Spectrophotometric Enzymatic Assay Research Articles

Thioalbamide inhibits FoF1-ATPase in breast cancer cells and reduces tumor proliferation and invasiveness in breast cancer in vivo models

ObjectiveThioalbamide is a ribosomally synthesized and post-translationally modified peptide (RiPP) belonging to the family of thioamitides, a rare class of microbial specialized metabolites with unusual post-translational modifications and promising biological activities. Recent studies have demonstrated the ability of thioalbamide to exert highly selective cytotoxic effects on tumor cells by affecting their energy metabolism, thus causing abnormal ROS production and triggering apoptosis. This study is aimed to investigate the molecular mechanisms underlying the antitumor activity of thioalbamide in order to identify its exact molecular target. MethodsWild type MCF-7 and MDA-MB-231 breast cancer cell lines as well as cancer cells deprived of mitochondrial DNA (ρ0 cells) were employed in order to assess thioalbamide effects on tumor bioenergetics. In this regard, metabolic profile was evaluated by a Seahorse XFe96 analyzer, and the activity of the enzyme complexes involved in oxidative phosphorylation was quantified by spectrophotometric assays. Thioalbamide effects on tumor invasiveness were assessed by gelatin zymography experiments and invasion assays. In vivo experiments were carried out on breast cancer xenograft and “experimental metastasis” mouse models. ResultsExperiments carried out on ρ0 breast cancer cells, together with Seahorse analysis and the application of spectrophotometric enzymatic assays, highlighted the ability of thioalbamide to affect the mitochondrial respiration process, and allowed to propose the FoF1-ATPase complex as its main molecular target in breast cancer cells. Additionally, thioalbamide-mediated OXPHOS inhibition was shown, for the first time, to reduce tumor invasiveness by inhibiting metalloproteinase-9 secretion. Furthermore, this study has confirmed the antitumor potential of thioalbamide in two different in vivo models. In particular, experiments on MCF-7 and MDA-MB-231 xenograft mouse models have confirmed in vivo its high anti-proliferative and pro-apoptotic activity, while experiments on MDA-MB-231 ″experimental metastasis” mouse models have highlighted its ability to inhibit breast cancer cell invasiveness. ConclusionsOverall, our results shed more light on the molecular mechanisms underlying the pharmacological potential of thioamidated peptides, thus reducing the gap that separates this rare class of microbial metabolites from clinical studies, which could validate them as effective tools for cancer treatment.

Alpha-Ketoglutarate: A Potential Inner Mitochondrial and Cytosolic Protector against Peroxynitrite and Peroxynitrite-Induced Nitration?

The generation of peroxynitrite (ONOO−) is associated with several diseases, including atherosclerosis, hypertension, neurodegeneration, cancer, inflammation, and sepsis. Alpha-ketoglutarate (αKG) is a known potential highly antioxidative agent for radical oxidative species such as peroxides. The question arises as to whether αKG is also a potential scavenger of ONOO− and a potential protector against ONOO−-mediated nitration of proteins. NMR studies of 1 mM αKG in 100 mM phosphate-buffered saline at pH 7.4 and pH 6.0 were carried out in the presence or absence of a final concentration of 2 mM ONOO−. An ONOO−–luminol-induced chemiluminescence reaction was used to measure the scavenging function of several concentrations of αKG; quantification of αKG was performed via spectrophotometric enzymatic assay of αKG in the absence or presence of 0, 1, or 2 mM ONOO−. The nitration of tyrosine residues on proteins was measured on ONOO−-treated bovine serum albumin (BSA) in the presence or absence of 0–24 mM αKG by an ELISA technique using a specific anti-IgG against nitro-tyrosine. The addition of ONOO− to αKG led to the formation of succinic acid and nitrite at pH 7.0, but not at pH 6.0, as αKG was stable against ONOO−. The absorbance of enzymatically estimated αKG at the time point of 30 min was significantly lower in favour of ONOO− (1 mM: 0.21 ± 0.03, 2 mM: 0.12 ± 0.05 vs. 0 mM: 0.32 ± 0.02; p < 0.001). The luminol technique showed an inverse logarithmic correlation of the ONOO− and αKG concentrations (y = −2 × 105 ln(x) + 1 × 106; r2 = 0.99). The usage of 4 mM αKG showed a significant reduction by nearly half in the chemiluminescence signal (284,456 ± 29,293 cps, p < 0.001) compared to the control (474,401 ± 18,259); for 20 and 200 mM αKG, there were further reductions to 163,546 ± 26,196 cps (p < 0.001) and 12,658 ± 1928 cps (p < 0.001). Nitrated tyrosine residues were estimated using the ELISA technique. A negative linear correlation was obtained by estimating nitrated tyrosine residues in the presence of αKG (r2 = 0.94): a reduction by half of nitrated tyrosine was estimated using 12 mM αKG compared to the control (326.1 ± 39.6 nmol vs. 844.5 ± 128.4 nmol; p < 0.001).

Intra-Laboratory Validation of Alpha-Galactosidase Activity Measurement in Dietary Supplements.

Introduction: Alpha-galactosidase (α-Gal) is an enzyme responsible for the hydrolyzation of glycolipids and glycoprotein commonly found in dietary sources. More than 20% of the general population suffers from abdominal pain or discomfort caused by intestinal gas and by indigested or partially digested food residuals. Therefore, α-Gal is used in dietary supplements to reduce intestinal gases and help complex food digestion. Marketed enzyme-containing dietary supplements must be produced in accordance with the Food and Drug Administration (FDA) regulations for Current Good Manufacturing Practice (cGMPs). Aim: in this work we illustrated the process used to develop and validate a spectrophotometric enzymatic assay for α-Gal activity quantification in dietary supplements. Methods: The validation workflow included an initial statistical-phase optimization of materials, reagents, and conditions, and subsequently a comparative study with another fluorimetric assay. A final validation of method performance in terms of specificity, linearity, accuracy, intermediate-precision repeatability, and system precision was then executed. Results and conclusions: The proven method achieved good performance in the quantitative determination of α-Gal activity in commercial food supplements in accordance with the International Council for Harmonisation of Technical Requirements for Pharmaceuticals (ICH) guidelines and is suitable as a rapid in-house quality control test.

Skeletal muscle maximal mitochondrial activity in ambulatory children with cerebral palsy.

To compare skeletal muscle mitochondrial enzyme activity and mitochondrial content between independently ambulatory children with cerebral palsy (CP) and typically developing children. Gracilis biopsies were obtained from 12 children during surgery (n=6/group, children with CP: one female, five males, mean age 13y 4mo, SD 5y 1mo, 4y 1mo-17y 10mo; typically developing children: three females, three males, mean age 16y 5mo, SD 1y 4mo, 14y 6mo-18y 2mo). Spectrophotometric enzymatic assays were used to evaluate the activity of mitochondrial electron transport chain complexes. Mitochondrial content was evaluated using citrate synthase assay, mitochondrial DNA copy number, and immunoblots for specific respiratory chain proteins. Maximal enzyme activity was significantly (50-80%) lower in children with CP versus typically developing children, for complex I (11nmol/min/mg protein, standard error of the mean [SEM] 1.7 vs 20.7nmol/min/mg protein, SEM 4), complex II (6.9nmol/min/mg protein, SEM 1.2 vs 21nmol/min/mg protein, SEM 2.7), complex III (31.9nmol/min/mg protein, SEM 7.4 vs 72.7nmol/min/mg protein, SEM 7.2), and complex I+III (7.4nmol/min/mg protein, SEM 2.5 vs 31.8nmol/min/mg protein, SEM 9.3). Decreased electron transport chain activity was not the result of lower mitochondrial content. Skeletal muscle mitochondrial electron transport chain enzymatic activity but not mitochondrial content is reduced in independently ambulatory children with CP. Decreased mitochondrial oxidative capacity might explain reported increased energetics of movement and fatigue in ambulatory children with CP. What this paper adds Skeletal muscle mitochondrial electron transport chain enzymatic activity is reduced in independently ambulatory children with cerebral palsy (CP). Mitochondrial content appears to be similar between children with CP and typically developing children.

Protein engineering of cellobiose dehydrogenase from Phanerochaete chrysosporium in yeast Saccharomyces cerevisiae InvSc1 for increased activity and stability

Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyper-glycosylation and the best S137 N mutant showed 2.2 times increased kcat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137 N mutant retained 30% of it’s original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM lactose compared to the wild type.

Comparative enzymatic studies using ion-selective electrodes. The case of cholinesterases

The application of traditional ion-selective electrodes for comparative enzymatic analysis was demonstrated for the first time in this study. A kinetic-potentiometric method based on the monitoring of the concentration of the ionic substrate involved in the enzymatic reaction catalyzed by different cholinesterases is used for this purpose. A comparative study was performed comprising both enzymatic assays using different ionic substrates and the corresponding inhibited reactions in presence of neostigmine (a synthetic anticholinesterase). The developed approach is used to obtain valuable comparative results through calculation of kinetic parameters, such as Michaelis and inhibition constants. Interesting results were obtained for acetylcholinesterase and butyrylcholinesterase enzymes, which were selected as proof-of-concept: (i) the binding affinity that these enzymes have for their natural substrates showed to be higher (acetylcholine and butyrylcholine respectively) than for their corresponding thiol derivatives (acetylthiocholine and butyrylthiocholine), which are traditionally used in spectrophotometric enzymatic assays; (ii) as expected, the maximum hydrolysis rate found in the assays of each enzyme was independent of the substrate used; (iii) acetylcholinesterase enzyme inhibition due to neostigmine was found to be higher (higher inhibition constant). Advantageously, the use of ion-selective electrodes permits to perform cholinesterases’ enzymatic assays using their natural substrates and under physiological conditions, unlike the traditional spectrophotometric methods used in routine enzymatic assays. Importantly, while well-known enzymes are use throughout this work, this approach can be extended to other types of enzymatic assays as a tangible alternative to traditional spectrophotometric methods.

Aqueous Extract of Nypa fruticans Wurmb. Vinegar Alleviates Postprandial Hyperglycemia in Normoglycemic Rats.

Nypa fruticans Wurmb. vinegar, commonly known as nipa palm vinegar (NPV) has been used as a folklore medicine among the Malay community to treat diabetes. Early work has shown that aqueous extract (AE) of NPV exerts a potent antihyperglycemic effect. Thus, this study is conducted to evaluate the effect of AE on postprandial hyperglycemia in an attempt to understand its mechanism of antidiabetic action. AE were tested via in vitro intestinal glucose absorption, in vivo carbohydrate tolerance tests and spectrophotometric enzyme inhibition assays. One mg/mL of AE showed a comparable outcome to the use of phloridzin (1 mM) in vitro as it delayed glucose absorption through isolated rat jejunum more effectively than acarbose (1 mg/mL). Further in vivo confirmatory tests showed AE (500 mg/kg) to cause a significant suppression in postprandial hyperglycemia 30 min following respective glucose (2 g/kg), sucrose (4 g/kg) and starch (3 g/kg) loadings in normal rats, compared to the control group. Conversely, in spectrophotometric enzymatic assays, AE showed rather a weak inhibitory activity against both α-glucosidase and α-amylase when compared with acarbose. The findings suggested that NPV exerts its anti-diabetic effect by delaying carbohydrate absorption from the small intestine through selective inhibition of intestinal glucose transporters, therefore suppressing postprandial hyperglycemia.

Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry

Isothermal titration calorimetry (ITC) was applied to determine enzymatic activity and inhibition. We measured the Michaelis–Menten kinetics for trypsin-catalyzed hydrolysis of two substrates, casein (an insoluble macromolecule substrate) and Nα-benzoyl-dl-arginine β-naphthylamide (a small substrate), and estimated the thermodynamic parameters in the temperature range from 20 to 37°C. The inhibitory activities of reversible (small molecule benzamidine) and irreversible (small molecule phenylmethanesulfonyl fluoride and macromolecule α1-antitrypsin) inhibitors of trypsin were also determined. We showed the usefulness of ITC for fast and direct measurement of inhibition constants and half-maximal inhibitory concentrations and for predictions of the mechanism of inhibition. ITC kinetic assays could be an easy and straightforward way to estimate Michaelis–Menten constants and the effectiveness of inhibitors as well as to predict the inhibition mechanism. ITC efficiency was found to be similar to that of classical spectrophotometric enzymatic assays.

Bile Acid Aspiration in Suspected Ventilator-Associated Pneumonia

The aims of this study were to measure the levels of bile acids in patients with suspected ventilator-associated pneumonia (VAP) and provide a possible pathway for neutrophilic inflammation to explain its proinflammatory effect on the airway. Bile acid levels were measured by spectrophotometric enzymatic assay, and liquid chromatography mass spectrometry was used to quantify the major bile acids. Alveolar cells were grown on modified air-liquid interface culture inserts, and bile acids were then employed to stimulate the cells. Reverse transcriptase polymerase chain reaction and Western blots were used to determine the involved gene expression and protein levels. The mean (+/- SE) concentration of total bile acids in tracheal aspirates was 6.2 +/- 2.1 and 1.1 +/- 0.4 mumol/L/g sputum, respectively, for patients with and without VAP (p < 0.05). The interleukin (IL)-8 level was significantly higher in the VAP group (p < 0.05). The major bile acid, chenodeoxycholic acid, stimulated alveolar epithelial cells to increase IL-8 production at both the messenger RNA and protein level through p38 and c-Jun N-terminal kinase (JNK) activation. The selective p38 and JNK inhibitors, as well as dexamethasone, successfully inhibited IL-8 production. These data suggest that early intervention to prevent bile acid aspiration may reduce the intensity of neutrophilic inflammation in intubated and mechanically ventilated patients in the ICU.

Exposure of Airway Epithelium to Bile Acids Associated With Gastroesophageal Reflux Symptoms: A Relation to Transforming Growth Factor-β1 Production and Fibroblast Proliferation

Gastroesophageal reflux (GER) is common in patients with various airway diseases. Airway epithelial cells can release growth factors that promote fibroblast proliferation. Exposure of airway epithelium to bile acids may induce a fibrotic response.To determine how bile acids interact with airway epithelium; particularly, whether transforming growth factor-beta1 secretion and fibroblast proliferation are affected.Induced sputum from patients with asthma, GER, or asthma associated with GER symptoms, or from healthy control subjects was collected. Total bile acids were measured by a spectrophotometric enzymatic assay. The major components of bile acids, chenodeoxycholic acid (CD) and glycochenodeoxycholic acid (GCD), were used to stimulate primary airway epithelial cells. Quantitative polymerase chain reaction and Western blotting were applied for messenger RNA expression and signal pathway analysis, respectively. Conditioned medium following CD stimulation was coincubated with fibroblasts for proliferation study.The amount of total bile acids in induced sputum was significantly higher in patients with GER and asthma-associated GER symptoms compared to that of healthy control subjects (p<0.005). CD, but not GCD, significantly induced TGF-beta1 production. TGF-beta1 messenger RNA expression was 2.5-fold increased compared to unstimulated cells. This occurred via p38 mitogen-activated protein (MAP) kinase and activating transcription factor-2 activation. Pretreatment with dexamethasone inhibited TGF-beta1 production at both messenger RNA and protein levels by inhibiting p38 MAP kinase phosphorylation. Conditioned medium from CD-treated epithelial cells enhanced fibroblast proliferation.Aspiration of bile acids may induce airway fibrosis through the production of TGF-beta1 and fibroblast proliferation. Early intervention to attenuate these processes may reduce fibrogenesis in various airway diseases associated with GER.

An in vitro method for evaluating vascular endothelial ADPase activity

Some xenobiotics, known to promote the development of thrombotic phenomena, affect vascular endothelium ADPase, a regulatory enzyme that inactivates vaso- and platelet-active adenine nucleotides. This proposed new experimental approach represents an improved method of evaluation of vascular endothelial ADPase activity which is assessed by measuring, at pre-established times, the degradation rate of exogenous ADP incubated with aortic bovine patches. The ADP dosage was performed by using a spectrophotometric enzymatic assay. Statistical analyses showed that the method is capable of highlighting the linearity of the ADPase activity time-course, thus indicating that the slopes of time-degradation curves of ADP are a valid index for this endothelial ectoenzyme activity. Results obtained with ADPase inhibiting or stimulating agent confirm that this in vitro method is an efficient tool for estimating the ability of xenobiotics or drugs to modify the nonthrombogenic properties of vascular endothelium.

Fatal neonatal liver failure and mitochondrial cytopathy: An observation with antenatal ascites

Mitochondrial cytopathies are multisystemic diseases of extremely variable expression caused by a deficiency in oxidative phosphorylation. Only five cases of neonatal liver failure in the context of mitochondrial cytopathy have been reported, with incomplete morphological data of the liver in three. In the case presented here, ascites had been diagnosed prenatally and liver failure was particularly severe (factor V < 15% with fatal coma the fourth day), Histologically there were incomplete cirrhosis, microvesicular steatosis, major canalicular cholestasis with proliferative neocholangioles, and bile duct thrombi. There were also some iron pigments in the periportal area and partial glycogen depletion. By electron microscopy, mitochondria in numerous hepatocytes appeared abnormal with occasional cristae in a fluffy matrix, some containing dense inclusions. Study of respiratory chain activity showed a defect in cytochrome c oxidase (complex IV), revealed by oxygraphic measurement on fresh muscle biopsy and confirmed by spectrophotometric enzymatic assays performed on muscle and liver homogenates. The association of neonatal liver failure with hyperlactacidemia warrants investigation into a deficiency in oxidative phosphorylation.

Rapid Spectrophotometric Determination of Plasma Carnitine Concentrations

A spectrophotometric enzymatic assay for plasma carnitine concentrations has been automated on the Monarch 2000. Prior to the assay, each plasma sample was divided into three fractions, ie, free carnitine, acid-soluble carnitine and total carnitine, in order to determine the concentration of both free and esterified carnitine. Using the method developed by Tachikawa et al (Seikagaku 56:998, 1984), each of the samples was then chromatographed on an anion exchange resin to eliminate those compounds which could adversely impact the accuracy of the enzymatic assay for carnitine. After the completion of these preparatory steps, 32 specimens were assayed in less than 16 min on the Monarch 2000 with a high degree of both accuracy and precision. The assay was linear over a wide concentration range (5.0-80 mumol/liter), with the lower limit of sensitivity being 5.0 mumol/liter. The coefficient of variation (CV%) of the within run precision was 2.1%, 2.8%, and 6.7% for the determinations of free carnitine, acid-soluble carnitine, and total carnitine, and 17.4% and 27.8% for the calculated values of short-chain and long-chain acylcarnitines, respectively. The CV% of the between run precision for the same fractions was 6.5%, 2.7%, 3.8%, 14.2%, and 14.3%, respectively. When authentic L-carnitine was added to the plasma, the mean recovery rate was 94.7 +/- 11.0%. Reference values were determined using plasma obtained from 40 healthy adult volunteers.

Spectrophotometric enzymatic assay for S-3-hydroxyisobutyrate

An enzymatic spectrophotometric end-point assay has been developed for determination of S-3-hydroxyisobutyrate in biological fluids. The assay measures NADH production at 340 nm after initiation of the reaction with rabbit liver 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31). The assay is not affected by R-3-hydroxyisobutyrate, lactate, malate, 3-hydroxybutyrate, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 3-hydroxy- n-valerate, 2-methyl-3-hydroxyvalerate, and 3-hydroxypropionate. The assay does measure 2-ethyl-3-hydroxypropionate, a minor metabolite produced by catabolism of alloisoleucine. Application of the method to measure S-3-hydroxyisobutyrate in plasma obtained from normal, 48-h starved, and mildly and severely diabetic rats gave levels of 28, 42, 112, and 155 μ m, respectively.

(R)-mevalonate excretion in human and rat urines.

(R)-Mevalonate was identified by radioenzymatic assay in human and rat urines. This was confirmed by spectrophotometric enzymatic assay of an ether extract of human urine. The average excretion rate of (R)-mevalonate in humans was 1.7 mumol/24 hr which corresponds to 29% of the glomerular filtration rate. In anesthetized rats the average rate of (R)-mevalonate excretion was 350 pmol/min, corresponding to 44% of the glomerular filtration rate. These rates were not affected by the sex of the subjects or animals. After bilateral nephrectomy, the concentration of (R)-mevalonate in rat serum increased within 2 hr to a new level that was 5 times that of sham-operated controls. Nephrectomized rats showed a decrease in their tolerance to an intravenous load of mevalonate. These data show that (R)-mevalonate is normally excreted in the urine of humans and rats. Urinary excretion of (R)-mevalonate should therefore be taken into account in in vivo studies on the metabolism of this compound.

Screening for flavonol 3-glycoside specific β-glycosidases in plants using a spectrophotometric enzymatic assay

A simple and rapid spectrophotometric assay for following the hydrolysis of flavonol 3-glycosides has been developed. The assay profits from the fact that peroxidase converts flavonol aglycones to their corresponding 2,3-dihydroxyflavanones, producing a large shift in UV absorption, whereas flavonol 3-glycosides are not attacked. The amount of liberated aglycone can therefore be calculated from the decrease of flavonol absorption at 350–380 nm. A horseradish peroxidase-H 2O 2 test system can be used to investigate the hydrolysis of most flavonol 3-glycosides, whereas quercetin 3-glycosides can be tested using a peroxidase preparation from Mentha sp. which uses O 2 as cofactor rather than H 2O 2. Flavonol 3-glycoside synthesis, e.g. with UDP-sugars as cofactors, may also be tested by this particular system. Various plants and plant cell cultures were screened for kaempferol and quercetin 3-glycoside specific β-glycosidases. However, in no case could any specific activity be detected.

Serum carnitine. An index of muscle destruction in man.

Carnitine was identified as a constituent of normal human serum by reaction with a specific enzyme and by thin-layer chromatography. A spectrophotometric enzymatic assay was adapted for use with human serum, based upon the liberation of coenzyme A from acetylcoenzyme A in the presence of carnitine and carnitine acetyltransferase. Normal human serum contains 2.3μmols to 7.0μmols/100ml. Serum content increased to 33.4μmols/100 ml during attacks of myoglobinuria but was not increased in patients with Duchenne dystrophy, liver disease, uremia, or acute myocardial infarction.