Kinetic analysis of the michaelis-menten mechanism in which the substrate and the product are unstable

• 1.1. A kinetic analysis of the Michaelis-Menten mechanism for the case in which both the substrate and the product are unstable, either spontaneously or as the result of the addition of a reagent, has been made.
• 2.2. The explicit time course equations of the immediate product and the species into which it subsequently is transformed have been derived under the conditions of rapid equilibrium and limiting substrate concentration.
• 3.3. The validity of these equations has been checked using numerical simulations.
• 4.4. The kinetic data analysis which we suggest is based on the time progress curves of the product or, in the case in which the product accumulation cannot be monitored experimentally, on the time progress curve of the species into which the immediate product is transformed.
• 5.5. This analysis allows the determination of the rate and the equilibrium constants if adequate experimental results are available.
• 6.6. We have chosen a numerical example, with which we illustrate the procedure of the kinetic data analysis by simulating some curves with assumed experimental errors.

     

Kinetics of porphyrin accumulation in cultured epithelial cells exposed to ALA

• 1.1. The kinetics of porphyrin accumulation in cultured mammalian epithelial cells (CNCM-I-221) during exposure to ALA was investigated.
• 2.2. The total porphyrin synthesized is a function of ALA concentration and the incubation time. The cellular porphyrin content exhibited a saturation pattern, reaching a plateau at about 0.04 fmol porphyrins/cell. A biphasic time-dependent increase in the total porphyrin synthesized was observed.
• 3.3. After 3 hr of exposure to ALA the rate of synthesis increased to ahnost twice the initial rate, reaching between 0.02 and 0.05 fmol porphyrins/cell/hr depending on serum concentration in the medium.
• 4.4. Two effects of FBS on ALA-stimulated porphyrin accumulation were observed. Greater total porphyrin synthesis was found when incubations were made in 10% FBS compared to those in 1% FBS.
• 5.5. The higher serum concentration also caused a greater release into the medium of the porphyrins generated in the cells with a calculated half-life of 24 min in 10% serum-supplemented medium compared with 62 min in 1% serum.
• 6.6. The results obtained from cell synchronization experiments suggest that there is little obvious cell cycle-dependent variation in the synthesis of porphyrins from ALA.
• 7.7. The small differences in the intracellular porphyrin content that were observed may be attributed to a slight reduction in the rate of loss of porphyrins in G2/M cells.

     

Changes in chemical composition of muscle in young hybrids between russian sturgeon Acipenser guldenstadti brandt × beluga Huso huso l. (Pisces; acipenseriformes) under different levels of salinity

• 1.1. The salinity tolerance in young RS × B hybrids increases as the fingerlings grow. The specimens weighing about 7 g are able to tolerate the direct transfer to the water salinity 18%..
• 2.2. Under hypo- and iso-osmotic water ion concentration in the hybrid muscle free amino acids, the exchange of taurine for β-alanine and glycine takes place.
• 3.3. Under hyperosmotic conditions within the first 2 days in the hybrid muscle the water quantity declines, the protein quantity also slightly decreases, the urea and free amino acids concentration (mostly alanine, aspartic and glutamic acids, leucine), and a portion of reserved lipids increase.
• 4.4. During the next 4 days the muscle moisture, protein quantity, and the concentration of urea and free amino acids return to control values, but the portion of reserved lipids declines below the original level.

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

Transient state isoelectric focusing. Experimental determination of apparent diffusion coefficients in polyacrylamide gels

A photoelectric scanning assembly utilizing uv absorption optics and an on-line digital data acquisition and processing system has been used to follow kinetically zone spreading during the defocusing stage (absence of electric field) of transient state isoelectric focusing (TRANSIF) in polyacrylamide gels. Measurement of the variance (σ²) of a diffusing zone as a function of time yields a linear relationship, the slope of which corresponds to the apparent diffusion coefficient (D) of the protein. A linear relationship is also obtained when the logarithm of the apparent diffusion coefficients (logD) are plotted vs acrylamide concentration (T). This relationship can be used to extrapolate D to zero gel concentration. The apparent diffusion coefficient measured in this way is significantly larger than the true diffusion coefficient. The slope of the plot logD vs T, designated C_R, is expected to be a measure of molecular size related to the retardation coefficient in polyacrylamide gel electrophoresis.     

Effects of recombinant salmon growth hormone on hypophysectomized male Fundulus heteroclitus

• 1.1. Recombinant salmon growth hormone at doses of 0.8 and 2.1 μg/g significantly enhanced linear growth in hypophysectomized male killifish, Fundulus heteroclitus, over that of controls and a significant regression was found between growth and the logarithm of dose.
• 2.2. Bovine growth hormone elicited significant growth enhancement at all three dosages tested (1,4 and 10 μg/g) and a significant log/dose relationship was also observed.
• 3.3. Observations on the relative weight of the gonads indicate that whole salmon pituitary extract (25 μg/g) possesses strong gonadotropic activity and that both bGH and rsGH had smaller but significant effects on the gonads.
• 4.4. It is suggested that growth hormone may play a subsidiary synergistic role to other pituitary hormones in gonadal development.

     

Effect of methyl methacrylate on mitochondrial function and structure

• 1.1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.
• 2.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.
• 3.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.
• 4.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.
• 5.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.
• 6.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.
• 7.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.

     

The effect of water loss upon the urate,urea and ammonia content of the egg of the Japanese quail Coturnix coturnix japonica

• 1.1. The urate, urea and ammonia content of the whole egg of the Japanese quail was measured in late incubation in eggs subject to different rates of water loss.
• 2.2. High rates of water loss substantially increased egg urate content, but had little or no effect on urea or ammonia content.
• 3.3. Allopurinol, an inhibitor of urate synthesis, reduced egg urate content to low levels, but produced no effect on urea content, and a small reduction in ammonia content.
• 4.4. The urea concentration of the embryo was lower than in allantoic fluid.
• 5.5. It is concluded that urate production by the avian embryo is primarily concerned with the modification of allantoic fluid composition.

     

Application of electrical analogs to metabolic control analysis: Linear pathways with multiple feedback

• 1.1. Using electrical analogs, we have presented a systematic procedure for calculating the flux control coefficients of linear metabolic pathways with multiple feedback loops.
• 2.2. In this method, an electrical analog circuit is constructed first for the unregulated pathway.
• 3.3. This circuit is subsequently modified in a step-by-step fashion to take into account the effect of each feedback loop in the pathway.
• 4.4. An analog circuit consists of resistances which are connected in series (or parallel) with a voltage (or current) source.
• 5.5. The flux control coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's law.
• 6.6. We have investigated the possible patterns in linear pathways with two feedback loops.
• 7.7. This is followed by an analysis of a linear pathway with an arbitrary pattern of feedback inhibition.

     

Changes in glycolytic and associated enzyme activities during rana ridibunda oocyte development

• 1.1. Most of glycolytic and associated enzymes in the oocytes of the frog Rana ridibunda exhibit a higher activity at the early growth stages; the activity declines by the time the oocyte reaches full growth. Citrate syntase follows a similar pattern.
• 2.2. Enzymes related to gluconeogenesis have non-detectable activity.
• 3.3. It is suggested that at the early stages of oocyte growth glycogen could contribute as a fuel mainly for the pentose phosphate pathway; in the full-grown oocyte glycogen could serve mainly as a fuel for the glycolytic pathway.

     

Variations of respiratory rate of Tisbe holothuriae humes (copepoda,harpacticoida) in relation to temperature,salinity and food type

• 1.1. The influence of temperature (14,19, 24°C), salinity (26,32, 38,44%.) and food type (artificial diets: Fryfood, Mytilus, Soya, Yeast, Spirulina) on the respiratory rate of Tisbe holothuriae has been studied.
• 2.2. Oxygen consumption decreased with decreasing temperature, but with a greater rate at supra- or subnormal salinities.
• 3.3. Multiple-regression analysis showed the quadratic effect of temperature and the linear effect of salinity to be the more important factors affecting respiration.
• 4.4. The food type also seems to exert an important effect on oxygen consumption.
• 5.5. A significant lowering of respiration was observed for all food tested when the animals were starved.

     

δ-Aminolevulinic acid dehydratase inactivation by uroporphyrin I in light and darkness

• 1.1. The action of uroporphyrin I on erythrocytic ALA-D activity under dark and light conditions was examined.
• 2.2. Photo and non-photoinactivation of ALA-D induced by uroporphyrin I were observed.
• 3.3. Both effects were dependent on uroporphyrin concentration, temperature and time of exposure of the protein to the porphyrin.
• 4.4. Light-dependent effect of uroporphyrin I is related with the phototoxicity of porphyrins and could be produced by primary amino acid photooxidation followed by secondary cross-linking of the protein.
• 5.5. Light-dependent effect of uroporphyrin I could be ascribed to a direct enzyme inhibition due to binding of the porphyrin to the protein inducing structural changes at or near its active site.

     

Acute toxicity and bioaccumulation of endosulfan in rotifer (Brachionus calyciflorus)

• 1.1. The acute toxicity of endosulfan was determined for the freshwater rotifer Brachionus calyciflorus.
• 2.2. The mean 24 hr lc₅₀ value for endosulfan was 5.15 ppm with a coefficient of variation of 14.7%.
• 3.3. Rotifers were exposed at two sublethal concentrations (1.5–2.0 ppm) of endosulfan for bioaccumulation experiments, for an exposure time of 24, 48, 72 and 96 hr. The rotifers were fed with Nannochloris oculata (5 × 10⁵cell/ml).
• 4.4. The highest accumulation of endosulfan was found 24 hr after the start of the exposure to 1.5 ppm of the toxicant. A steady-state concentration in rotifer was reached between 24–48 hr, followed by a gradual decrease until 96 hr.

     

The flow theory of enzyme kinetics: Role of solid geometry in the control of reaction velocity in live animals

• 1.1. When blood flows, membranes are bombarded with ions etc., whose entry creates an ATP demand proportional to flow rate. Also proportional to flow rate is ATP production from oxidation of substrates [S] from the same blood volume.
• 2.2. O₂ is limiting and reaction velocity at rest (metabolic rate) is determined by flow rate, F, but not by [S].
• 3.3. Since resting blood O₂ A-V difference is about 5 vol%, 11 circulated produces about 0.25 kcal in mammals, birds or warm reptiles.
• 4.4. Where O₂ is not limiting, as in most amino acid deaminations, V = K F[S] with K a constant unrelated to K_m.
• 5.5. At equal blood vol/kg, solid geometry dictates that the average cross-sectional area of major vessels/kg will be an inverse function of body mass. The smaller the animal, the shorter the vessels, the “thicker” the vessels/kg body wt, and at any one blood pressure, the higher the flow/kg/hr. If a man's major vessels were equal in cross-section/kg to those of a shrew, it would take 2241 of blood to fill them.
• 6.6. Growth decreases flow/kg (and therefore metabolic rate), by decreasing vessel cross-section/kg without changing blood pressure or linear velocity of flow.
• 7.7. Surface area/g, body wt to some power, average vessel length/kg, circulation time and average major vessel cross-sectional area are all related mathematically.

     

Hormonal regulation of human apolipoprotein E gene expression in HepG2 cells

• 1.1. Hormonal regulation of apolipoprotein E (apoE) gene expression by insulin and thyroid hormone was studied in a human hepatoma cell line, HepG2.
• 2.2. Changes at the mRNA level, mRNA translation, in vivo synthesis and secretion were monitored.
• 3.3. Both insulin and triiodothyronine were found to have no significant effect on apoE mRNA levels.
• 4.4. Insulin treatment caused an inhibition of: (a) the in vitro translation of endogenous apoE mRNA in a HepG2 cell-free system (25%), and (b) the incorporation of radioactivity into newly-synthesized apoE in an in vivo pulse-chase labeling experiment (32%).
• 5.5. Interestingly, apoE secretion rate was found to be significantly reduced with insulin (84%) suggesting that a major portion of newly-synthesized apoE may be shunted into a degradative pathway.
• 6.6. Using a similar experimental approach, triiodothyronine showed no significant effect on the rate of apoE synthesis or translation (6–15% decrease), however a slight reduction (20%) in secretion rate was shown.
• 7.7. Overall, apoE gene expression does not appear to be influenced by triiodothyronine significantly but is modulated by insulin at the translational and post-translational level.

     

Glucose metabolism in a kangaroo (Macropus robustus erubescens) and a similar size eutherian herbivore,the feral goat

• 1.1. Glucose pool size, space, entry rate, and turnover time were estimated from the specific radioactivity vs time curves of [³H] and [¹⁴C]glucose administered as a single injection in the euro (Macropus robustus erubescens) and the sympatric feral goat (Capra hircus).
• 2.2. Digestible energy intake was greater (P < 0.05 ± SE) in the goat than in the euro (798 ± 64 vs 624 ± 31kJ/kg^0.75 × day).
• 3.3. However, there were no significant differences between the two species in parameters of glucose metabolism.
• 4.4. The use of an implantable osmotic infusion pump to deliver isotopic glucose showed promise as a means of avoiding the stress involved with the single injection technique.

     

Age-dependent proteins in eyes of annual killifishes Pterolebias longipinnis detected by 2-dimensional electrophoresis

• 1.1. Eye proteins of Pterolebias longipinnis have been analyzed by 2-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis during aging from adolescence until normal death.
• 2.2. The protein pattern on the gels changed gradually with progressing age.
• 3.3. In senescent eyes, three protein spots appeared for a time and 36 disappeared from the pattern.
• 4.4. The isoelectric points of the proteins in the presence of urea and the molecular weights in an unreducing buffer are presented.

     

Secretory oedema in diabetic submandibular glands during parasympathetic nerve stimulation: Relationship to microvascular abnormalities in streptozotocin-treated rats

• 1.1. Submandibular secretion during parasympathetic stimulation (5 Hz) was examined in streptozotocin-diabetic and age-matched control rats.
• 2.2. At 3 weeks, but not 3 and 6 months, flow rate was initially greater than in controls, but it declined rapidly after 30 min.
• 3.3. The reduction in flow rate was associated with oedema of the gLond.
• 4.4. At 3 months, graded stimulation revealed a tendency to oedema at frequencies of 10 Hz and above.
• 5.5. Morphologically, submandibular capillary density was increased in diabetic rats.
• 6.6. Thus, in diabetes the submandibular gland appears less able to withstand continuous parasympathetic stimulation, due in part to an increase in tissue capillary area.

     

A relationship between circulating natural glucocorticoids and the mechanical responses of the heart in atricial and precocial rodents

• 1.1. A comparison was made of the mechanical performance of heart muscle from mouse, an atricial mammal, with corticosterone as glucocorticoid and spiny mouse (Acomys cahirinus), a precocial mammal, with cortisol as glucocorticoid.
• 2.2. Force-frequency responses were negative in mouse and positive in spiny mouse.
• 3.3. During recovery, there was a gradual increase and an overshoot in the mouse, while in the spiny mouse there was an initial enhanced response, diminishing gradually with time.
• 4.4. High calcium concentration inhibited contractile tension in mouse heart, while it was positively inotropic in spiny mouse heart. Changes in the concentration of calcium did not change the patterns of force-frequency response.
• 5.5. Lowering the experimental temperature increased the time course and amplitude of the tension curve. However, various parameters exhibited different temperature sensitivity.
• 6.6. There was a significant difference in the levels of circulating cortisol between male and female spiny mice.
• 7.7. It is proposed that the differences in the mechanical responses of mouse and spiny mouse hearts may be explained in terms of the effects of the specific glucocorticoid hormone on the development of the sodium-calcium exchanger.