Creatine kinase and ATPase in human seminal fluid and prostatic fluid

A creatine kinase assay based on estimation of creatine liberated from creatine phosphate was accurate and reproducible for use with seminal or prostatic fluid, after allowance was made for acid phosphatase interference. Comparison of this method with one which relies on enzymic coupling of ATP formation to NADP+ oxidation shows that the latter under-estimates creatine kinase activity by a factor of about 3. This discrepancy could be due to the high ATPase activity found in prostatic and seminal fluid. Uncritical use of the NADP+ assay might account for different seminal creatine kinase values reported in the literature. Interrelationships between ATPase, creatine kinase and zinc suggest that seminal ATPase is a prostatic secretory product while creatine kinase may be multiglandular in origin.     

Removal of protein interference in the Fiske-Subbarow method by sodium dodecyl sulfate.

In kinetic studies on erythrocyte membrane ATPase, the activity is assayed by following the release of orthophosphate from ATP by the Fiske-SubbaRow method. The deproteinization of the samples, which is essential before assay by this method, is an obstacle to the quick and efficient assay of large numbers of samples. Nonionic detergents, e.g., Lubrol, which are used in the purification of Na⁺-K⁺-ATPase from plasma membranes, also interfere strongly with the Fiske-SubbaRow method.It was found that the addition of sodium dodecyl sulfate to the samples before the orthophosphate assay solubilized much of the membrane protein, allowing orthophosphate to be determined by the Fiske-SubbaRow method without the deproteinization step. Sodium dodecyl sulfate and the solubilized proteins did not interfere with the determination. The addition of sodium dodecyl sulfate was also found to abolish the interference of nonionic detergents with the Fiske-SubbaRow determination. As a result, the assay of erythrocyte membrane ATPase could be greatly simplified.     

A simplified method for inorganic phosphate determination and its application for phosphate analysis in enzyme assays

A simplified method for inorganic phosphate determination has been developed. The method is sensitive, easy, economic, and applicable for estimation of phosphate released in both enzymatic and nonenzymatic reactions. A mixture of hydrazine sulfate and ascorbic acid was used as the reducing agent and the conditions for the development of the molybdenum blue color were optimized. Thus in the 4.0 ml assay system, 0.4 ml of the reducing agent solution containing 20 mg each of hydrazine sulfate and ascorbic acid per milliliter of 1.0 N H2SO4 gave a rapid optimum color development with absorption maximum at 820 nm. Color development showed a linear relationship up to 10 microg Pi concentration. Thus the method has a 2.5x higher range of Pi estimation than that of the Bartlett method. The molar extinction coefficient at 820 nm was higher than that obtained in the Bartlett procedure. Also the molybdenum blue color formed was stable up to 24 h. Under the standard assay conditions, interference from acid-labile phosphate as in the case of Na+,K+ ATPase was at the minimum. The applicability of the method for assay of microsomal Na+,K+ ATPase and glucose-6-phosphatase was checked in microassays (final volume 0.1 ml) in comparison to the conventional procedures which use 3-4 times higher volumes. Likewise the applicability of the method for phospholipid analysis was compared with that of the conventional Bartlett method. Under both test systems the results obtained by the micromethod were identical to those obtained by the conventional methods. In general the method, which rapidly produces quantitatively molybdenum blue color, not only is rapid economical, and convenient but also has wide applicability.     

Streptococcus pneumononiae gyrase ATPase: development and validation of an assay for inhibitor discovery and characterization

The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors.     

The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity.

The purpose of this study was to examine the effects of lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpio L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10 degrees C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca(2+)-activated ATPase activity was assessed by an ATP regenerating assay that coupled the myofibrillar ATPase to pyruvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85%. Lactate had no effect on myofibrillar ATPase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not to the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

利用荧光素酶生物发光体系测定Mg~(2+)-ATP酶结合和水解活性

本文应用LKB公司的ATP测液建立了Mg~(2 )-ATP酶的ATP结合及水解活性的测定方法;利用国产荧光素酶粗品在连串反应体系中建立测定Mg~(2 )-ATP酶结合活性的方法,并与水解活性相比较.对Mg~(2 )-ATP酶的去脂样品,Mg~(2 )-ATP酶与卵磷脂复合物以及微粒体样所做的测定表明,上述两种方法是可靠、简便的,尤其是利用国产荧光素酶粗品建立的ATP结合活性的测定方法,能避免水解对结合活性测定的干扰,刘其它的酶-底物的结合研究有参考价值.     

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.     

Bioluminometric method for real-time detection of ATPase activity

We have developed a real-time, simple, and sensitive method for the detection of ATP hydrolysis activity (ATPase) of apyrase (EC 3.6.1.5). The assay is based on the continuous monitoring of the ATP hydrolysis reaction using the firefly luciferase system. The method is sensitive and yields linear responses between 0.7 and 70 mU for the Solanum tuberosum apyrase. The detection limit was found to be 0.7 mU apyrase. We used the method to study the inhibitory effects of various compounds on the ATPase activity of potato apyrase, measured with 500 nM ATP. The concentrations of azide, AMP, Pi, fluoride, and ADP, which inhibit the ATPase activity by 50% (IC50), were found to be approximately 100, 0.25, 0.125, 0.04, and 0.035 mM, respectively. Under our assay conditions, vanadate inhibited about 98% of the ATPase activity of the potato apyrase at a concentration of 250 microM. The possibility of using the new method for other applications is discussed.     

An improved assay of inorganic phosphate in the presence of extralabile phosphate compounds: application to the ATPase assay in the presence of phosphocreatine.

Two methods of determining inorganic phosphate in the presence of labile phosphate compounds using the catalyst polyvinylpyrrolidone (PVP) are described. The first method provides a simple assay of ATPase activity at room temperature without deproteinization. The second method minimizes the decomposition of phosphocreatine during the assay to 1%. An application to the measurement of myofibrillar ATPase in the presence of phosphocreatine is given, as well as the advantages of the methods.     

Demonstration of an ATPase at the cell surface of intact normal and neoplastic human cells in culture

An ATPase reaction has been studied at the surface of intact normal and neoplastic human cells in culture. For this purpose a sensitive method has been developed which permits repeated monitoring of enzyme activity without interference with cellular viability. Experiments could be performed with the cells cultured in a single dish. The cells were firmly attached to the supporting medium throughout the experiment. The incubation medium could easily be separated from the cells at the end of the reaction. There was no diffusion of the surface-located ATPase into the incubation medium. Another advantage was the possibility of microscopic control of the appearance of the cells during the reaction. High ATPase activity was found in glia-like cells derived from normal adult brain cells while lines from gliomas had very low activity. One SV40 transformed glia line had an extremely low ATPase activity in contrast to the uninfected cells. Normal fibroblasts and sarcoma cells had about the same low activity as the glioma cells.     

An automated assay for Na-K ATPase kinetics

We have developed an automated assay for the Na and K activated ATPase which has been used to determine the enzyme activity in a sample of unknown enzymatic activity or the dependence of the initial rate of reaction on ligand concentration where identical samples of enzyme are used. The interference of nucleotides on the color development of the phosphomolybdate complex has been eliminated by the addition of MgCl₂ to the acid molyb-date solution. Ways of handling the microsomal Na and K stimulated ATPase have been found which insure the stability of the enzyme and facilitate washing through autoanalyzer tubing. Finally, a modification of normal autoanalyzer procedures permits kinetic analysis to be carried out in an automated fashion.     

Measurement of sarcoplasmic reticulum Ca2+ ATPase activity using high-performance liquid chromatography

The goal of this investigation was to develop an assay whereby we could measure changes in ATP, ADP, and phosphocreatine (PCr) during stimulation of the sarcoplasmic reticulum (SR) Ca2+ ATPase. After stopping the enzyme reaction, compounds were extracted by perchloric acid and separated by reversed-phase high-performance liquid chromatography (HPLC). Absorbance of ATP and ADP was monitored at 260 nm, and detection of PCr was done at 205 nm. Chromatograms show that peaks associated with each compound are clearly separated and easily detected. The SR Ca2+ ATPase assay was run for various time periods and using varying free [Ca2+]. The changes in ATP and ADP contents were linear with increasing time and varied as expected with increasing free [Ca2+]. The ATPase activities determined using changes in ATP and ADP were nearly identical to those determined using previously established assays. When PCr was added to the assay, we were able to confirm that the Ca2+ ATPase uses ATP that is synthesized locally from PCr via creatine kinase (CK). The results indicate that this is a valid and reliable method for examining SR Ca2+ ATPase activity and for investigating its interaction with CK.     

The alkaline adenosine triphosphatase activity of 30S dynein after modification of the SH groups. Possible involvement of some of the most reactive SH groups.

An apparent 'triphasic' alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and susequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively. The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 microM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mM largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again. This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 microM NEM was not stable under the assay conditions (pH 10-10.2 at 25 degrees C), whereas modification with 0.3 mM NEM stabilized 30S dynein against the assay conditions. The possible significance of the 10 microM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.     

A comparison of hydraulic and laser capture microdissection methods for collection of single B cells,PCR, and sequencing of antibody VDJ

A new procedure for measuring ATPase activity in which gamma-(33)P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.     

Characterization studies on the membrane-bound adenosine triphosphatase (ATPase) of Azotobacter vinelandii.

The adenosinetriphosphatase (ATPase) (EC 3.6.1.3) activity in Azotobacter vinelandii concentrates in the membranous R3 fraction that is directly associated with Azotobacter electron transport function. Sonically disrupted Azotobacter cells were examined for distribution of ATPase activity and the highest specific activity (and activity units) was consistently found in the particulate R3 membranous fraction which sediments on ultracentrifugation at 144 000 X g for 2 h. When the sonication time interval was increased, the membrane-bound ATPase activity could neither be solubilized nor released into the supernatant fraction. Optimal ATPase activty occurred at pH 8.0; Mg2+ ion when added to the assay was stimulatory. Maximal activity always occurred when the Mg2+:ATP stoichiometry was 1:1 on a molar ratio at the 5 mM concentration level. Sodium and potassium ions had no stimulatory effect. The reaction kinetics were linear for the time intervals studied (0-60 min). The membrane-bound ATPase in the R3 fraction was stimulated 12-fold by treatment wiTH TRypsin, and fractionation studies showed that trypsin treatment did not solubilize ATPase activity off the membranous R3 electron transport fraction. The ATPase was not cold labile and the temperature during the preparation of the R3 fraction had no effect on activity; overnight refrigeration at 4 degrees C, however, resulted in a 25% loss of activity as compared with a 14% loss when the R3 fraction was stored overnight at 25 degrees C. A marked inactivation (although variable, usually about 60%) did occur by overnight freezing (-20 degrees C), and subsequent sonication failed to restore ATPase activity. This indicates that membrane reaggregation (by freezing) was not responsible for ATPase inactivation. The addition of azide, ouabain, 2,4-dinitrophenol, or oligomycin to the assay system resulted in neither inhibition nor stimulation of the ATPase activity. The property of trypsin activation and that ATPase activity is highest in the R3 electron transport fraction suggests that its probable functional role is in coupling of electron transport to oxidative phosphorylation.     

A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein

P-glycoprotein (P-gp) is a multidrug transporter responsible for resistance to anticancer chemotherapy and physiologically involved in absorption, distribution, and excretion of a large number of hydrophobic xenobiotics. P-gp exhibits both an ATPase activity correlated with its drug transport function and a basal ATPase activity in the absence of any drug. We have developed a high-throughput screening test to detect specific interactions between drugs and P-gp. We took into account the existence of multiple binding sites on P-gp to propose and validate an optimized strategy, based on the modulation of the basal ATPase activity of P-gp and of the ATPase activity stimulated by three reference substrates (verapamil, vinblastine, and progesterone). The ATPase activity measurements were performed on P-gp-containing membrane vesicles from actinomycin-D-selected hamster DC-3F lung fibroblasts by a spectrophotometric method based on continuous monitoring of ADP formation, regenerated in ATP by a coupled enzyme system. This assay may be performed on 96- or 384-well microtiter plates. When applying this ATPase assay to 41 compounds known from the literature for their interaction with P-gp, 95% of them were found to be positive, whereas only 78% were positive when considering solely the modulation of the basal activity.     

Fluorometric determination of phosphatidylcholine as a measure of phospholipid methylation.

The successive methylation of phosphatidylethanolamine to phosphatidylcholine (phospholipid methylation) has been measured by the incorporation of S-[methyl-3H]adenosylmethionine or colorimetric assay of phosphatidylcholine extracted from adipocyte plasma membranes. A fluorometric assay for phosphatidylcholine was developed to measure phospholipid methylation. This assay is 10 times more sensitive than the colorimetric assay and demonstrates no significant interference with other methylated phospholipids. The fluorometric assay was used to determine a biphasic insulin dose response in adipocyte plasma membranes. This fluorometric assay for phosphatidylcholine represents an alternative method for monitoring phospholipid methylation, especially when increased sensitivity is required.     

A modified colorimetric method for the determination of orthophosphate in the presence of high ATP concentrations.

We describe a modified colorimetric method that quantitates inorganic phosphate linearly up to 60 nmol, with high stability of the developed color and with a low interference by ATP concentration (up to 30 mM). This method is very suitable for use in ATPase enzymatic assays, especially with enzymes that have low specific activities and (or) high Km values for ATP.     

Development of a high-throughput screening assay for the discovery of small-molecule SecA inhibitors

A major pathway for bacterial preprotein translocation is provided by the Sec-dependent preprotein translocation pathway. Proteins destined for Sec-dependent translocation are synthesized as preproteins with an N-terminal signal peptide, which targets them to the SecYEG translocase channel. The driving force for the translocation reaction is provided by the peripheral membrane ATPase SecA, which couples the hydrolysis of ATP to the stepwise transport of unfolded preproteins across the bacterial membrane. Since SecA is essential, highly conserved among bacterial species, and has no close human homologues, it represents a promising target for antibacterial chemotherapy. However, high-throughput screening (HTS) campaigns to identify SecA inhibitors are hampered by the low intrinsic ATPase activity of SecA and the requirement of hydrophobic membranes for measuring the membrane or translocation ATPase activity of SecA. To address this issue, we have developed a colorimetric high-throughput screening assay in a 384-well format, employing an Escherichia coli (E. coli) SecA mutant with elevated intrinsic ATPase activity. The assay was applied for screening of a chemical library consisting of ∼27,000 compounds and proved to be highly reliable (average Z′ factor of 0.89). In conclusion, a robust HTS assay has been established that will facilitate the search for novel SecA inhibitors.     

Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase

The continuous bioluminescent assay of ATP has been adapted to the study of Mg²⁺-dependent ATPases, including the (Na⁺,K⁺) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg²⁺-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.