Human liver glycogen phosphorylase. Kinetic properties and assay in biopsy specimens.
(17/3500)
1. The two forms of glycogen phosphorylase were purified from human liver, and some kinetic properties were examined in the direction of glycogen synthesis. The b form has a limited catalytic capacity, resembling that of the rabbit liver enzyme. It is characterized by a low affinity for glucose 1-phosphate, which is unaffected by AMP, and a low V, which becomes equal to that of the a form in the presence of the nucleotide. Lyotropic anions stimulate phosphorylase b and inhibit phosphorylase a by modifying the affinity for glucose 1-phosphate. Both enzyme forms are easily saturated with glycogen. 2. These kinetic properties have allowed us to design a simple assay method for total (a + b) phosphorylase in human liver. It requires only 0.5 mg of tissue, and its average efficiency is 90% when the enzyme is predominantly in the b form. 3. The assay of total phosphorylase allows the unequivocal diagnosis of hepatic glycogen-storage disease caused by phosphorylase deficiency. One patient with a complete deficiency is reported. 4. The assay of human liver phosphorylase a is based on the preferential inhibition of the b form by caffeine. The a form displays the same activity when measured by either of the two assays. (+info)
Site-directed mutagenesis of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase.
(18/3500)
Site-specific mutants have been produced in order to investigate the role of proline 204 in the 'hinge' region of yeast phosphoglycerate kinase (PGK). This totally conserved proline has been shown to be the only cis-proline in the high resolution crystal structures of yeast, B. stearothermophilus, T. brucei and T. maritima PGK, and may therefore have a role in the independent folding of the two domains or in the 'hinge' bending of the molecule during catalysis. The residue was replaced by a histidine (Pro204His) and a phenylalanine (Pro204Phe), and the resulting proteins characterised by differential scanning calorimetry (DSC), circular dichroism (CD), tryptophan fluorescence emission and kinetic analysis. Although the secondary and tertiary structure of the Pro204His protein is generally similar to that of the wild-type enzyme as assessed by CD, the enzyme is less stable to heat and guanidinium chloride denaturation than the wild-type. In the denaturation experiments two transitions were observed for both the wild-type and the Pro204His mutant, as have been previously reported for yeast PGK [Missiakas, D., Betton, J.M., Minard, P. & Yon, J.M. (1990) Biochemistry 29, 8683-8689]. The first transition is accompanied by an increase in fluorescence intensity leading to a hyperfluorescent state, followed by the second, corresponding to a decrease in fluorescence intensity. However, for the Pro204His mutant, the first transition proceeded at lower concentrations of guanidinium chloride and the second transition proceeded to the same extent as for the wild-type protein, suggesting that sequence-distant interactions are more rapidly disrupted in this mutant enzyme than in the wild-type enzyme, while sequence-local interactions are disrupted in a similar way. The Michaelis constants (K(m)) for both 3-phospho-D-glycerate and ATP are increased only by three or fourfold, which confirms that, as expected, the substrate binding sites are largely unaffected by the mutation. However, the turnover and efficiency of the Pro204His mutant is severely impaired, indicating that the mechanism of 'hinge' bending is hindered. The Pro204Phe enzyme was shown to be significantly less well folded than the wild-type and Pro204His enzymes, with considerable loss of both secondary and tertiary structure. It is proposed that the proline residue at 204 in the 'hinge' region of PGK plays a role in the stability and catalytic mechanism of the enzyme. (+info)
Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.
(19/3500)
The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions. (+info)
The crystal growth technique--a laboratory evaluation of bond strengths.
(20/3500)
An ex vivo study was carried out to determine differences in the bond strengths achieved with brackets placed using a crystal growth technique compared with a conventional acid-etch technique. A solution of 37 per cent phosphoric acid was used for acid-etching and a commercially available polyacrylic acid gel, Crystal-lok for crystal growth. A heavily-filled composite resin was used for all samples to bond brackets to healthy premolar teeth extracted for orthodontic purposes. Polycrystalline ceramic and stainless steel brackets were used and tested to both tensile and shear failure using an Instron Universal Testing machine. The tensile and shear bond strengths were recorded in kgF. In view of difficulties experienced with previous authors using different units to describe their findings, the data were subsequently converted to a range of units in order to facilitate direct comparison. The crystal growth technique produced significantly lower bond strengths than the acid-etch technique for ceramic and stainless steel brackets, both in tensile and shear mode. The tensile bond strength for stainless steel brackets with crystal growth was 2.2 kg compared with 6.01 kg for acid-etch, whilst with ceramic brackets the tensile bond strengths were 3.9 kg for crystal growth and 5.55 kg for acid-etch. The mean shear bond strength for stainless steel brackets with crystal growth was 12.61 kg compared with 21.55 kg for acid-etch, whilst with ceramic brackets the shear bond strengths were 7.93 kg with crystal growth compared with 16.55 kg for acid-tech. These bond strengths were below those previously suggested as clinically acceptable. (+info)
Transport characteristics of the apical anion exchanger of rabbit cortical collecting duct beta-cells.
(21/3500)
To functionally characterize transport properties of the apical anion exchanger of rabbit beta-intercalated cells, the mean change in anion exchange activity, dpHi/dt (where pHi is intracellular pH), was measured in response to lumen Cl- replacement with gluconate in perfused cortical collecting ducts (CCDs). beta-Cell apical anion exchange was not affected by 15-min exposure to 0.2 mM lumen DIDS in the presence of 115 mM Cl-. In contrast, apical anion exchange was significantly inhibited by 0.1 mM lumen DIDS in the absence of Cl-. beta-Cell apical anion exchange was unchanged by 15 mM maleic anhydride, 10 mM phenylglyoxal, 0.2 mM niflumic acid, 1 mM edecrin, 1 mM furosemide, 1 mM probenecid, or 0.1 mM diphenylamine-2-carboxylate. However, beta-cell apical anion exchange was inhibited by alpha-cyano-4-hydroxycinnamic acid, with an IC50 of 2.4 mM. Substitution of either sulfate or gluconate for lumen Cl- resulted in a similar rate of alkalinization. Conversely, pHi was unchanged by substitution of sulfate for lumen gluconate, confirming the lack of transport of sulfate on the beta-cell apical anion exchanger. Taken together, the results demonstrate a distinct "fingerprint" of the rabbit CCD beta-cell apical anion exchanger that is unlike that of other known anion exchangers. (+info)
Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.
(22/3500)
We have determined two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex, providing snapshots of different steps on the catalytic pathway. One of the structures is of a complex with a nonhydrolyzable analog of ATP and is thus a "substrate" complex. The other structure contains a bound sulphate ion that sits in a position equivalent to that occupied by the phosphate ion produced after ATP hydrolysis, thereby mimicking a "product" complex. In both complexes, the protein is monomeric. Large and distinct conformational changes occur on binding DNA and the nucleotide cofactor. Taken together, these structures provide evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism. (+info)
Factors affecting dimensional instability of alginate impressions during immersion in the fixing and disinfectant solutions.
(23/3500)
To clarify the factors determining the dimensional stability of alginate impressions during immersion in disinfectant and fixing solution, the weight change of impressions in solutions of glutaraldehyde (GA), NaClO, Na2SO4, K2SO4, CaCl2, and ZnSO4 was measured. In the nonelectrolytic solution, GA, the weight decreased in proportion to concentration, possibly due to the gradient of osmotic pressure between the impression and solution. In monovalent metallic salt solutions the weight change decreased with increased concentration. Especially at lower concentrations the rate of weight loss was high. A chemical action of the solution might also be involved, in addition to the osmotic pressure difference. The weight loss in divalent metallic salt solutions was greater than in monovalent solutions, implicating crosslinking reactions between the impression and solution. (+info)
Role of methanogens and other bacteria in degradation of dimethyl sulfide and methanethiol in anoxic freshwater sediments.
(24/3500)
The roles of several trophic groups of organisms (methanogens and sulfate- and nitrate-reducing bacteria) in the microbial degradation of methanethiol (MT) and dimethyl sulfide (DMS) were studied in freshwater sediments. The incubation of DMS- and MT-amended slurries revealed that methanogens are the dominant DMS and MT utilizers in sulfate-poor freshwater systems. In sediment slurries, which were depleted of sulfate, 75 micromol of DMS was stoichiometrically converted into 112 micromol of methane. The addition of methanol or MT to DMS-degrading slurries at concentrations similar to that of DMS reduced DMS degradation rates. This indicates that the methanogens in freshwater sediments, which degrade DMS, are also consumers of methanol and MT. To verify whether a competition between sulfate-reducing and methanogenic bacteria for DMS or MT takes place in sulfate-rich freshwater systems, the effects of sulfate and inhibitors, like bromoethanesulfonic acid, molybdate, and tungstate, on the degradation of MT and DMS were studied. The results for these sulfate-rich and sulfate-amended slurry incubations clearly demonstrated that besides methanogens, sulfate-reducing bacteria take part in MT and DMS degradation in freshwater sediments, provided that sulfate is available. The possible involvement of an interspecies hydrogen transfer in these processes is discussed. In general, our study provides evidence for methanogenesis as a major sink for MT and DMS in freshwater sediments. (+info)