Evaluation of the enzyme thermistor as a specific detector for chromatographic procedures

The successful application of a simple flow calorimeter, called the enzyme thermistor, to the specific monitoring of different enzymes in the eluants from some common chromatographic procedures is described. The enzyme thermistor enables a specific enzyme to be continuously determined even in optically dense solutions where spectrophotometric procedures cannot be used. The instrument can be operated either on-line or it can be used for discrete samples. The sensitivity is in the order of 0.1 IU/ml for most enzymes.     

Distribution of acid hydrolases in subcellular fractions of proliferating vs non-proliferating fibroblasts

We have employed colloidal silica (Percoll) density-gradient subcellular fractionation technique to examine the distribution of lysosomal hydrolases between intermediate vesicles (primary lysosomes) and secondary lysosomes in contact-inhibited non-proliferating vs proliferating chicken embryo fibroblasts. We find that the activities of lysosomal specific enzymes from both phases of growth are distributed within two peaks; however, the relative amounts differ markedly. In normal, non-proliferating cells approx. 60% of the total activities of cathepsin B, beta-mannosidase, alpha-fucosidase, beta-galactosidase and hexosaminidase is recovered in the heavier density fraction corresponding to secondary lysosomes, while less than 9% of the enzyme activities are recovered in the light-density peak. With transformed cells, between 16 and 22% of activity for these enzymes are recovered in the lighter density intermediate vesicle fraction, when less than 40% of the enzyme activities recovered in the heavy density fraction. beta-Glucuronidase distribution was different from that of the above enzymes. First, a more even distribution between the two lysosomal fractions was found with non-proliferating normal cells (33% in heavy-density fraction and 21% in light-density fraction), whereas more than 40% of the total enzyme activity was recovered in the lighter density fraction from transformed cells. Also, the amount of cathepsin B contained in the vesicle fractions is increased severalfold relative to that of contact-inhibited normal cells. However, the apparent differences in enzyme distribution between confluent normal and transformed cells are not found when vesicles are prepared from subconfluent, actively proliferating cultures. We have also compared the Percoll density gradient patterns of membrane vesicles from proliferating and non-proliferating human fibroblasts, since most earlier studies utilized this system. Again, we find that the majority of beta-hexosaminidase activity (41%) of contact-inhibited, confluent cells is recovered in the heavier density fraction with less than 15% in the lighter density fraction. Also, the distribution of beta-hexosaminidase between the heavy density and light density vesicle fractions is altered in homogenates from exponentially growing cells, being 22% and 26% respectively. We conclude that the distribution of lysosomal hydrolases between the two vesicle populations is growth-phase dependent and is markedly heterogeneous in proliferating cells.     

Galactose oxidase: applications of the covalently immobilized enzyme in a packed bed configuration.

Galactose oxidase (E.C. 1.1.3.9) was covalently immobilized to chemically modified porous silica particles by reaction of the native enzyme with pendant benzoyl azide groups on the carrier. The enzyme loading on the carrier was 100-150 units per milliliter. The immobilized enzyme was incorporated into a hardware assembly suitable for the determination of galactose or lactose concentrations in complex biological fluids. The prototype instrument as described is suitable for continuous, on-line monitoring or discrete sample analysis. Reaction conditions can be readily provided which maintain global first order kinetics within the reactor and strict linearity of the procedure over a wide range of sample concentrations. Auto-inactivation of the immobilized enzyme can be prevented by K3Fe(CN)6 and long-term reactor stability can be achieved by the periodic application of the reagent to the enzyme reactor in situ.     

Serial estimation of serum angiotensin converting enzyme activity during and after pregnancy in a woman with sarcoidosis.

Serum angiotensin converting enzyme activities were estimated during pregnancy and the puerperium in a woman with sarcoidosis and a series of normal women. In the patient with sarcoidosis angiotensin converting enzyme activity was raised during pregnancy, particularly at 21 weeks'' gestation, yet she remained well with no symptoms to suggest relapse of sarcoidosis. Serum angiotensin converting enzyme activity may not be of value in monitoring sarcoidosis activity during pregnancy.     

Efficient protoplast isolation from fungi using commercial enzymes

Several commercial polysaccharases have been compared for their ability to liberate protoplasts from fungi. These enzymes were found to contain side activities capable of hydrolysing fungal cell walls. Protoplasts have been commonly isolated from fungi using enzyme systems prepared by workers in their own laboratories. However, these procedures are time consuming and considerable variation may be found between different batches of enzyme. The present study shows that high yields of protoplasts can be prepared from a variety of fungi using relatively cheap commercial enzymes. The yields obtained were normally as good as or better than those previously produced.     

A spectrophotometric method for the assay of phospholipase D activity

Phosphatidylcholine phosphatidohydrolase (EC 3.1.4.4, phospholipase D) catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid and choline. We have developed a spectrophotometric assay for phospholipase D using choline kinase, pyruvate kinase, and lactate dehydrogenase to couple the release of choline with the oxidation of NADH. The assay was linear both with time and with enzyme concentration. The assay should prove useful for continuous monitoring of enzyme activity, determination of initial rates of reaction, and detailed kinetic studies of phospholipase D. The method is limited to analysis of purified preparations of phospholipase D lacking competing activities to the coupled system.     

The use of biological activities to monitor the removal of fuel contaminants—perspective for monitoring hydrocarbon contamination: a review

Soil biological activities are vital for the restoration of soil contaminated with hydrocarbons. Their role includes the biotransformation of petroleum compounds into harmless compounds. In this paper, the use of biological activities as potential monitoring tools or bioindicators during bioremediation of hydrocarbon-contaminated soil are reviewed. The use of biological activities as bioindicators of hydrocarbon removal in soil has been reported with variable success. This variability can be attributed partially to the spatial variability of soil properties, which undoubtedly plays a role in the exposure of organisms to contaminants. Widely used bioindicators have been enzyme activities, seed germination, earthworm survival and microorganisms or microbial bioluminescence. A mixture of some successful utilization of biological activities and several failures, and inconsistencies reported, show that at this stage there is no general guarantee of successful utilization of biological activities as monitoring tools. Wherever possible, the use of biological activities as bioindicators of hydrocarbon removal must be used to complement existing traditional monitoring tools.     

A transformer of molecular beacon for sensitive and real-time detection of phosphatases with effective inhibition of the false positive signals

Molecular beacons (MBs) have shown great potential in measurement of enzyme activities. However, currently available methods for monitoring of phosphatases only use MBs as a signal reporter. Extra substrates for the phosphatases are needed to hybridize to the MB either as a primer or as a template. Moreover, few MB-based methods have been used to detect enzyme activities in real biological samples due to insufficient sensitivity or false positive interference signals caused by nonspecific nucleases. In this work, a novel type of fluorescent probe was designed and synthesized for monitoring of phosphatases by integrating the DNA substrate and the signaling structures into a single molecule. Such a new design not only significantly simplified the probing system and greatly enhanced the sensitivity, but also offered a practical way to guard against the false-positive signal problems in the application to real samples. The unique design of the assay format should be widely applicable to many other enzymatic assays using oligonucleotide fluorescent probes.     

Biochemical changes as enzymatic defense system of phenanthrene exposure in olive flounder, Paralichthys olivaceus

The objective of this study was to investigate early biological response in olive flounder exposed to sub-lethal concentrations of waterborne phenanthrene (0.5, 1 or 2 microM). The fish were exposed for 4 weeks and we analyzed their enzymatic defense system, antioxidant and phase II enzyme activities, to evaluate the chronic exposure toxicity of phenanthrene. Waterborne phenanthrene affected antioxidant enzymes and glutathione-mediated detoxification as enzyme defense system. Hepatic, gill and kidney glutathione reductase as well as glutathione S-transferase, and catalase activities were markedly elevated after two or four weeks of exposure. These enzymes activities of olive flounder, Paralichthys olivaceus seem to be a convenient tool for monitoring pollution in coastal areas against PAHs pollution including phenanthrene.     

Lysophospholipase and transacylase activities of rat gastric mucosa.

A transacylase that converts 1-palmitoyl lysophosphatidylcholine to dipalmitoyl phosphatidylcholine was demonstrated in the rat gastric mucosa. This enzyme required neither ATP or CoA nor bile salt and detergent for its activity. The enzyme preparation also exhibited powerful lysophospholipase activity. The transacylase and lysophospholipase were both located in the cytosol fraction, and their activities remained associated at a constant ratio throughout the purification steps, including the isoelectrofocusing procedure. They responded similarly with respect to the addition of metal ions, bile salt, detergent, and heat treatment. Both enzyme activities also exhibited similar apparent K_m values for lysophosphatidylcholine. These observations suggest that both the lysophospholipase and transacylase activities may reside in the same enzyme.     

Perturbation of hepatic glutathione level and glutathione-related enzyme activities by repeated administration of aminopyrine in rats

The influence of repeated administration of aminopyrine on the tissue glutathione level and related enzyme activities was investigated in rats. Reduced glutathione level in the liver was not changed after 5 days of treatment but a significant increase was seen after 15 days of aminopyrine treatment. Oxidized glutathione level was unaltered throughout the experiment. Repeated administration of aminopyrine for 5 days caused a marked increase in gamma-glutamyl transpeptidase activities in liver whole homogenates as well as in the nuclear fraction, but not in liver microsomes. These results suggest that gamma-glutamyl transpeptidase located in plasma membrane may be induced by repeated administration of aminopyrine for 5 days. The activities of cytosolic glutathione peroxidase, which modulates glutathione level, were also significantly increased by aminopyrine treatment. Under the same conditions, glutathione peroxidase activity with H2O2 as a substrate was unaltered, while a time-dependent increase in the activity was found when cumene hydroperoxide was used as a substrate, even after a single administration of aminopyrine. The intracellular cysteine level was increased accompanying the increased gamma-glutamyl transpeptidase activities. Therefore, induced gamma-glutamyl transpeptidase may play a role in the reclamation of extracellular oxidized glutathione.     

Rapid continuous monitoring of enzyme activity in tissue sections: experience with the M85A and Zeiss UMSP-30 systems

We have previously described methods for the continuous monitoring of formazan deposition in tissue sections with a system based on the Vickers M85A microdensitometer. However, this instrument only allows the monitoring of a single field in each section. We have now developed a new system based on the Zeiss UMSP-30 microspectrophotometer. This machine is entirely computer controlled and by virtue of its fast-scanning stage allows the rapid (less than 0.5 s) sequential monitoring of multiple fields (up to 35) in each section. Thus a number of cell types may be studied simultaneously and work which used to take a full working day with the M85A system now can be performed in 45 min. As with the M85A the Zeiss system has full capability for data analysis (i.e. calculation of initial velocity rates, etc.). We have found that continuous monitoring of tissue sections by microdensitometry is a precise, sensitive and biochemically valid method of studying enzyme activity within the cellular matrix.     

Activity and distribution of lysosomal enzymes during collagenous matrix-induced cartilage,bone, and bone marrow development

The appearance of the lysosomal enzymes acid phosphatase, arylsulfatase, and β-glucuronidase was studied during endochondral bone and bone marrow formation induced by implantation of demineralized bone matrix. The activities of acid phosphatase and β-glucuronidase gradually increased from the stage of mesenchymal cell proliferation on Day 3 onward to reach a peak on Day 13, during maximal bone remodeling. However, arysulfatase activity exhibited a sharp increase on Day 9, associated with the onset of cartilage hypertrophy and chondrolysis. The peak of arylsulfatase activity was also attained on Day 13. The activities of all three enzymes declined on Day 15 but acid phosphatase again exhibited an increase during hematopoietic bone marrow differentiation on Days 19–21. Histochemical and ultrastructural studies revealed intense lysosomal enzyme activity in macrophage-like cells on Day 7 and thereafter. During chondrolysis and bone remodeling, these cells were present in a perivascular location. Osteoclasts also exhibited strong reactivity for the lysosomal enzymes. Due to its characteristic temporal appearance during development of endochondral bone, arysulfatase may be used as a marker enzyme for chondrolysis and bone resorption.     

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

A continuous spectrofluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent Km (Kapp) values for protoporphyrinogen IX were measured as 3.8+/-0.3, 3.6+/-0.5, and 1.0+/-0.1 microM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The Ki for acifluorfen, a diphenylether herbicide, was measured as 0.53 microM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h-1/mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min-1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.     

Studies on the action of porphyrinogenic trace metals on the activity of hepatic uroporphyrinogen decarboxylase

Trace metals which produce experimental uroporphyrinuria in animals during prolonged exposure inhibit uroporphyrinogen (uro) decarboxylase in rat liver extracts $\text{in}\text{,}\text{vitro}$. Inhibitory effects are prevented by sulfhydryl reagents, suggesting metal binding to sulfhydryl groups of the enzyme as the likely mode of action. Mercury, the most potent of the metals tested with respect to sulfur binding kinetics, produces the greatest inhibition of enzyme activity. In contrast, iron, which is considered to play a role in the etiology of porphyria cutanea tarda (PCT) via inhibition of uro decarboxylase, did not inhibit the enzyme in the present test system, suggesting an indirect mode of action $\text{in}\text{,}\text{vivo}$. These results suggest that direct inhibition of uro decarboxylase underlies uroporphyrinuria produced by prolonged trace metal exposure. Experimental inhibition of uro decarboxylase by trace metals may serve as a model for studying metal-induced uroporphyrinuria and PCT in humans.     

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).     

Urinary N-acetyl- beta-D-glucosaminidase activities in patients with renal disease.

Urinary N-acetyl-beta-D-glucosaminidase (NAG) activities were assayed in every urine void throughout 24 hours in 17 normal people and in four patients with renal disease. The variation in NAG activity due to changing rates of urine flow was almost eliminated by factoring enzyme activity by the urinary creatinine concentration. Random samples of urine may thus be used for assay. The results of NAG assay in 36 patients with acute and chronic renal diseases showed that NAG was a sensitive indicator of renal damage. This simple test may be valuable in detecting or monitoring renal disease.     

Soil microbial activities in a constructed soil reed-bed under cheese-dairy farm effluents

Soil microbial activities in a reed-bed used for effluent purification of a small cheese-dairy farm under a Mediterranean climate were described and studied. This work aims to demonstrate (i) whether certain enzyme activities used as bioindicators of dairy waste degradation (beta-galactosidase and protease) vary over time, which might influence organic matter degradation and (ii) whether specific microbial communities are selected through contact with the discarded effluent using community level catabolic profiles (CLCPs). beta-galactosidase and protease activities were followed in a 14-month monitoring experiment. These enzyme activities were strongly expressed during the whey-discarding period from February to May. CLCPs using Biolog Ecoplate showed great microbial diversity, as described by Shannon-Weaver index, and no difference was observed in microbial diversity between areas at the receiving end of the reed-bed (where effluent was discarded) and those at the opposite end. This may be explained by successive environmental factors which made enzyme activities vary: whey discarded from February to May and Mediterranean climate conditions (drying-rewetting effects on summer). Microbial enumeration using epifluorescence microscopy also showed a pattern linked to Mediterranean conditions with a drastic decrease in biomass during summer drought. These results on functional biodiversity were correlated with high purification yields: the minimum decrease in Biological Demand in Oxygen was 84% and that in suspended solids was 75%.     

Parameters of biological freezing damage in simple solutions: Catalase: II. Demonstration of an optimum-recovery cooling-rate curve in a membraneless system

Seeded solutions of catalase in neutral 10 mM potassium phosphate buffer exhibited characteristic rate dependencies for freeze-thaw damage: Damage increased as the cooling rate was increased, and as the warming rate was decreased. The pattern of warming-rate dependence was independent of the prior cooling rate and also of the addition of KCl or of NaCl to the buffer. In contrast, the cooling-rate curve became almost flat upon addition of 0.1 M KCl, suggesting increased damage from concentrating solute at low cooling rates. In the presence of added NaCl, frank optimum-recovery cooling-rate curves were generated. At low NaCl levels (less than 10 mM) the optimum occurred at 0.5 °C/ min; at 27 and 81 mM NaCl, the optimum shifted to 5 and 20 °C/min, respectively. By comparison with KCl, it appears that the major factor causing damage at low cooling rates in NaCl is acidification. The factor causing damage at high cooling rates remains obscure. The argument that it is due to the trapping of the enzyme molecules at interfaces at high dilution, to be subsequently damaged by shearing stress or dehydration during the recrystallization attending slow warming, is mitigated by the finding that inactivation remains a function of the initial enzyme concentration at all cooling rates. The possibility that a particular conformational state is trapped in an unfavorable temperature zone was also considered: Three simple models were formulated, and the relative order of recovery was deduced for the possible sequences of fast and slow cooling and warming. The permutation observed for catalase was inconsistent with any of these three mechanisms, although they may be pertinent for the red cell and other systems. A final possibility, not yet explored, is that rapid cooling causes damage by producing nonequilibrium freezing, with large deviations of pH and/or solute concentration from those expected at equilibrium.