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D Job

Publications and source records attributed to D Job.

At least 127 records · Page 7Linked to original sources
PubMed1982

Recycling of cold-stable microtubules: evidence that cold stability is due to substoichiometric polymer blocks.

A substantial subpopulation of mammalian brain crude extract microtubules is resistant to cold-temperature disassembly. We propose here that microtubules are rendered cold stable by rare substoichiometric blocks. Mild shearing of rat brain cold-stable microtubules makes them largely cold labile. In addition, cold-stable microtubules can be destabilized by exposure to low concentrations of calmodulin (5 microM) in the presence of calcium at 0 degree C. Cold-disassembled microtubule protein, obtained from sheared or calmodulin-treated cold-stable preparations, re-forms a cold-stable subpopulation upon reassembly. These observations allow strategies for the recycling purification of cold-stable microtubules. Comparison of purified cold-labile and cold-stable material by gel electrophoresis shows enrichment for a few unique polypeptides, of 135, 70-82, and 56 kilodaltons, in the cold-stable preparation. The 64-kilodalton "switch protein", previously identified as uniquely dephosphorylated in cold-stable microtubules, is equally represented in recycled cold-stable and cold-labile microtubule preparations. Furthermore, when disassembled, cold-stable microtubule proteins are passed through a calmodulin affinity column on which the polypeptides characteristic of cold-stable microtubules are specifically retained, the breakthrough (unbound) material repolymerizes into cold-labile microtubules only. Based on the above data, a model is presented in which microtubules are rendered cold stable by the presence of substoichiometric, calmodulin-sensitive blocks that randomly reshuffle upon reassembly of cold-stable microtubules.

Animals↗
PubMed1982

Enzymatic properties and cooperative effects in the kinetics of wheat-germ RNA polymerases. A comparative study of the three nuclear enzyme classes.

Some of the enzymatic properties of the three classes of RNA polymerase purified from wheat germ were studied. Although the four enzyme species exhibited different template specificities using synthetic polydeoxyribonucleotides, poly(dC) was the most efficiently transcribed. Furthermore, with this matrix all enzyme forms had nearly the same specific activity (approximately equal to 5500 units/mg). A comparative kinetic study of RNA synthesis catalyzed by the wheat germ RNA polymerases lead to the following results: when rate measurements were effected as a function of the concentration of purine nucleoside triphosphates, non-linear double-reciprocal plots were obtained for polymerases I and IIB, whereas linear plots were obtained for RNA polymerases IIA and III. The reaction rates were also measured as a function of UTP concentration (a nucleoside triphosphate which can only be used in the elongation step): the kinetics of the reactions catalyzed by RNA polymerases IIA and III can be accounted for by a simple ping-pong kinetic model; in contrast, negative cooperativity was obtained for enzymes I and IIB. This kinetic behaviour may signify that RNA polymerases I and IIB are allosterically regulated enzymes.

Cell Nucleus↗
PubMed1981

Ionic control of immobilized enzymes. Kinetics of acid phosphatase bound to plant cell walls.

When an enzyme is bound to an insoluble polyelectrolyte it may acquire novel kinetic properties generated by Donnan effects. It the enzyme is homogeneously distributed within the matrix, a variation of the electrostatic partition coefficient, when substrate concentration is varied, mimics either positive or negative co-operativity. This type of non-hyperbolic behaviour may be distinguished from true co-operativity by an analysis of the Hill plots. If the enzyme is heterogeneously distributed within the polyelectrolyte matrix, an apparent negative co-operativity occurs, even if the electrostatic partition coefficient does not vary when substrate concentration is varied in the bulk phase. If the partition coefficient varies, mixed positive and negative co-operativities may occur. All these effects must be suppressed by raising the ionic strength in the bulk phase. Attraction of cations by fixed negative charges of the polyanionic matrix may be associated with a significant decrease of the local pH. The magnitude of this effect is controlled by the pK of the fixed charges groups of the Donnan phase. The local pH cannot be much lower than the value of this pK. This effect may be considered as a regulatory device of the local pH. Acid phosphatase of sycamore (Acer pseudoplatanus) cell walls is a monomeric enzyme that displays classical Michaelis-Menten kinetics in free solution. However, when bound to small cell-wall fragments or to intact cells, it has an apparent negative co-operativity at low ionic strength. Moreover a slight increase of ionic strength apparently activates the bound enzymes and tends to suppress the apparent co-operativity. At I0.1, or higher, the bound enzyme has a kinetic behavior indistinguishable from that of the purified enzyme in free solution. These results are interpreted in the light of the Donnan theory. Owing to the repulsion of the substrate by the negative charges of cell-wall polygalacturonates, the local substrate concentration in the vicinity of the bound enzyme is smaller than the corresponding concentration in bulk solution. The kinetic results obtained are consistent with the view that there exist at least three populations of bound enzyme with different ionic environments: a first population with enzyme molecules not submitted to electrostatic effects, and two other populations with molecules differently submitted to these effects. The theory allows one to estimate the proportions of enzyme belonging to these populations, as well as the local pH values and the partition coefficients within the cell walls.

Acid Phosphatase↗
PubMed1981

Cyclic nucleotide independent casein kinase (G type) in bovine adrenal cortex: purification and properties of two molecular forms.

Two soluble cyclic nucleotide independent protein kinase (ATP: protein-phosphotransferase, EC 2.7.1.37) activities have been purified from bovine adrenal cortex cytosol. Both purified enzymes exhibit the best affinity for acidic substrates such as casein and can use GTP as well as ATP as phosphoryl donor. They can thus be classified as casein kinase of the G type as previously proposed (Cochet C. et al., (1980) Endocrinology 106, 750-757). Whereas the two moieties could be separated using their different affinities toward a phosphocellulose resin, both purified enzymes appeared indistinguishable on the basis of several molecular and catalytic properties. Both G type casein kinase moieties have an identical sedimentation behavior (5.5 S in the presence of 0.5 M NaCl), yield similar patterns upon electrophoresis under denaturing conditions with three major protein components (42 000, 38 000 and 27 000), and show an ability to undergo self-phosphorylation mostly on the 27 000 component. Both enzymes have the same protein and nucleotide (ATP and GTP) substrate specificity, show similar increases in activity in the presence of polyamines and Mg2+ (optimum at 50 mM) and similar inhibition by NaCl above 0.2 M. The only difference between the two forms of casein kinase (i.e., affinity for phosphocellulose) could not be explained by a different degree of self-phosphorylation or by a limited proteolytic process during handling and purification. These results suggest that the two active moieties may represent isoenzymatic forms of the G type casein kinase activity in bovine adrenal cortex cytosol.

Adenosine Triphosphate↗
PubMed1981

Rapid disassembly of cold-stable microtubules by calmodulin.

Purified cold-stable microtubules from the rat brain are insensitive to podophyllotoxin and to millimolar concentrations of free calcium. However, in the presence of calmodulin at concentrations substoichiometric to that of tubulin, calcium causes rapid microtubule disassembly. The half-maximal effective calcium concentration in the presence of calmodulin is 100 microM. With 800 microM free calcium, the half-maximal effective concentration of calmodulin is 1.0 microM (or one-tenth the tubulin concentration). Calmodulin is without effect in the absence of calcium. Troponin C is approximately one-fifth as effective as calmodulin, and parvalbumin is totally ineffective. Troponin I partially inhibits the calcium/calmodulin-induced disassembly of microtubules in the crude extract and blocks the calcium/calmodulin effect on purified cold-stable microtubules. A 5-fold excess of trifluoperazine does not inhibit the calcium/calmodulin-induced disassembly.

Animals↗
PubMed1980

Peroxidase content of soybean root nodules.

A peroxidase has been isolated from soybean nodules and its main characteristics have been determined. Its molecular weight (48 000) and spectral properties are similar to those of usual plant peroxidases. Its activity is comparable to that of low-efficiency plant peroxidases. The rate constant of the reaction with H2O2 is 3 x 10(5) M-1 x s-1. In this reaction, nodule peroxidase yields an oxidized intermediate analogous to the compound I species of peroxidases already studied. A comparison is made with the pseudoperoxidatic activity of soybean leghemoglobin components. Leghemoglobins a and c react with H2O2 with rate constants of 5 x 10(3) and 2.5 x 10(3) M-1 x s-1, respectively, yielding the leghemoglobin (IV) species. During these reactions leghemoglobins are inactivated.

Kinetics↗
PubMed1980

Kinetic studies of the reaction of ferric soybean leghemoglobins with hydrogen peroxide, cyanide and nicotinic acid.

A kinetic study of the reaction of two soybean leghemoglobins (components a and c) with hydrogen peroxide to form the oxidized compound (leghemoglobin IV) has been carried out over the pH range 2.5--10. Three different ionization processes of leghemoglobins with pKa values of 3,4.7 +/- 0.2 and 8.2 +/- 0.1 are required to explain the rate/pH profiles. Protonation of the former group and ionization of the latter cause a decrease in the rate of reaction of the hemoproteins with H2O2. The results are compared to those obtained for the reactions of plant peroxidases and myoglobin with H2O2. The results obtained from the kinetic study of cyanide binding to soybean leghemoglobins indicate that CN- is the reactive species. Two ionization processes of leghemoglobins with pKa values of 4.7 +/- 0.2 and 8.2 +/- 0.1 affect the reaction rates. The association and dissociation rate constants corresponding to nicotinic acid binding to leghemoglobins a and c have been measured over the pH range 2.5--7. The dissociation rate constant is affected by ionization of a group with pKa less than 2.5 for both leghemoglobin-nicotinate complexes. In this pH range the association rate constant is only affected by ionization of a group with pKa value of 4.7 +/- 0.2. The analysis of these results shows that both ionization processes corresponding to ring nitrogen atom of the ligand (pKa approximately equal to 4.9) and to a heme-linked group (pKa approximately equal to 4.7 +/- 0.2) influence the association rate constant. Furthermore, it appears that in the binding site of leghemoglobins the pKa value corresponding to ionization of the ring nitrogen atom of nicotinic acid is shifted from the normal value of 4.9 to a value of less than 2.5. This pecularity might explain the exceptional reactivity of leghemoglobins for nicotinic acid, over a large pH range. For both cyanide and nicotinic acid binding reactions, the ionizable group of leghemoglobins with pKa value of 4.7 +/- 0.2 seems to act as an electrostatic gate. When the group is deprotonated, it restricts the access of anion ligands to the heme pocket. For all the three reactions studied, leghemoglobin a reacts about twice as fast as leghemoglobin c.

Cyanides↗
PubMed1979

Delayed progression of diabetic retinopathy by divided insulin administration: a further follow-up.

We have previously reported a slower rate of progression in the number of microaneurysms in insulin dependent diabetic patients treated by divided insulin injections when compared to patients receiving a single daily insulin injection [Diabetes 25, 463--469 (1976)]. This observation is strengthened by the present analysis which uses complementary data obtained after a further year follow up: the mean yearly increase in the number of microaneurysms was 9 +/- 1 in the single injection group and 3 +/- 1 in the multiple one (p less than 0.001).

Aneurysm↗
PubMed1978

Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases.

A kinetic study of the reaction of two turnip peroxidases (P1 and P7) with hydrogen peroxide to form the primary oxidized compound (compound I) has been carried out over the pH range from 2.4 to 10.8. In the neutral and acidic pH regions, the rates depend linearly on hydrogen peroxide concentration whereas at alkaline pH values the rates display saturation kinetics. A compound is made with the cyanide binding reaction to peroxidases since the two reactions are influenced in the same manner by ionization of groups on the native enzymes. Two different ionization processes of peroxidase P1 with pKa values of 3.9 and 10 are required to explain the rate pH profile for the reaction with H2O2. Protonation of the former group and ionization of the latter causes a decrease in the rate of reaction of the enzyme with H2O2. In the case of peroxidase P7 a minimum model involves three ionizable groups with pKa values of 2.5, 4 and 9. Protonation of the former two groups and ionization of the latter lowers the reaction rate. In the pH-independent region, the rate of formation of compound I was measured as a function of temperature. From the Arhenius plots the activation energy for the reaction was calculated to be 2.9 +/- 0.1 kcal/mol for P1 and 5.4 +/- 0.3 kcal/mol for P7. However, the rates are independent of viscosity in glycerol-water mixtures up to 30% glycerol.

Glycerol↗