Effect of Alloxan Diabetes and Subsequent Insulin Treatment on Temperature Kinetics Properties of Succinate Oxidase Activity in Rat Kidney Mitochondria

Early and late effects of alloxan diabetes and subsequent treatment with insulin on the temperature kinetics properties of succinate oxidase (SO) activity in rat kidney mitochondria were examined. In diabetic animals SO activity increased significantly and the increase was more pronounced at the late stage. Insulin treatment partially restored SO activity. However, the effect was temperature-dependent. In diabetic animals the energy of activation in the low temperature range (E_L) increased significantly while that in the high temperature range (E_H) decreased. The latter seems to be responsible for improving catalytic efficiency in the diabetic state. Insulin treatment normalized E_H only in the 1-month diabetic group. The phase transition temperature (Tt), decreased in diabetic animals. Insulin treatment caused an increase beyond the control value in Tt in 1-month diabetic animals. The results suggest that insulin status-dependent modulation of SO activity is a complex process.     

Insulin-status-dependent modulation of FoF1 ATPase activity in rat kidney mitochondria

The early and late effects of alloxan-diabetes and insulin treatment on kinetic properties of mitochondrial FoF1 ATPase were examined. Diabetic state resulted in significant decrease in the activity while insulin treatment caused hyper-stimulation. In control animals the enzyme activity resolved in three kinetic components. In diabetic condition only component I and II were present. With insulin treatment component III was restored but component II was abolished. Diabetic state and insulin treatment had varied effects on Km values of the three components, whereas the Vmax values were generally on the higher side. Evaluation of the AppKcat/Km values revealed that diabetic state resulted in increased catalytic efficiency; insulin treatment brought back these values to normality. Temperature kinetics studies indicated that the phase transition temperature decreased significantly in the diabetic and insulin-treated diabetic animals. The energy of activation in low temperature range increased in the diabetic animals. Insulin treatment corrected the Arrhenius pattern at early stage of diabetes; at late stage the pattern was reversed. The results are suggestive of subtle insulin-status-dependent alterations in membrane structure - function relationships.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

Natural Triterpenoids Isolated from Akebia trifoliata Stem Explants Exert a Hypoglycemic Effect via α-Glucosidase Inhibition and Glucose Uptake Stimulation in Insulin-Resistant HepG2 Cells

The inhibition of α-glucosidase activity is a prospective approach to attenuate postprandial hyperglycemia in the treatment of type 2 diabetes mellitus (T2DM). Herein, the inhibition of α-glucosidase by three compounds T₁ – T₃ of Akebia trifoliata stem, namely hederagenin ( T₁ ), 3-epiakebonoic acid ( T₂ ), and arjunolic acid ( T₃ ) were investigated using enzyme kinetics and molecular docking analysis. The three triterpenoids exhibited excellent inhibitory activities against α-glucosidase. T₁ – T₃ showed the strongest inhibition with IC₅₀ values of 42.1±5.4, 19.6±3.2, and 11.2±2.3 μM, respectively, compared to the acarbose positive control (IC₅₀=106.3±8.2). Enzyme inhibition kinetics showed that triterpenoids T₁ – T₃ demonstrated competitive, mixed, and noncompetitive-type inhibition against α-glucosidase, respectively. The inhibition constant (K_i) values were 21.21, 7.70, and 3.18 μM, respectively. Docking analysis determined that the interaction of ligands T₁ – T₃ and α-glucosidase was mainly forced by hydrogen bonds and hydrophobic interactions, which could result in improved binding to the active site of the target enzyme. The insulin resistant (IR)-HepG2 cell model used in this study (HepG2 cells exposed to 10⁻⁷ M insulin for 24 h) and glucose uptake assays showed that compounds T₁ – T₃ had no cytotoxicity with concentrations ranging from 6.25 to 25 μM and displayed significant stimulation of glucose uptake in IR-HepG2 cells. Thus, triterpenoids T₁ – T₃ showed dual therapeutic effects of α-glucosidase inhibition and glucose uptake stimulation and could be used as potential medicinal resources to investigate new antidiabetic agents for the prevention or treatment of diabetes.     

Influence of ration level and rearing temperature on hepatic GHR1 and 2, and hepatic and intestinal TRα and TRβ gene expression in late stages of rainbow trout embryos

The study examined whether the early life-history temperature experience of rainbow trout Oncorhynchus mykiss embryos affects subsequent growth and expression of growth-related genes in the growing-up juveniles in response to variations in ration levels. Embryos were reared in a Heath incubator at either 8·5° C (E_8·5) or 6·0° C (E_6·0) until hatching, at which time they were transferred to grow-up tanks supplied with water at 8·5° C. At swim-up, the late stage embryos were subsequently fed a salmonid starter diet at levels of 5, 2 or 0·5% of live body mass per day. The body mass and proximate composition of the juveniles was examined when yolk absorbance was complete (21 days after the fish commenced feeding). Quantitative RT-PCR was used to examine the expression of mRNA encoding for growth hormone receptors 1 and 2 (GHR1 and GHR2) in the liver, and the two isoforms of thyroid hormone receptor (TRα and TRβ) in the liver and intestinal tract. Final body mass and total length, liver and intestinal masses, and total lipid content of the E_8·5 treatment group were directly related to increased ration size. These variables in the E_6·0 treatment group fed the 5% ration were significantly lower than for the comparable E_8·5 treatment group, suggesting an effect of embryo rearing temperature on the subsequent growth of these late stage embryos as they undergo the transition from embryo to early juvenile. Intestinal TRα and TRβ mRNA abundance was directly related to ration size in the E_8·5 treatment group, but not in the E_6·0 treatment group. Conversely, hepatic TRα and TRβ mRNA abundance was significantly affected by ration size only in the E_6·0 group, with TRβ and TRα abundance showing direct and inverse relationships with ration size, respectively. Hepatic GHR1 mRNA abundance was significantly and directly related to ration size in both the E_8·5 and E_6·0 treatment groups, but there were no differences in the abundance of hepatic GHR2 mRNA among any treatments.     

Thyroid Hormone Treatments Differentially Affect the Temperature Kinetics Properties of FoF1 ATPase and Succinate Oxidase as well as the Lipid/Phospholipid Profiles of Rat Kidney Mitochondria: A Correlative Study

Effect of thyroidectomy (Tx) and subsequent treatment with 3,5,3′-triiodo-l-thyronine (T₃) or replacement therapy (T_R) with T₃ + l-thyroxine (T₄) on the temperature kinetics properties of FoF₁ adenosine triphosphatase (ATPase, ATP synthase, H⁺-translocating ATP synthase EC 3.6.3.14) and succinate oxidase (SO) and on the lipid/phospholipid makeup of rat kidney mitochondria were examined. Tx lowered ATPase activity, which T₃ treatment restored. SO activity was unchanged in Tx but decreased further by T₃ treatment. T_R restored both activities. The energies of ATPase activation in the high and low temperature ranges (E _H and E _L) increased in the Tx and T₃ animals with decrease in phase transition temperature (Tt). T_R restored E _H and E _L but not Tt to euthyroid levels. E _H and E _L of SO decreased in Tx animals. T₃ and T_R restored E _H whereas E _L was restored only in the T_R group; Tt increased in both groups. Total phospholipid and cholesterol contents decreased significantly in Tx and T₃-treated animals. In Tx animals, sphingomyelin (SPM) and phosphatidylcholine (PC) components decreased, while phosphatidylserine (PS) and diphosphatidylglycerol components increased. T₃ and T_R treatments caused decreases in SPM, phosphatidylinositol and PS. PC and phosphatidylethanolamine (PE) increased in the T₃ group. T_R resulted in increased lysophospolipids and PE. Changes in kinetic parameters of the two enzymes were differently correlated with specific phospholipid components. Both T₃ and T_R regimens were unable to restore normal membrane structure-function relationships.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Activation of brain steroidogenesis and neurogenesis during the gonadal differentiation in protandrous black porgy,Acanthopagrus schlegelii

The early brain development, at the time of gonadal differentiation was investigated using a protandrous teleost, black porgy. This natural model of monosex juvenile fish avoids the potential complexity of sexual dimorphism. Brain neurogenesis was evaluated by histological analyses of the diencephalon, at the time of testicular differentiation (in fish between 90 and 150 days after hatching). Increases in the number of both Nissl‐stained total brain cells, and Pcna‐immunostained proliferative brain cells were observed in specific area of the diencephalon, such as ventromedialis thalami and posterior preoptic area, revealing brain cell proliferation. qPCR analyses showed significantly higher expression of the radial glial cell marker blbp and neuron marker bdnf. Strong immunohistochemical staining of Blbp and extended cellular projections were observed. A peak expression of aromatase (cyp19a1b), as well as an increase in estradiol (E₂) content were also detected in the early brain. These data demonstrate that during gonadal differentiation, the early brain exhibits increased E₂ synthesis, cell proliferation, and neurogenesis. To investigate the role of E₂ in early brain, undifferentiated fish were treated with E₂ or aromatase inhibitor (AI). E₂ treatment upregulated brain cyp19a1b and blbp expression, and enhanced brain cell proliferation. Conversely, AI reduced brain cell proliferation. Castration experiment did not influence the brain gene expression patterns and the brain cell number. Our data clearly support E₂ biosynthesis in the early brain, and that brain E₂ induces neurogenesis. These peak activity patterns in the early brain occur at the time of gonad differentiation but are independent of the gonads. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 121–136, 2016     

How could the Gompertz-Makeham law evolve

In line with the origin of life from the chemical world, biological mortality kinetics is suggested to originate from chemical decomposition kinetics described by the Arrhenius equation k=A*exp(−E/RT). Another chemical legacy of living bodies is that, by using the appropriate properties of their constituent molecules, they incorporate all their potencies, including adverse ones. In early evolution, acquiring an ability to use new molecules to increase disintegration barrier E might be associated with new adverse interactions, yielding products that might accumulate in organisms and compromise their viability. Thus, the main variable of the Arrhenius equation changed from T in chemistry to E in biology; mortality turned to rise exponentially as E declined with increasing age; and survivorship patterns turned to feature slow initial and fast late descent making the bulk of each finite cohort to expire within a short final period of its lifespan. Numerical modelling shows that such acquisition of new functions associated with faster functional decline may increase the efficiency of investing resources into progeny, in line with the antagonistic pleiotropy theory of ageing. Any evolved time trajectories of functional changes were translated into changes in mortality through exponent according to the generalised Gompertz-Makeham law μ=C(t)+Λ*exp[−E(t)], which is reduced to the conventional form when E(t)=E₀−γt and C is constant. The proposed model explains the origin of the linear mid-age functional decline followed by its deceleration at later ages and the positive correlation between the initial vitality and the rate of ageing.     

Effect of cerebrocrast on the lymphocyte blast transformation activity in normal and streptozotocin‐induced diabetic rats

Both IDDM and NIDDM are characterized by deviations in peripheral T and B lymphocyte count, T_helper:T_suppressor ratio, as well as by impaired T_suppressor function. These abnormalities may promote insulin antibody and other antibody production, contributing to overt diabetes mellitus development in early stage of the disease. In the present study we explored the effects of cerebrocrast (1,4‐dihydropyridine derivative) administration on Con A‐ and IL‐2‐stimulated tissue lymphocyte blast transformation activity and on the thymus and lymph node mass in normal and streptozotocin (STZ)‐induced diabetic rats. It was established that cerebrocrast, administered four times at the doses of 0·05 and 0·5 mg kg⁻¹, has long‐term (up to 14 days) effects on the immune system and protects against the toxic effect of STZ in STZ‐induced diabetic rats, preventing thymus and lymph node mass loss. We conclude that cerebrocrast administration leads to the increase in number and activity of T_helper and T_suppressor lymphocytes. Glycolysis and DNA synthesis in these cells is augmented under the influence of cerebrocrast administration. We propose that the increase in lymphocyte suppressive activity caused by cerebrocrast administration may prevent the development of IDDM and NIDDM in patients with pre‐diabetes, but in patients with early and overt diabetes mellitus the drug administration may prevent the overexpression of insulin antibodies and other antibodies. The effect of cerebrocrast on the de novo production of insulin and IL‐2 receptors may be beneficial for IDDM and NIDDM patients. Copyright © 1999 John Wiley & Sons, Ltd.     

The role of lipid-protein interactions in NADH-cytochrome c reductase (rotenone-insensitive) of rat liver mitochondria

The phospholipid depletion of rat liver mitochondria, induced by acetone-extraction or by digestion with phospholipase A₂ or phospholipase C, greatly inhibited the activity of NADH-cytochrome c reductase (rotenone-insensitive). A great decrease of the reductase activity also occurred in isolated outer mitochondrial membranes after incubation with phospholipase A₂. The enzyme activity was almost completely restored by the addition of a mixture of mitochondrial phospholipids to either lipid-deficient mitochondria, or lipid-deficient outer membranes. The individual phospholipids present in the outer mitochondrial membrane induced little or no stimulation of the reductase activity. Egg phosphatidylcholine was the most active phospholipid, but dipalmitoyl phosphatidylcholine was almost ineffective. The lipid depletion of mitochondria resulted in the disappearance of the non-linear Arrhenius plot which characterized the native reductase activity. A non-linear plot almost identical to that of the native enzyme was shown by the enzyme reconstituted with mitochondrial phospholipids. Triton X-100, Tween 80 or sodium deoxycholate induced only a small activation of NADH-cytochrome c reductase (rotenone-insensitive) in lipiddeficient mitochondria. The addition of cholesterol to extracted mitochondrial phospholipids at a 1 : 1 molar ratio inhibited the reactivation of NADH-cytochrome c reductase (rotenone-insensitive) but not the binding of phospholipids to lipid-deficient mitochondria or lipid-deficient outer membranes.These results show that NADH-cytochrome c reductase (rotenone-insensitive) of the outer mitochondrial membrane requires phospholipids for its activity. A mixture of phospholipids accomplishes this requirement better than individual phospholipids or detergents. It also seems that the membrane fluidity may influence the reductase activity.     

Pressure effects on lipid-protein interactions in (Na+ + K+)-ATPase

The (Na⁺ + K⁺)-stimulated ATPase activity decreases with increasing pressure and a plot of the logarithm of the activity versus pressure shows a change in slope at a defined breakpoint pressure (P_b). The value of P_b increases linearly with increasing temperature. A $\text{d}\text{T}\text{d}\text{P}$ value of 27.7 ± 0.4 (S.D.) K/1000 atm is obtained. This is in very good agreement with the pressure shift for the melting transitions in phospholipids and aliphatic chains. This strongly indicates that an aliphatic chain melting process is involved in the breakpoint in the Arrhenius plot and pressure dependence of (Na⁺ + K⁺)-ATPase. The p-nitrophenyl phosphatase activity of this enzyme also decreases with pressure. In this case the plot of the logarithm of the activity versus pressure is linear without a break-point. The temperature dependence for (Na⁺ + K⁺)-ATPase was also studied in the presence of fluidizing drugs: desipramine and benzylalcohol. The presence of these drugs had no effect on the inflection point in the Arrhenius plot.     

Dynamics of Docosahexaenoic Acid Metabolism in the Central Nervous System: Lack of Effect of Chronic Lithium Treatment

Using a method and model developed in our laboratory to quantitatively study brain phospholipid metabolism, in vivo rates of incorporation and turnover of docosahexaenoic acid in brain phospholipids were measured in awake rats. The results suggest that docosahexaenoate incorporation and turnover in brain phospholipids are more rapid than previously assumed and that this rapid turnover dilutes tracer specific activity in brain docoshexaenoyl-CoA pool due to release and recycling of unlabeled fatty acid from phospholipid metabolism. Fractional turnover rates for docosahexaenoate within phosphatidylinositol, choline glycerophospholipids, ethanolamine glycerophospholipids and phosphatidylserine were 17.7, 3.1, 1.2, and 0.2 %.h^–1, respectively. Chronic lithium treatment, at a brain level considered to be therapeutic in humans (0.6 mol.g^–1), had no effect on turnover of docosahexaenoic acid in individual brain phospholipids. Consistent with previous studies from our laboratory that chronic lithium decreased the turnover of arachidonic acid within brain phospholipids by up to 80% and attenuated brain phospholipase A₂ activity, the lack of effect of lithium on docosahexaenoate recycling and turnover suggests that a target for lithium's action is an arachidonic acid-selective phospholipase A₂.     

Thermodynamic analysis of the physical state of water during freezing in plant tissue, based on the temperature dependence of proton spin-spin relaxation

Multi-proton spin-echo images were collected from cold-acclimated winter wheat crowns (Triticum aestivum L.) cv. Cappelle Desprez at 400 MHz between 4 and ?4 °C. Water proton relaxation by the spin-spin (T2) mechanism from individual voxels in image slices was found to be mono-exponential. The temperature dependence of these relaxation rates was found to obey Arrhenius or absolute rate theory expressions relating temperature, activation energies and relaxation rates, Images whose contrast is proportional to the Arrhenius activation energy (E_a), Gibb's free energy of activation (ΔG^?), and the entropy of activation (ΔS^?) for water relaxation on a voxel basis were constructed by post-image processing. These new images exhibit contrast based on activation energies rather than rules of proton relaxation. The temperature dependence of water proton T₂ relaxation rates permits prediction of changes in the physical state of water in this tissue over modest temperature ranges. A simple model is proposed to predict the freezing temperature kof various tissue in wheat crowns. The average E_a and ΔH^? for water proton T₂ relaxation over the above temperature range in winter wheat tissue were ?6.4 ± 14.8 and ?8.6 ± 14.8kj mol^?1, respectively. This barrier is considerably lower than the E_a for proton translation in ice at 0°C, which is reported to be between 46.0 and 56.5 kj mol^?1     

Hepatic Circadian-Clock System Altered by Insulin Resistance,Diabetes and Insulin Sensitizer in Mice

Circadian rhythms are intrinsic rhythms that are coordinated with the rotation of the Earth and are also generated by a set of circadian-clock genes at the intracellular level. Growing evidence suggests a strong link between circadian rhythms and energy metabolism; however, the fundamental mechanisms remain unclear. In the present study, neonatal streptozotocin (STZ)-treated mice were used to model the molecular and physiological progress from insulin resistance to diabetes. Two-day-old male C57BL/6 mice received a single injection of STZ and were tested for non-obese, hyperglycemic and hyperinsulinemic conditions in the early stage, insulin resistance in the middle stage, and diabetes in the late stage. Gene expression levels of the hepatic circadian-clock system were examined by real-time quantitative PCR. Most of the components of the hepatic circadian-clock gene expression system, such as the mRNAs of Bmal1 (brain and muscle Arnt-like protein-1), Per2 (period 2) and Cry1 (cryptochrome 1), were elevated, and circadian patterns were retained in the early and middle stages of insulin-resistant conditions. The insulin sensitizer, rosiglitazone, returns the physiological and molecular changes associated with the diabetic phenotype to normal levels through peroxisome proliferator-activated receptor γ (PPARγ) rather than PPARα. Early and chronic treatment with rosiglitazone has been shown to be effective to counter the diabetic condition. Over time, this effect acts to attenuate the increased gene expression levels of the hepatic circadian-clock system and delay the severity of diabetic conditions. Together, these results support an essential role for the hepatic circadian-clock system in the coordinated regulation and/or response of metabolic pathways.     

Temperature Dependence of the Sodium Pump is Altered in the Cerebral Cortex of CCK<Subscript>2</Subscript> Receptor-Deficient Mice

Previously we have shown that the temperature dependence of the sodium pump (Na⁺,K⁺-ATPase) is altered under different neuropathological conditions. In this study we compared temperature dependence of the Na⁺,K⁺-ATPase in the fronto-parietal cortex of CCK₂ receptor-deficient (homo- and heterozygous) and normal (wild-type) mice. The Arrhenius plot for Na⁺,K⁺-ATPase from wild-type brain is non-linear with a breakpoint at 20.3 ± 0.4°C. In case of the brain cell membrane of CCK₂ receptor-deficient mice (homo- and heterozygous) the breakpoint on Arrhenius plot was detected at 26.0 ± 1.1°C and 25.4 ± 0.4°C, respectively. The shift of the breakpoint on the Arrhenius plot established in CCK₂ receptor-deficiency as well as in case of some other pathological conditions confirms that such kind of alteration in the Na⁺,K⁺-ATPase temperature dependence is likely related to the homeostatic adjustment of altered function of the sodium pump.     

The bilayer enhances rhodopsin kinetic stability in bovine rod outer segment disk membranes

Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (T_m) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the T_m as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (E_act) was calculated from the scan rate dependence of the T_m and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The E_act as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the E_act to rhodopsin denaturation.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.     

Phospholipase C digestion of rat liver plasma membrane stimulates adenylate cyclase

Brief treatment of rat liver plasma membranes with phospholipase C of Clostridium welchii increased both the ratio of saturated to unsaturated fatty acids and the ratio of cholesterol to phospholipids. Using 5-doxylstearic acid spin probes two breaks at 29 and 19.6 °C could be observed in the order parameter, S_A, vs temperature curve for untreated membranes. Upon phospholipase C digestion the lower phase transition temperature was shifted to 23 °C, while the higher phase transition temperature could not be detected up to 40 °C. The order parameter, S_A, was consistently higher at all temperatures in the phospholipase C-treated membranes. As phospholipase C is known to attack the outer lamella, these results can be interpreted as indicating an increase in ordering (i.e., decrease in fluidity) of the outer membrane lamella. On the other hand, an increase in basal activity of adenylate cyclase of the treated membranes was observed with an apparent reduction of the activation energies both below and above the break (at 20 °C) in the Arrhenius plot of enzyme activity. Phospholipase C treatment did not affect the temperature of the break in Arrhenius kinetics of the enzyme. The results are discussed in terms of the role of the ordering state of membrane lipids in adenylate cyclase activity.