Stability of Some Serum Enzymes in Sheep,Cattle, and Swine During Storage at Different Temperatures

Owing to the increased use of serum enzyme determinations in veterinary diagnostic work, greater knowledge about the keeping qualities of different animal sera under various storing conditions seems desirable. The present paper deals with the stability of serum aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (A1AT = GPT), lactate dehydrogenase (LDH), and a-hydroxybutyrate dehydrogenase (HBD) in cattle, sheep, and swine. Sera from 14—16 animals of each species were analysed daily for 5 days after storage at room temperature (22°C) and in the refrigerator (4°C). Samples kept in the deep-freezer (—20°C) were reanalysed once after 32—38 days. Significant differences of serum activity were found between individuals for all enzymes in the three species. Great variations were found in the stability of enzyme activities of different species. To summarize, it may be said that the changes of transferase activities were less pronounced under the different storing conditions than those of the dehydrogenases investigated. Pig serum in particular showed heavy losses of the latter enzymes already after 1 day, more pronounced at refrigerator than at room temperature. As a consequence of the results obtained, practical recommendations for analytical work on these enzymes are suggested.     

Effect of preservation at low temperature (4 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

When polymorphonuclear neutrophils were stored at 4 °C for up to 2 weeks, the maintenance of the integrity of PMNs was examined by determining changes in enzyme activity, enzyme release, stimulated superoxide anion generation, and sensitivity to hypotonicity. Until at least 3-day storage, no changes were observed in enzyme activity, enzyme release, and stimulated superoxide anion generagion. After 1-week storage, the ability of PMNs to generate superoxide anions decreased considerably and the extracellular release of lactate dehydrogenase (LDH) was observed. After 2 weeks of storage, this LDH release and inhibition of O₂^?-generating ability of PMNs increased further, although enzyme activities were only slightly affected except for acid p-nitrophenyl phosphatase. The resistance of PMNs to hypotonic solutions decreased with increasing preservation time at 4 °C.     

野生鸡枞菌种长期保存方法比较

野生鸡枞菌种质资源的有效保存是对野生鸡枞加以保护和利用的前提。以自行分离的5个野生鸡枞菌株作为研究对象,采用蒸馏水保藏法和-80°C冻结保藏法对野生鸡枞菌种长期保存的方法进行了实验研究,蒸馏水法分别保存于室温和4°C,-80°C冻结保藏同时采用程控降温法和泡沫盒降温法,保存20个月后对4种不同方法保存的5个菌株的保存效果进行比较。实验结果表明:蒸馏水室温保存法菌种存活率为100%,萌发期较短,为4-10 d,是一种简便、实用、有效而成本低廉的长期保存方法;-80°C冻结保藏法的存活率为56%-76%,萌发期7-16 d,泡沫盒降温法可以很好地控制降温速度,是一种简便有效的控温方法。     

Multiple immobilized enzyme reactors: determination of pyruvate and phosphoenolpyruvate concentrations using immobilized lactate dehydrogenase and pyruvate kinase

Lactate dehydrogenase (LDH) and pyruvate kinase (PK), immobilized on solid glass beads by diazotization, were used in packed bed reactors to analyze for both pyruvate (PYR) and phosphoenolpyruvate (PEP) through the disappearance of β-nicotinamide adenine dinucleotide (NADH) monitored spectrophotometrically at 340 nm. Packed bed reactors containing PK and/or LDH were also capable of monitoring continuously varying concentrations of adenosine-5′-diphosphate (ADP), PEP, and PYR. The immobilized enzymes (～40 μg/g glass) retained about 4% of the activity of the soluble enzymes. Preparations of immobilized LDH and PK exhibited enhanced stability when maintained in the presence of β-mercaptoethanol and NADH or EDTA, respectively, and were shown to regain 75% of the original activity after four months storage at 4°C.     

Evaluation of functional stability of quercetin as a raw material and in different topical formulations by its antilipoperoxidative activity

The present study evaluates the antioxidant activity of the flavonol quercetin, and its functional stability as a raw material and when added in formulations. The iron-chelating activity was determined using the bathophenanthroline assay, and the functional stability was evaluated with the antilipoperoxidative assay. Raw material presented concentration-dependent antilipoperoxidative and iron-chelating activities. The initial antilipoperoxidative activity of the raw material, cream and gel-cream were 63%, 78%, and 69%, respectively. There was no detectable loss of activity during 182 days (6 months) of storage at all tested temperatures (4°C, room temperature [RT], 37°C, and 45°C) for the raw material. Considering the method variability of 10%, activity loss greater than 10% for nonionic cream was detected after 126 days at 4°C (20.1%), decreasing thereafter to 22.2% after 182 days. At 45°C, the loss of activity started after 182 days (13.2%). For the anionic gel-cream, activity loss started after 84 days (28.4%, 45°C), decreasing after 182 days to 40.3% at 45°C. At 37°C, activity loss was detected after 182 days (12%). In conclusion, the results suggest that the activity of quercetin depends on iron chelation, and its posible usefulness as a topical antioxidant to prevent oxidative stress-induced skin damage depends on maintaining its antilipoperoxidative activity stored at RT, which avoids special storage conditions. Published: February 3, 2006     

Electrophoretic study of heating in situ on proteins and enzymes in germinated Arachis hypogaea cotyledons: Comparison with dormant seed

-Cotyledons from 5-day germinated seed of Arachis hypogaea were heated in a moisturized chamber at temperatures from 25 to 121°. Proteins were extracted in phosphate buffer and analyzed with horizontal starch gel electrophoresis to determine the effect of heat on migration patterns of soluble proteins, malate dehydrogenase, glutamate dehydrogenase, leucine aminopeptidase, peroxidases and nonspecific esterases. The intensity of staining of soluble proteins from 5-day cotyledons began decreasing at 80–90°; very little staining occurred at 100° with the exception of a distinct band at R_f 1·0. Glutamate dehydrogenase and benzidine peroxidase retained some activity at 80° but other enzymes were inactivated at temperatures near 65°. Differential heat sensitivities of isoenzymes were obvious. Heat did not alter the R_f values of the bands of soluble proteins or enzymes but influenced the intensity of staining. Two-year storage at 4° of viable seed and 33-month storage at -10° of frozen extracts from dormant seed had no influence upon migration patterns of soluble proteins and enzymes assayed.     

Additional lactate dehydrogenase (LDH) isoenzymes in normal testis and spermatozoa of adult man

Summary Adult human testicular tissue contains up to six previously undescribed lactate dehydrogenase (LDH) isoenzymes in addition to the five LDH isoenzymes normally found and the sixth found in spermatogenic cells and spermatozoa, LDH-X. Additional LDH isoenzymes were also found in spermatozoa but not in seminal fluid or in serum. After electrophoresis one additional LDH isoenzyme of testicular tissue was localized between LDH-1 and LDH-2, two between LDH-2 and LDH-3, two between LDH-3 and LDH-4, and two between LDH-4 and LDH-5. These localizations indicate that the additional LDH isoenzymes are tetramers combining the A and B subunits of the five normal LDH isoenzymes and the C subunit of LDH-X. The additional LDH isoenzymes may be important in the metabolism of spermatogenic germ cells and spermatozoa.     

Selective inactivation of lactate dehydrogenase of rat tissues by sodium deoxycholate.

Soluble lactate dehydrogenase (EC 1.1.1.27) extracted from brain, skeletal and cardiac muscle and liver of rats, and purified isoenzymes LDH-1 and LDH-5, were incubated with sodium deoxycholate. Deoxycholate almost totally inactivated isoenzyme LDH-5 (A4), whereas it left isoenzyme LDH-1 (B4) unaffected. Tissue lactate dehydrogenase was inactivated to different degrees depending on the origin of the enzyme. Electrophoretic isoenzyme studies of tissue lactate dehydrogenase showed the loss of activity to be quantitatively related to the overall percentage of subunit A distributed among the homotetramer LDH-5 and the heterotetramers LDH-2, LDH-3 and LDH-4. It was concluded that subunit A of lactate dehydrogenase interacts selectively with deoxycholate, irrespective of its association with subunit B. Distinct changes in electrophoretic mobilities of deoxycholate-treated isoenzymes strongly indicated an indiscriminate binding of deoxycholate by all LDH isoenzymes, probably through hydrophobic interactions. The results suggest that the inactivation of the enzyme is non-competitive, but the basis of the selectivity of deoxycholate towards subunit A is not known at present.     

Changes in the lactate dehydrogenase isoenzyme spectrum during T-lymphocyte differentiation

The pattern of lactate dehydrogenase isoenzyme spectrum changes on different stages of T-lymphocyte differentiation was studied An enriched population of stem cells has LDH-5, 4 and 3 isoenzymes, and much less LDH-2 activity. The isoenzyme pattern of thymic cell precursors consists of LDH-5, 4, 3 and 2. All the five LDH isoenzymes were found in cortical thymocytes. Medullary thymocytes reveal LDH-5, 4 and 3 isoenzymes. T-lymphocytes of peripheral lymphoid organs contain mainly LDH-5 and in a lesser degree LDH-4 activity.     

Lactate dehydrogenase activity and its isoenzyme spectrum in the human pancreas in the prenatal period

Lactate dehydrogenase (LDH) activity and the character of its isoenzyme distribution in pancreas of the human embryos and feti of the 5th-13th-week development were studied. It is shown that LDH activity was rather high already in early periods of the embryonic development, peaks of the enzymic activity were observed after 7-8 and 12-13 weeks. The isoenzymic LDH spectrum was characterized by the presence of four isoenzymes: LDH1, LDH2, LDH3, LDH5. Isoenzyme LDH4 was absent in the human pancreas in all the studied periods of embryonic development. The data obtained evidence for intensity of the glycolysis processes at LDH reaction level in the prenatal ontogenesis period and they reflect most probably the processes of development and differentiation ox cellular populations in the given organ.     

Methods of short-term storage of cultures of the blowfly <Emphasis Type="Italic">Calliphora Vicina</Emphasis> R.-D. (Diptera,Calliphoridae)

Three methods of short-term storage of the blowfly Calliphora vicina strains are considered based on the experimental study of 21 strains originating from different parts of the species range. The colony can be preserved as diapausing adults at 6° and darkness for 2–3 months or more, depending on the geographical origin of the population. During the first five days of adult life the flies should be kept at 12° and short day on a sugar diet, after which they should be transferred into a refrigerator. During artificial hibernation the flies also require periodic sugar feeding every 20 days (3–4 h at 20°C) to maintain their vital functions. The combination of temperatures of 20–23°C and a protein diet terminates reproductive diapause, and oviposition starts in 10–17 days. The fly strain may be preserved as reproductive females at 6°C and darkness with sugar feeding. Flies also require periodic sugar feeding at 20°C (3–4 hours). In this case the flies start laying eggs 2–3 days after being transferred to 20–23°C. The preservation of diapausing larvae is a more reliable method of prolonged strain storage. In this case the flies of maternal generation are maintained at 20–23°C on sugar and protein diet. The egg rafts laid during 5–6 hours are then transferred into 12°C and short day until hatchment. The hatched larvae should be immediately placed into a refrigerator (2–3 or 5–6°C), where they feed during 1–1.5 months and enter diapause. For strain restoration, the diapausing larvae should be transferred into 20–25°C, where they pupariate in 3–5 days and the flies emerge in nearly 10 days.     

Stability evaluation of 6-phosphogluconate dehydrogenase immobilized on amino-functionalized magnetic nanoparticles

Abstract

In this study, 6-phosphogluconate dehydrogenase was covalently immobilized onto the N-2-aminoethyl-3-aminopropyltriethoxysilane (APTES) modified core-shell Fe₃O₄@SiO₂ magnetic nanoparticles (ASMNPs) using glutaraldehyde (GA). Immobilization of 6PGDH on ASMNPs was confirmed using fourier transform-infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. The NADP⁺ conversion ratio, the reusability, thermal, and storage stability of the immobilized 6PGDH were determined and compared with those of the free enzyme. The maximum retention of enzyme activity reached to 96% when the enzyme was immobilized on ASMNPs activated with monomer form of GA. Although the thermal stability of free and immobilized enzymes was similar, at 30?°C, the immobilized 6PGDH showed the improved thermal stability at 40?°C and 50?°C compared with free 6PGDH. While the free 6PGDH only converted 33% of NADP⁺ in reaction medium upon 480?s, the immobilized 6PGDH performed 56% conversion of NADP⁺ at same time. The immobilized 6PGDH retained 62% of its initial activity up to the fifth cycle and 35% of its initial activity after 22?days of storage at 4?°C.     

外源钙对根际低氧胁迫下黄瓜幼苗ADH、LDH活性和同工酶的影响

以‘新泰密刺’黄瓜为材料,采用营养液栽培,外源使用Ca2+、钙离子通道抑制剂La3+与钙调素拮抗剂三氟拉嗪(TFP),研究了钙对根际低氧胁迫下黄瓜幼苗根系ADH、LDH活性和同工酶的影响。结果表明,低氧胁迫诱导产生了新的ADH和LDH同工酶条带。低氧胁迫下,ADH、LDH同工酶丰度和活性显著高于对照;外源增施Ca2+有利于Ca2+信号的形成和逆境信号的传递,营养液添加CaCl2缓解了低氧胁迫对黄瓜植株的伤害,ADH、LDH同工酶丰度和活性接近对照水平;La3+抑制Ca2+的吸收和体内运输,营养液添加LaCl3显著抑制了ADH和LDH同工酶丰度和酶活性,黄瓜幼苗植株生长受到抑制,生物量显著低于低氧处理,表明La3+加重了低氧胁迫对黄瓜幼苗植株的伤害;TFP抑制了低氧逆境胁迫信号的传递,营养液添加TFP抑制了ADH和LDH同工酶丰度和酶活性,ADH和LDH同工酶丰度和酶活性显著低于低氧处理,黄瓜幼苗植株生长受到抑制,黄瓜植株的低氧耐性降低。暗示外源Ca2+参与了低氧胁迫下黄瓜根系无氧呼吸代谢的调节,增强了Ca2+向植物体内的运输,缓解了低氧胁迫对黄瓜幼苗植株的伤害,增强了植物对低氧的耐性。     

A mild methods of general use for covalent coupling of enzymes to chemically activated collagen films

Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable ? COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.     

Effect of temperature and ripening stages on membrane integrity of fresh-cut tomatoes

The shelf-life of fresh-cut tomatoes mainly depends on loss of tissue integrity and firmness that occurs also in intact fruits after long-term cold storage due to chilling injury. Round-fruit tomatoes (Solanum lycopersicum L.) cv. Jama were stored in 1.1-L plastic (polyethylene) fresh-cut produce containers as 10.0-mm-thick tomato slices and as intact tomatoes at 4 ± 0.5 °C. The aim of this work was to study the loss of membrane integrity and biochemical processes involved in membrane disruption. Electrolyte leakage and lipid peroxidation were studied at different stages of maturity: mature green, pink (PK), fully ripe and two different storage temperatures: 4 and 15 °C. The tomato slices of PK stage stored at 4 °C did not show changes for both parameters, while significant increase in membrane leakage and lipid peroxidation was observed at 15 °C, especially after 24 h of storage. The enzymes showed a simultaneous increase in their activities with a rise in electrolyte leakage and lipid peroxidation after 7 days of storage. Finally, phospholipase C (PLC) and phospholipase D (PLD) were investigated for intact fruit and tomato slices stored at 4 °C. The PLC had higher activity compared with PLD. In conclusion, the loss of membrane integrity in fresh-cut tomatoes is mainly affected by ripening stages, storage temperature and duration. The wounds enhance the PLC and PLD activities and they play a role late during storage.     

Amino acid activation of 3-phosphoglycerate dehydrogenase from peas

When l-methionine was added to an extract of etiolated pea epicotyls activation of 3-phosphoglycerate dehydrogenase occurred and was complete in 20–30 min. Storage of extracts at 5° led to a loss in the ability of l-methionine to activate the enzyme and after 24 hr storage almost no activation was observed. On the basis of tests with 16 compounds the ability to activate 3-phosphoglycerate dehydrogenase was restricted to l-amino acids with intermediate-length side chains. There appears to be no requirement for a reactive group in the side chain. Gel-filtration showed that the higher levels of 3-phosphoglycerate dehydrogenase activity obtained after treatment with l-methionine are relatively stable.     

Changes in lactate and malate dehydrogenase isoenzymes in salmonella under the effect of potassium dichloroisocyanurate

The method of enzyme-electrophoresis in agar gel according to Wieme (1959) was used for the study of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) isoenzymes of 24-hour and 48-hour Salmonella cultures exposed to a 0.02% solution of potassium dichloroisocyanurate (PDIC). Severe repression of LDH and MDH isoenzymes was observed immediately after the exposure of the culture to the disinfectant solution. A significant decrease in the content of the isoenzyme LDH1 and of the cytoplasmic fraction (C1) of MDH simultaneously with the appearance of the fractions LDH4, LDH1a and LDH1b were established in the strains cultured on MPA in the course of 24 hours following the exposure. A tendency to a decrease in the LDH1 content was preserved in the experimental cultures after 48 hours, but the spectrum of MDH isoenzymes showed almost no differences in comparison with that of MDH isoenzymes in 48-hour cultures of the control strains.     

Properties of the testicular lactate dehydrogenase isoenzyme.

1. Studies were carried out with pure lactate dehydrogenase isoenzymes C4 (LDH isoenzyme X), B4, (LDH isoenzyme 1) and A4 (LDH isoenzyme 5) isolated from mouse testis, heart and muscle tissue respectively; with LDH isoenzyme X purified from pigeon testes and with crude lysates of spermatozoa from man, bull and rabbit. 2. LDH isoenzyme X from all species showed greater ability than the other isoenzymes to catalyse the NAD+-linked interconversions of 2-oxobutanoate into 2-hydroxybutanoate and of 2-oxopentanoate into 2-hydroxypentanoate. 3. Mouse LDH isoenzyme X presented the broadest spectrum of substrate specificity. It exhibited very similar Km values for a variety of 2-oxo acids: 2-oxopropanoate (pyruvate), 2-oxobutanoate, 2-oxo-3-methylbutanoate, 2-oxopentanoate, 2-oxo-3-methylpentanoate, 2-oxo-4-methylpentanoate, 2-oxohexanoate and 2-oxo-3-phenylpropanoate (phenylpyruvate). The corresponding 2-hydroxy acids were also readily utilized in the reverse reaction. A strong inhibition by substrate and product was demonstrated for the direct reaction. 4. Intracellular distribution of LDH isoenzyme X was investigated in mouse testes. LDH isoenzyme X activity was located in the fraction of "heavy mitochondria" and in the soluble phase. 5. A possible functional role for LDH isoenzyme X is proposed: the redox couple-2-oxo acid-2-hydroxy acid could integrate a shuttle system transferring reducing equivalents from cytoplasm to mitochondria.     

Bioluminescent flow sensors: L-lactate dehydrogenase activity determination in serum

The catalytic activity of serum L -lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L -lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37°C and from 3 to 2000 U/l at 25°C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.