Effects of Berberineon Cardiac Function and ATPase Activity of Postischemic Myocardial Stunned Rat Hearts

This is a report of a study on the protective effect of berberine(Ber) on postischemic myocardial stunning and the role it plays in ATPase activity. Isolated working rat hearts were used with global ischemia for 30 min followed by reperfusing for 40 min. Both systolic and diastolic functions of stunned myocardium were significantly decreased. The recovery of LVSP×HR and CO was 52%±8% and 40%±8% respectively; LVEDP and T were elevated; while both Na +, K +ATPase activity and Ca 2+ , Mg 2+ ATPase activity of myocardial membrane and mitochondria were depressed. Berberine(25 mg·kg -1 ·d -1 , ip, 3 d, and 10 μmol L -1 for isolated heart perfusion) was able to enhance the percent recovery of LVSP×HR and CO to 85%±12% and 75%±11%, respectively, and reduce LVEDP from 298%±64% to 166%±44%, with an improvement in myocardial membrane Na +, K +ATPase activity and mitochondria Ca 2+ , Mg 2+ ATPase activity. This study suggested that berberine can protect cardiac function from ischemia reperfusion stunning injury by preserving ATPase activity in ischemic myocardium.

C0818,a novel curcumin derivative,interacts with Hsp90 and inhibits Hsp90 ATPase activity

The aims of the present study were to estimate the affinity between 3,5-(E)-bis(3-methoxy-4-hydroxybenzal)-4-piperidinone hydrochloride(C0818) and heat shock protein 90 (Hsp90) and to investigate the inhibitory effects of this compound on Hsp90 ATPase activity. Fluorescence spectroscopy was used to examine the affinity between varying concentrations of C0818 and Hsp90, N-Hsp90, MHsp90 and C-Hsp90. Fluorescence intensities were recorded in the range of 290–510 nm at 293, 303 and 310 K, respectively. A colorimetric assay for inorganic phosphate(based on the formation of a phosphomolybdate complex and the subsequent reaction with malachite green) were used to examine the inhibitory effects of C0818 on Hsp90 ATPase activity. The equilibrium dissociation constant K_D value of C0818 was found to be 23.41270.943 μmol/L. The interaction between C0818 and Hsp90 was driven mainly by electrostatic interactions. C0818 showed the strongest affinity with C-Hsp90. These results conclusively demonstrate the inhibitory activity of C0818 on the activity of Hsp90 ATPase.

An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method, using a known myosin Ⅱ ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate(ATP) and calcium chloride, p H, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L^(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.

Na^+-K^+-ATPase activity of hepatocyte plasma membranes,reversible and irreversible alterations due to bile acids

Background Chenodeoxycholic acid (CDC) is an apolar bile salt and damages hepatocytes, whereas ursodeoxycholic acid (UDC) is a more polar bile salt and protects liver cell against toxic bile salts. We therefore investigated the activity of membrane associated Na + K + ATPase, a coenzyme of the sodium dependent bile salt carrier. Methods Liver plasma membranes (LPM) were isolated from rat livers according to the method of Song et al (J Cell Biol 1969; 41:124). The LPM were incubated with bile salts (TUDC, UDC, TCDC, CDC) in concentrations of 0.1 2 mmol/L for 0 30 minutes at room temperature. To study reversibility of the effect of CDC, LPM were diluted with buffer 50 times of volume after incubation. The activity of membrane associated Na + K+ ATPase was determined enzymatically at 37℃ and the phospholipid (PL) release into the supernatant was measured. Results CDC and TCDC both showed a dose dependent inhibition of the enzyme activity (P<0.01 vs control). Gastroenterology, Center of Internal Medicine, University Hospital, D 60590 Frankfurt Main, Germany (You T, Guldutuna S, Bhatti S and Leuschner U)Initially TCDC induced an increase in enzyme activity at concentrations of 0.1 1 mmol/L, however, after 3 minutes activity repidly decreased to less than 30% of controls. Up to a concentration of 1 mmol/L CDC the inhibition of enzyme activity could be reversed by diluting the bile salt in the incubation medium. At a concentration of 2 mmol/L CDC activity was only partially restored and at this concentration a marked PL release into the supernatant was observed, indicating solubilization of the membranes. UDC did not decrease the enzyme activity at concentrations of 0.1 2 mmol/L. At a concentration of 2 mmol/L TUDC inhibited the Na + K + ATPase by about 20%. Solubilization of membrane PL was not observed. Conclusion CDC and TCDC inhibited Na + K + ATPase. Dilution of the bile salt with buffer reversed the inhibitory effect up to concentrations of 1 mmol/L CDC. The inhibitory effect is probably due to alteration of the plasma membrane. 2 mM CDC caused irreparable membrane damage. Physiological concentrations of UDC and TUDC did not affect membrane ATPase.

Kinetics of the H＋-ATPase from Dry and 5-Hours- Imbibed Maize Embryos in Its Native, Solubilized, and Reconstituted Forms

Membranes undergo recovery upon rehydration in seed germination. Previous work has described that the plasma membrane H＋-ATPase from maize embryos adopts two different forms at 0 and 5 h of imbibition. We investigated how the kinetics of these two forms could be affected by alterations in the plasma membrane （PM）. In comparison to the O-h, PMs from the 5-h imbibed embryos showed changes in glycerophospholipid composition, decrease in leakage, and increase in fluidity. Kinetics of the PM H^-ATPase from 0 and 5-h imbibed embryos showed negative cooperativity, With the removal of the membrane environment, the activity of the enzymes shifted to a more complex kinetics, displaying two enzyme components. Lipid reconstitution produced one component with positive cooperativity. In all cases, enzymes from 0 and 5-h imbibed embryos presented similar kinetics with some quantitative differences. These results indicate that the two enzyme forms have the potential ability to respond to changes in the membrane enyiror^rpent, but the fact that they do not show differences in the native membranes at 0 or 5 h implies that modifications in the membrane are not drastic enough to alter their kinetics, or that they are able to preserve their boundary lipids or associated proteins and thus retain the same kinetic behavior.

Expression and characterization of a new valosin-containing protein from silkworm

Valosin-containing protein （VCP） is a type-II adenosine triphosphatase （ATPase） wih extensive biological function in organisms. Silkworm is the second in- sect model for genetic studies and a bioreactor for proteinaceous drugs and biomaterials. In this paper, a new VCP-like gene was amplified from the fat body of silkworm follow- ing genome prediction and spliced expressed sequence tag sequences, using both reverse transcription polymerase chain reaction （RT-PCR） and 3＇-RACE （rapid amplification of complementary DNA ends） methods. Bioinformatical analysis showed that the translated amino acid sequence contained a highly conserved domain of VCPs similar to that of many insects. This domain consists of the conserved structure motifs of the ATP binding site and the catalytical center, which is closely related to the insect VCPs in a phylogenetic tree. The silkworm VCP-like gene was successfully inserted into the plasmid and transformed into Escherichia coli cells to express VCP-like protein with ATPase activity. The expression of silkworm VCP-like protein was also confirmed by Western blotting and mass spectromet- ric analyses. Distribution of the VCP-like gene in various tissues of the silkworm was also studied by real-time PCR. Results showed that the messenger RNA （mRNA） of VCP-like protein is widely expressed in fat body, reproductive organs （testis or ovary）, silk gland, head, Malpighian tubule, epidermis and midgut. Among them, fat body has the highest mRNA expression level of the VCP-like gene, while the midgut has the lowest expression level. This study provides groundwork for further study on the structure and function of the new VCP-like protein.

Cu(Ⅱ), Fe(Ⅲ) and Mn(Ⅱ) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron（Fe（Ⅲ））, manganese（Mn（Ⅱ）） and copper（Cu（Ⅱ）） ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu2＋–Fe3＋combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atp D mutant（with defective FoF1-ATPase） MS116. Addition of Mn2＋–Fe3＋combinations within the same concentration range had no effects on growth compared to control（without heavy metals）. ATPase activity was increased in the presence of Mn2＋–Fe3＋, while together with0.2 mmol/L N,N′-dicyclohexylcarbodiimide（DCCD）, ATPase activity was decreased compared to control（when only 0.2 mmol/L DCCD was present）. These results indicate that heavy metals ion combinations probably affect the FOF1-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential.The effects observed when Fe3＋was added separately disappeared in both cases, which might be a result of competing processes between Fe3＋and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria,and could be applied for regulation of stress response patterns in the environment.