BMY-28864 to the cations and the solubility of the chloride salts are explained by the electronegativity of these cations. The UV-Vis and CD spectra of these solutions show isosbestic points that correspond to an exchange process in the cations' binding to BMY-28864. The results provide experimental support for the hypothesis that there is a common mechanism underlying their cations interactions with the antibiotic. The CD study also provided evidence about the stoichiometric relation of the divalent metal ions to the antibiotic, 1:2, which was obtained by NMR analyses.

Anthracyclines↗
PubMed1987

Isolation and characterization of a cDNA encoding rat cationic trypsinogen.

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.

Amino Acid Sequence↗
PubMed1981

Role of metal cofactors in enzyme regulation. Differences in the regulatory properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate specific malic enzyme, depending on whether magnesium ion or manganese ion serves as divalent cation.

A number of differences in the kinetic and physical properties of the Escherichia coli nicotinamide adenine dinucleotide phosphate (NADP+) dependent malic enzyme have been found, depending upon whether Mg2+ or Mn2+ served to fulfill the divalent cation requirement. The velocity-NADP+ and velocity-cation saturation curves exhibit a simple hyperbolic response in the presence of either metal cofactor, but the affinity for NADP+ (and malate) as well as the Vmax is increased in the presence of Mn2+. The high affinity of the enzyme for Mn2+ coupled with the increased affinity for substrates indicates that Mn2+ is the preferred cofactor in vitro. With either Mg2+ or Mn2+ as cation, the velocity-malate saturation curves in the absence of effectors are complex at pH 7.45, indicating varying combinations of apparent positive and negative cooperative behavior. Greater initial positive cooperative behavior between malate binding sites is observed with Mg2+ as cation. The enzyme appears to be equally sensitive to inhibition by the allosteric inhibitors reduced nicotinamide adenine dinucleotide (NADH) and oxaloacetic acid (OAA) in the presence of either cation, but the interaction between malate binding sites, in the presence of effectors, varies significantly with the choice of metal cofactor. The inhibitor NADH increases the interaction between malate binding sites in the presence of Mn2+ but has little effect on subunit interaction in the presence of Mg2+. The inhibitor OAA increases the interaction between malate binding sites in the presence of both cations, with increased positive cooperativity observed with Mn2+ but increased negative cooperativity with Mg2+. The kinetic data can be explained by a model involving sequential ligand-induced conformational changes of the enzyme, resulting in a mixture of apparent positive and negative cooperative behavior. Alternative explanations involving different classes of noninteracting binding sites or different enzyme forms are also considered. The metal cofactors, Mg2+ and Mn2+, appear to stabilize two distinct conformational states of the enzyme which differ in response to varying substrate and effector concentrations. Altered conformational states of the enzyme in the presence of the two cations are further substantiated by proteolytic digestion studies with the homogeneous enzyme. The results are strikingly similar to previous results reported on the nicotinamide adenine dinucleotide (NAD+) dependent malic enzyme and the NAD+-dependent isocitrate dehydrogenase, supporting the suggestion that metal cofactors function as regulatory entities.

Escherichia coli↗
PubMed1976

Dual divalent cation requirement for activation of pyruvate kinase; essential roles of both enzyme- and nucleotide-bound metal ions.

Rabbit muscle pyruvate kinase requires two divalent cations per active site for catalysis of the enolization of pyruvate in the presence of adenosine 5'-triphosphate (ATP). One divalent cation is bound directly to the enzyme and forms a second sphere complex with the bound ATP (site 1). The second divalent cation is directly coordinated to the phosphoryl groups of ATP and does not interact with the enzyme (site 2). The essential role of the divalent cation at site 1 is shown by the requirement for Mg2+ or Mn2+ for the enolization of pyruvate in the presence of the substitution inert Cr3+-ATP complex. The rate of detritiation of pyruvate shows a hyperbolic dependence of Mn2+ concentration in the presence of high concentrations of enzyme and Cr3+-ATP. A dissociation constant for Mn2+ from the pyruvate kinase-Mn2+-ATP-Cr3+-pyruvate complex of 1.3 +/- 0.5 muM is determined by the kinetics of detritiation of pyruvate and by parallel Mn2+ binding studies using electron paramagnetic resonance. The essential role of the divalent cation at site 2 is shown by the sigmoidal dependence of the rate of detritiation of pyruvate on Mn2+ concentration in the presence of high concentrations of enzyme and ATP yielding a dissociation constant of 29 +/- 9 muM for Mn2+ from site 2. This value is similar to the dissociation constant of the binary Mn-ATP complex (14 +/- 6 muM) determined under similar conditions. The rate of detritiation of pyruvate is proportional to the concentration of the pyruvate kinase-Mn2+-ATP-Mn2+-pyruvate complex, as determined by parellel kinetic and binding studies. Variation of the nature of the divalent cation at site 1 in the presence of CrATP causes only a twofold change in the rate of detritiation of pyruvate which does not correlate with the pKa of the metal-bound water. Variation of the nature of the divalent cation at both sites in the presence of ATP causes a sevenfold variation in the rate of detritiation or pyruvate that correlates with the pKa of the metal-bound water. The greater rate of enolization observed with CrATP fits this correlation, indicating that the electrophilicity of the nucleotide bound metal (at site 2) determines the rate of enolization of pyruvate.

Adenosine Triphosphate↗