Accuracy of alternative representations for integrated biochemical systems

The Michaelis-Menten formalism often provides appropriate representations of individual enzyme-catalyzed reactions in vitro but is not well suited for the mathematical analysis of complex biochemical networks. Mathematically tractable alternatives are the linear formalism and the power-law formalism. Within the power-law formalism there are alternative ways to represent biochemical processes, depending upon the degree to which fluxes and concentrations are aggregated. Two of the most relevant variants for dealing with biochemical pathways are treated in this paper. In one variant, aggregation leads to a rate law for each enzyme-catalyzed reaction, which is then represented by a power-law function. In the other, aggregation produces a composite rate law for either net rate of increase or net rate of decrease of each system constituent; the composite rate laws are then represented by a power-law function. The first variant is the mathematical basis for a method of biochemical analysis called metabolic control, the latter for biochemical systems theory. We compare the accuracy of the linear and of the two power-law representations for networks of biochemical reactions governed by Michaelis-Menten and Hill kinetics. Michaelis-Menten kinetics are always represented more accurately by power-law than by linear functions. Hill kinetics are in most cases best modeled by power-law functions, but in some cases linear functions are best. Aggregation into composite rate laws for net increase or net decrease of each system constituent almost always improves the accuracy of the power-law representation. The improvement in accuracy is one of several factors that contribute to the wide range of validity of this power-law representation. Other contributing factors that are discussed include the nonlinear character of the power-law formalism, homeostatic regulatory mechanisms in living systems, and simplification of rate laws by regulatory mechanisms in vivo.     

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of hen oviduct microsomal signal peptidase

Hen oviduct signal peptidase requires only two proteins for proteolysis of fully synthesized secretory precursor proteins in vitro: one with a molecular mass of 19 kilodaltons (kDa) and one which is a glycoprotein whose mass varies from 22 to 24 kDa depending on the extent of glycosylation. Purified signal peptidase has been analyzed both as part of an active catalytic unit and after electroelution of the individual proteins out of a preparative polyacrylamide gel. The multiple forms of the glycoprotein component of signal peptidase bind to concanavalin A and are shown to be derived from the same polypeptide backbone. Removal of their oligosaccharides by digestion with N-glycanase converts these proteins to a single 19.5-kDa polypeptide. The glycoproteins all exhibit very similar profiles following individual digestion with trypsin and separation of the resulting peptides by reverse-phase high-performance liquid chromatography. In addition, sequence analysis of selected peptides from corresponding regions in chromatograms representing each form of the glycoprotein reveals the same amino acid sequences. The 19-kDa signal peptidase protein does not bind concanavalin A, has a distinct tryptic peptide map from that of the glycoprotein, and appears to share no amino acid sequences in common with the glycoprotein. Its copurification on a concanavalin A-Sepharose column indicates that it must interact directly with the glycoprotein subunit.     

Human cytochrome c oxidase isoenzymes from heart and skeletal muscle; purification and properties

Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme.     

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Receptor-mediated degradation of insulin in isolated rat adipocytes. Formation of a degradation product slightly smaller than insulin

More than 90% of the radioactivity associated with isolated rat adipocytes incubated with [TyrA14-125I]monoiodoinsulin represented at steady state iodoinsulin possessing full binding affinity. In contrast, about half of the radioactivity dissociating from the cells was [125I]monoiodotyrosine. The other half was of a molecular size similar to that of iodoinsulin as judged from gel-filtration chromatography. However, the descending limb of the 'insulin' peak (i.e., the smaller molecules) possessed a reduced binding activity compared with native iodoinsulin, material from the ascending limb, or a similar fraction isolated from dissociation medium from IM-9 lymphocytes, a cell type devoid of receptor-mediated insulin degradation. The cells, thus, release an intermediary degradation product.     

Structural elements of bactopterin from Pseudomonas carboxydoflava carbon monoxide dehydrogenase

Bactopterin is a novel pterin occurring in bacterial molybdoenzymes as the organic portion of the molybdenum cofactor. Its structure is investigated here. The compound contains a single pterin ring and carries a side chain at carbon atom 6 of the pterin nucleus as indicated by the formation of pterin-6-carboxylic acid upon alkaline permanganate oxidation. Studies with phosphate-cleaving enzymes revealed the presence of two monophosphoric acid monoesters. The affinity of reduced bactopterin for thiol-Sepharose points to the presence of thiol(s) in active bactopterin.     

The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.     

The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.