Development Of Enzyme Activity Research Articles

Effect of Bromelain-Based Enzymatic Debridement on Skin Cells.

Several reports have concluded that enzymatic debridement based on Bromelain (NX) is selective and efficient. Although clinical trials showed that viable tissue is not damaged at the macroscopic level, the effect on the cellular level is largely unknown. The current study is meant to close this gap by evaluating whether NX has an effect on vital cells of the human dermis on a cellular level. In an experimental in vitro study design, the effect of NX on human keratinocytes, fibroblasts, and macrophages was analyzed. Enzymatic treatment was performed for 4 hours by using either cell culture medium or phosphate-buffered saline as diluting agent for NX. Cell viability and relative cell number in relation to untreated control cells were determined using a resazurin-based assay. In addition, the development of enzyme activity during clinical treatment was analyzed: wound fluid collected from a burn wound at different points of debridement was applied on collagen-elastin disks to prove enzymatic digestion activity. Both keratinocytes and fibroblasts were damaged by NX even at low concentrations. Both cell types showed improved survival when a medium was used for dissolving NX. Macrophages appeared to resist NX treatment more efficiently than the other cell types. In the clinical trial, NX activity in the wound fluid decreased clearly following 4 hours of enzymatic debridement. NX induces toxicity of vital skin cells in vitro. However, macrophages appear to be more resistant against NX treatment in vitro. The inflammatory responses of vital cells in the burn wound itself are likely to inhibit NX activity. The effect of this inflammatory process on NX activity will have to be investigated in future studies.

Evidence for an extracellular l–amino acid oxidase in nitrogen-deprived Phaeodactylum tricornutum (Bacillariophyceae) and inhibition of enzyme activity by dissolved inorganic carbon

T.A.V. Rees and V.J. Allison. 2006. Evidence for an extracellular l–amino acid oxidase in nitrogen-deprived Phaeodactylum tricornutum (Bacillariophyceae) and inhibition of enzyme activity by dissolved inorganic carbon. Phycologia 45: 337–342. DOI: 10.2216/04-92.1Nitrogen-deprived cells of the marine diatom Phaeodactylum tricornutum possessed extracellular l–amino acid oxidase (EC 1.4.3.2) activity. No enzyme activity was detectable in nitrogen-replete cells. When nitrogen-deprived cells were provided with 100 μM alanine, the amino acid was metabolised, with both pyruvate and H2O2 accumulating stoichiometrically in the medium; this accumulation was largely prevented if the cells were preincubated with the membrane-impermeable protease Proteinase K. Following the transfer of nitrogen-replete cells to nitrogen-free medium, development of enzyme activity lagged, with little or no enzyme activity detectable until 4 h after the onset of nitrogen deprivation. This lag was followed by rapid development of enzyme activity during the ensuing 20 h. In addition to alanine, methionine, methionine sulfoximine, leucine, glutamate and valine, at the same initial concentration of 100 μM, were exclusively metabolised by an amino acid oxidase. Histidine was partially metabolised by an amino acid oxidase, whereas arginine and lysine were transported into the cell with little or no metabolism by an amino acid oxidase. Neither serine nor proline was used. In general, the neutral nonpolar and acidic polar amino acids were used via an extracellular amino acid oxidase, whereas the basic polar amino acids were transported. l–Amino acid oxidase activity was inhibited in darkness and in light in the presence of the photosynthetic inhibitor DCMU (3-[3,4-dichlorophenyl]-1,1-dimethylurea). In addition, enzyme activity decreased on addition of bicarbonate to cells in light. Together these observations suggest that extracellular l–amino acid oxidase activity was inhibited by dissolved inorganic carbon.

Effects of Pulses of Higher Temperature on the Development of Enzyme Activity During Malting

The increase of temperature at the beginning, in the middle and at the end of malting has been evaluated in terms of quality parameters (malting losses, index of acrospire development, friability, HWE, viscosity, SNR) and enzyme (β-glucanase and α-amylase) development, in a good quality malting barley (Otis) and a higher protein-higher β-glucan content barley used for feed (Extra). A shift from 15 to 20°C at the beginning of malting was shown to increase acrospire development, friability, HWE and SNR and to reduce viscosity, without significantly affecting malting losses. This effect was related to higher β-glucanase and α-amylase activities within each variety. However, the same enzyme activities were not directly related to a better malting quality when the two genotypes were compared. This confirms previous indications that diversity in malting performance between genotypes cannot simply be traced back to differences in enzyme activities; but, indeed, it suggests that, for a defined barley lot, changes in the levels of enzyme activities following different malting procedures may have a direct effect on malt quality.

Variability of laccase activity in the white-rot basidiomycetePleurotus ostreatus

The production of laccase in liquid cultures of the white-rot fungus Pleurotus ostreatus was highly variable. During the first days of cultivation, the relative variability was as high as 80-100% and it decreased to 30% in the course of cultivation. The main source of variability was assumed to be the independent development of enzyme activity in individual cultures. Cultures with high laccase production showed also high production of the other ligninolytic enzyme--Mn-dependent peroxidase. The variability was probably due to the source of inoculum, deactivation of the enzyme in culture liquid and genetic variations among the cultures. Variability of laccase activities was lower during solid-state fermentation on wheat straw and during the growth in nonsterile soil.

A developmental study of lactate dehydrogenase isozyme and aspartate aminotransferase activity in organotypic rat hippocampal slice cultures and primary cultures of mouse neocortical and cerebellar neurons

The development of enzyme activity and isozyme distribution of lactate dehydrogenase (LDH) was studied in murine organotypic hippocampal slice cultures and dissociated cultures of neocortical neurons and cerebellar granule cells and compared with that of the respective brain regions invivo. In the hippocampal slice cultures and the hippocampus invivo, the activity of aspartate aminotransferase (AAT) was also measured. During development in culture the specific activity of LDH increased in all types of cultures reaching values similar to that found in the corresponding brain areas invivo. However, significant differences in the isozyme distribution were observed between the preparations in vitro and invivo. During development invivo, the LDH isozyme pattern changed from a preferential M-subunit composition to a preferential H-subunit composition regardless of the brain area. This shift was not observed in the respective cultures where the M4-isozyme prevailed at all culture periods examined accounting for 30–45% of the total LDH activity. The cultured cerebellar granule cells did not express the H4-isozyme at all, while in the hippocampal slice cultures and the cultured neo-cortical neurons this isozyme accounted for about 5% of the total LDH activity. The activity of AAT in the hippocampal organotypic slice cultures increased considerably during the culture period in parallel with the increase in AAT activity during postnatal development of hippocampus invivo. The activity of AAT in the slice cultures was, however, consistently lower than the corresponding activity invivo. The results show that perinatal hippocampal tissue and neurons from neocortex or cerebellum retain their immature LDH-isoenzyme distribution when grown as organotypic slice cultures or dissociated cell cultures, respectively. This lack of shift from the M4 to the H4 LDH isozyme which is typical of tissues relying on aerobic glycolysis has several implications but does not apparently interfere with the otherwise normal differentiation of morphological, biochemical and electrophysiological characteristics of these preparations.

The influence of inclusion of either an antibiotic or a probiotic in the diet on the development of digestive enzyme activity in the pig

The aim of the present experiment was to determine the influence of either probiotic or antibiotic inclusion in the diets of pigs from birth on the development of enzyme activity in the small intestine. Pigs were fed on creep feed and grower diets containing either a probiotic, an antibiotic or no added growth promoter. At 7, 17, 42 and 80 d of age pigs from each treatment group were sampled to investigate the development of carbohydrase and peptidase activity in the mucosa at five sites along the small intestine. Inclusion of either the probiotic or antibiotic had a significant effect on the development of sucrase (sucrose alpha-D-glucohydrolase; EC 3.2.1.48), lactase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) and tripeptidase (EC 3.4.11.4) activities before weaning but had no effect on depeptidase (EC 3.14.13.11) activity. The study of the distribution of enzyme activity along the small intestine showed significant differences between the proximal and distal sections associated with weaning.

The influence of inclusion of a probiotic in the diet on the development of enzyme activity in the small intestine of the pig

The objective of the present study was to investigate the development of hydrolase enzyme activity at six sites along the small intestine of the pig before and after weaning and the influence inclusion of a probiotic preparation had upon this. The enzymes studied lactase, dipeptidase and tripeptidase are primarily associated with the brush border of the enterocyte cell and expression of hydrolase activity has been shown to be affected by manipulation of the microbial population of the gut.

Spectrofluorimetric assays for hydrolytic activity in germinating wheat

Two rapid and sensitive spectrofluorimetric methods for detecting hydrolytic activity in flour suspensions from germinated wheat are described. The methods are based upon the enzymically-catalysed hydrolysis of non-fluorescent 4-methylumbelliferyl heptanoate (MUH) and fluorescein dibutyrate (FDB) to the highly-fluorescent compounds 4-methylumbelliferone and fluorescein respectively. MUH hydrolysis was best correlated with lipase activity (using olive oil as substrate) and with α-amylase activity, all three assays showing an exponential development of enzyme activity with increasing germination. Hydrolytic activity, measured using the FDB assay, and esterase activity (using ethyl butyrate as substrate) displayed similar development curves during germination, but at a much lower rate. The fluorimetric assays are rapid, sensitive and simple to perform, and were able to detect hydrolytic activity as early as 6–12 h after germination at 15°C of grain previously steeped for 24 h at 15°C.

SOME BARLEY GRAIN AND GREEN MALT PROPERTIES AND THEIR INFLUENCE ON MALT HOT-WATER EXTRACT: I. β-GLUCAN, β-GLUCAN SOLUBILASE AND ENDO-β-GLUCANASE

A study has been made of the variation between varieties in some properties of barley and malt and how this variation relates to malt hot water extract (HWE). The development of enzyme activity along the grain during germination was investigated. In this first paper we have examined β-glucan-related characters and found significant varietal variation in maximum enzyme activities and in the activities in different sections of grain during germination. Varietal variation was greater than environmental variation for each character. The fraction of β-glucan soluble in acid was the character most highly correlated with HWE.

Changes in phenylalanine ammonia‐lyase levels in excised potato tuber tissue: effects of the gaseous environment and desensitization to cinnamate repression by in situ pre‐incubation

Abstract. The rise in phenylalanine ammonia‐lyase (PAL) activity following excision of potato tuber discs is antagonized by increasing partial pressures of CO2 This inhibition is potentiated by depleting the atmospheric ethylene level. We suggest that the previously observed suppression of PAL appearance by in situ incubation of excised discs in reassembled tubers may be related to an internal atmosphere relatively rich in CO2 and of low ethylene content. The transition to an oscillatory time course of PAL activity that follows transfer of discs from in situ incubation to air appears to be accompanied by the development of enzyme activity becoming desensitized to repression by exogenous cinnamate. The concentration dependence of cinnamate uptake is not significantly altered by in situ pre‐incubation of tuber discs.

Influence of adrenal epinephrine on postnatal development of tyrosine hydroxylase activity in the superior cervical ganglion.

The effects of bilateral adrenalectomy on postnatal development of tyrosine hydroxylase (TH) activity in the superior cervical ganglion were examined in the rat. Adrenalectomy on day 12 blocked further developmental rise of TH activity, while adrenalectomy on day 7 did not affect normal development of enzyme activity when examined on day 12. The effect of adrenalectomy could be reversed by replacement with daily injections of epinephrine, but not by similar replacement with corticosterone. Chronic administration of SKF 64139, a specific inhibitor of adrenal phenylethanolamine N-methyl-transferase activity that depletes circulating epinephrine levels, also led to a cessation of normal development of TH activity. Similar to adrenalectomy, the effect of SKF 64139 could be observed only after 2 weeks of postnatal development. From these data, it appears that after 2 postnatal weeks, further developmental rise of TH activity in the sympathetic ganglion is an epinephrine-dependent process.

Development of Δ5-3 β-hydroxysteroid dehydrogenase activity in the interrenal gland of Rana catesbeiana

A quantitative, histochemical analysis of the development of Δ 5-3 β-hydroxysteroid dehydrogenase activity in the interrenal gland of Rana catesbeiana from larval stages to young frogs was investigated. The quantitation of enzyme activity was evaluated by scanning reaction products in the histochemically prepared tissue with a computerized microscope spectrophotometer. The results indicate that Δ 5-3 β-HSD activity during the course of development exhibits a tendency to follow the progress of metamorphic events. The activity begins to appear weakly in cortical cells of the embryo at Shumway stage 23 and then increases with the differentiation of the interrenal in the larva. Development of enzyme activity is slow during premetamorphic and prometamorphic stages, after which the activity increases rapidly to reach a peak at the end of metamorphosis. However, the interrenals of young frogs show a sharp decline in enzyme activity. This developmental pattern of Δ 5-3 β-HSD activity is discussed from the viewpoint of its relations to thyroxine and ACTH.

Regulation of enzyme levels in phenylpropanoid biosynthesis: Characterization of the modulation by light and pathway intermediates

The accumulation of chlorogenic acid in disks of potato ( Solanum tuberosum cv King Edward) tuber tissue is controlled by rapid, interrelated changes in the activity levels of the biosynthetic enzymes phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11), and hydroxycinnamoyl-CoA:quinic acid hydroxycinnamoyl transferase (EC 2.3.1.-). The modulation of enzyme levels by light and by the pathway intermediates cinnamic acid and p-coumaric acid has been characterized by (a) effectiveness at various stages during the development of enzyme activity, (b) the kinetics of the regulatory responses, and (c) application of the inhibitors cordycepin, cycloheximide, and actinomycin-D. Using these criteria, clear mechanistic differences between modulation of enzyme levels by light and modulation by pathway intermediates have been observed. Photomodulation of enzyme levels occurs by a relatively slow, actinomycin-D-sensitive mechanism. In contrast, modulation by pathway intermediates occurs by a rapid, post-transcriptional mechanism acting at the rate-limiting step in the development of enzyme activity. This mechanism is consistent with the hypothesis that the coordination of enzyme levels is maintained by modulation by pathway intermediates.

Repetitive counterelectrophoresis on agar gel for the immunological identification of esterases produced by strains of Lancefield's group A, B, and C streptococci

A method, repetitive counterelectrophoresis (RCE), was devised for detecting specific antibody to streptococcal esterase (STE). Reference antisera were prepared by immunizing rabbits with STE of streptococcal strains as follows: SS379 (group A, type 40), 69882 (group A, type 49), and H36B (group B, type Ib). Some human sera, derived from patients with scarlet fever, were also used as references. By this method, we have confirmed the immunological specificity of the STE produced by strains SS379 (STE-AI), 69882 (STE-AII), H36B (STE-B), and Austin (STE-C, group C) and have shown that the STE produced by strain 10706 (group C) is immunologically identical with STE-AI. Each STE presented a distinct colored line with the respective homologous antibody upon development of enzyme activity except for STE-AII, which formed a round spot with the homologous antibody. Horse activating factor (Hayano and Tanaka, 1973) formed a round spot with each STE. The factor in serum that reacted specifically with STE seemed to correspond to gamma globulin.

STUDIES ON THE INTERACTIONS BETWEEN THE EMBRYONIC AXIS AND THE COTYLEDONS DURING GERMINATION IN PISUM SATIVUM L.: THE INFLUENCE OF INCUBATION CONDITIONS

SummaryThe effects of removal of the embryonic axis on the development of enzyme activities in the cotyledons of peas have been studied. Excised cotyledons incubated aseptically in large culture vessels show greater increases in acid ribonuclease and acid phosphatase activities than do cotyledons of intact seeds. In excised cotyledons incubated aseptically in Petri dishes, neither enzyme exhibits the normal increase in activity observed to intact seedlings. The significance of these results is discussed.

The induction of enzyme activity in the endosperm of germinating castor-bean seeds.

Endosperm extracts were prepared at various times during germination from intact castor-bean seeds and from seeds from which the embryos had been removed. The sterilized seeds were incubated either on solid water agar or on agar containing 0.3 mM-gibberellic acid. 2. Isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase had very low activities in the mature seeds, but increased 44-fold and 27-fold respectively during germination. In contrast, the extracts of mature seeds had considerable acid and alkaline lipase activity and this only increased two- to three-fold during the incubation period. 3. Incubation of the seeds with gibberellic acid accelerated the rate of appearance of isocitrate lyase and 3-hydroxyacyl-CoA dehydrogenase. It also increased the total activity attained. However, the application of hormone had, in comparison, little effect on the development of lipase activity. 4. The removal of the embryo had little influence on the development of enzyme activity in the endosperm tissue; only with isocitrate lyase was a decrease in activity observed in the absence of the embryo.

Gonadal factors regulating the activity of the 17beta-hydroxysteroid oxidoreductase in rat liver.

ABSTRACT The activity of the 17β-hydroxysteroid oxidoreductase (17β-HSOR), catalysing the oxidoreduction of oestradiol-17β and oestrone, has been studied in the cytosol fraction of rat liver under various conditions. The activity of the enzyme increased during maturation and reached a plateau at 100 days in females and at 180 days in males. In adult male rats, the activity of the 17β-HSOR was about 60% higher than in adult female rats. When female animals were castrated, the development of enzyme activity was similar to that observed in male rats; there was no difference in enzyme activity between adult castrated female rats and normal male rats. In normal female rats the activity of the 17β-HSOR was high during metoestrus and dioestrus, and low during pro-oestrus and oestrus. These findings show that oestrogens have a repressing effect on the activity of a key enzyme of steroid metabolism in rat liver.

Studies on the adaptive synthesis of rat liver fatty acid synthetase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of 3H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [ 14C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.

Hypoxanthine-guanine-phosphoribosyl-transferase from rat brain (purification, kinetic properties, development and distribution).

AbstractHypoxanthine‐guanine‐phosphoribosyltransferase (HGPR Tase; ECC 2.4.2.8) has been purified from rat brain 650‐fold to about 50 per cent purity by conventional methods. An isoenzyme pattern of at least three components is observed on DEAE‐cellulose chromatography. On polyacrylamide disc electrophoresis only one sharp band of enzyme activity can be detected. The apparent Km‐value determined for phosphoribosylpyrophosphate (PRPP) is about 0.2 mM. The product, GMP, and also GDP, GTP, UMP, CMP, AMP and ATP are competitive inhibitors with respect to PRPP. Inhibition by a number of other nucleotides has also been investigated. Studies on the development of enzyme activity in the brain of the young rat show that a rapid increase occurs during the first 15‐20 days of life and reaches a plateau thereafter. The regional distribution of HGPRTase activity in adult rat brain is more homogenous than that reported for human brain. The enzyme is predominantly a constituent of the soluble supernatant fraction, but can also be found in carefully washed synaptosomes. An antiserum against rat brain HGPRTase obtained from rabbits inhibits this enzyme to about 30 per cent of control activity, but does not crossreact with HGPRTases from rabbit or human erythrocytes.

Perinatal regulation of mitochondrial and cytoplasmic enzymes in rabbit liver.

Extract: The control of postnatal development of the activities of cytoplasmic and mitochondrial malate dehydrogenases and of the purely mitochondrial enzyme, cytochrom oxidase, has been studied in rabbit liver. Each of the three enzymes increased 1.6–1.8-fold in activity during the first 2 days of life. Delivery of animals at varying stages of maturation demonstrated (table II) that the increase in all cases occurred when delivery was effected and was a result of some aspect of the process of birth itself. Maturation affects cytoplasmic malate dehydrogenase (MDH) differently than the two mitochondrial enzymes. Cytoplasmic MDH is relatively constant in activity in utero and increases approximately 80% whenever delivery is effected (table II, figure 1). In contrast, both the mitochondrial (20–40%) following premature delivery is considerably smaller than the large (1.5-fold) increase following postmature delivery. Using the artificial feeding procedure developed for this study, newborn animals were maintained for 48 h in an hypoxic environment. Preliminary results suggested that development of enzyme activity was affected by hypoxia. Speculation: Normal alterations in perinatal enzyme activity are probably a result of various aspects of maturation and the birth process. Varying the time of delivery and the extrauterine environment makes possible experimental study of the physiologic control of this and other perinatal processes.