Purification and properties of three pig erythrocyte carbonic anhydrases

Three distinct forms of the zinc containing enzyme carbonic anhydrase were isolated from pig erythrocytes. One low activity type enzyme and two genetic variants of the high activity type enzyme with identical CO₂ hydratase activities which were 8 times as high were isolated from Danish Black and White Swine. In the isolation procedure described, the hemoglobin was eliminated by precipitation with chloroform-ethanol, and the isoenzymes were separated by DEAE-Sephadex chromatography. A number of enzymatically active minor components were separated. They were apparently all genetically linked to one of the three major components. The three purified isoenzymes behaved as homogeneous components during isoelectric focusing and electrophoresis at different pH values. They were characterized in terms of molecular weight, isoelectric pH, zinc content, amino acid composition, and enzymatic activity against CO₂, p-nitrophenyl acetate, and β-napthyl acetate. The circular dichroism of the enzymes in the ultraviolet region was studied. The properties of the enzymes were similar to those of carbonic anhydrases of corresponding types isolated from other mammalian species. Sulphur containing amino acid residues were absent in the low activity type enzyme. The amino acid composition of the two high activity mutants deviated only in that an arginine residue in the most widespread genetic variant was replaced by a histidine residue in the less frequent variant. Otherwise the two mutants showed identical properties.Keyword: carbonic anhydrase, pig, erythrocyte, isoenzymes, genetic variants, DEAE-Sephadex, isoelectric pH, amino acid composition, CO₂ hydratase activity, esterase activity, circular dichroism     

Activity and properties of alkaline phosphatase in the plasma and various organs (kidney, liver, small intestine mucosa, bone) of the swine]

There was a high activity of alkaline phosphatase in the blood plasma of piglets during the first few days of live; enzyme obtained at this time had high heat stability and was readily inhibited by L-phenylalanine (5 mM). The enzyme in blood was inhibited to a greater extent than alkaline phosphatase from intestinal mucosa. With increasing age there was a fall in heat stability and in the ease with that the enzyme could be inhibited by phenylalanine. The proportion of alkaline phosphatase derived from bone and present in blood plasma increased with increasing age. Two isoenzymes were detected in liver, kidney, lung, intestinal mucosa and endometrial mucosa by electrophoresis in polyacrylamide gel. Heat lability and inhibition by phenylalanine were good criteria for differentiating different types of alkaline phosphatase in pigs. In the case of alkaline phosphatase in blood plasma, disodium phenylphosphate was split more readily than p-nitrophenyl phosphate and very much more readily than phenolphthalein diphosphate and beta-glycerophosphate.     

Properties and isoenzymes of acid phosphatase in erythrocytes and various tissues (kidney, liver, spleen, small intestine mucosa, testis, myocardial and skeletal musculature) of cattle]

The substrate that was split most rapidly by acid phosphatase was p-nitrophenylphosphate. Two peaks of activity were obtained at pH 4.6-4.8 and 5.1-5.4. The enzyme remained stable for a long time when refrigerated. It was inhibited strongly by urea and tartrate, and slightly by fluoride and L-phenylalanine. Mercaptoethanol elicited pronounced activation of the enzyme. Four different forms of isoenzyme, giving rise to 11 phenotypes, were identified. A suitable analytical technique was electrophoresis on polyacrylamide gel with phosphate-citrate buffer. Mean activity was 3.15 +/- 0.41 units per gramme of haemoglobin haemolysate. Some of the isoenzyme preparations showed considerable variation in activity. There was no change in enzyme activity after temporary hypomagnesaemia. Acid phosphatase activity was high in testis, kidney and intestinal mucosa; myocardium, liver and spleen showed moderate activity. Five isoenzymes were demonstrable in a starch column and six in PAA gel.     

Lactate dehydrogenase isoenzymes in the lungs of sheep with acute and chronic pneumonia

Total lactate dehydrogenase and the absolute and percentage levels of its isoenzymes were measured in lung lesions and macroscopically normal areas of lung from lambs with chronic proliferative exudative pneumonia and acute pasteurella pneumonia. Lung lesions had a higher total enzyme activity which was associated mainly with increases in the activity of the LDH4 and LDH5 isoenzymes, particularly in chronic pneumonia, and gave lung lesions a considerable potential for altering the serum isoenzyme distribution. Thus, the nature of any changes in the serum isoenzyme distribution will depend on whether the isoenzymes are released from abnormal or normal areas of lung. This appears to be the first report on lactate dehydrogenase isoenzymes in ovine pneumonia.     

Lactate dehydrogenase isoenzymes in the lungs of sheep with acute and chronic pneumonia

Total lactate dehydrogenase and the absolute and percentage levels of its isoenzymes were measured in lung lesions and macroscopically normal areas of lung from lambs with chronic proliferative exudative pneumonia and acute pasteurella pneumonia. Lung lesions had a higher total enzyme activity which was associated mainly with increases in the activity of the LDH₄ and LDH₅ isoenzymes, particularly in chronic pneumonia, and gave lung lesions a considerable potential for altering the serum isoenzyme distribution. Thus, the nature of any changes in the serum isoenzyme distribution will depend on whether the isoenzymes are released from abnormal or normal areas of lung. This appears to be the first report on lactate dehydrogenase isoenzymes in ovine pneumonia.     

Isoenzymes of alkaline phosphatase in serum of lambs and ewes.

Electrophoresis in polyacrylamide gels was used to identify isoenzymes of serum alkaline phosphatase in sheep. Skeletal isoenzyme predominated in the serum of lambs and liver, skeletal and intestinal isoenzymes were found in the serum of adult ewes. In r ewes (R-r-i blood group system) intestinal isoenzyme activity was 67 per cent of total serum activity; in R ewes intestinal activity was only 36 per cent of total activity. Relative activities of the three isoenzymes differed greatly within individual ewes and between individual ewes during pregnancy and lactation.     

Alkaline phosphatase isoenzymes in feline serum using an agarose gel alkaline phosphatase kit method.

Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercially available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats (< 1 year of age) have a greater proportion of the bone isoenzyme in the serum.     

Canine serum alkaline phosphatase isoenzymes detected by polyacrylamide gel disk electrophoresis

Serum alkaline phosphatase (ALP) isoenzymes were studied in normal dogs using a commercially available polyacrylamide gel disk electrophoresis kit (PAG/disk kit). Serum samples taken from the dogs were incubated with neuraminidase, after which most showed ALP isoenzymes as two characteristic stained bands. To determine the origin of each band, ALP isoenzymes of serum and tissue extracts (liver, intestine and bone) were characterized by heating, wheat germ agglutinin (WGA) and levamisole treatments. The results suggested that the band detected on the anode was liver ALP (LALP) and that the band detected on the cathode represented bone ALP (BALP), and both were corticosteroid-induced ALP (CALP). The percentage of each ALP isoenzyme to total ALP activity was estimated by densitometry. The percentage of BALP was the highest in young dogs (age<1 year, 64.7% ), and this value decreased with age. In contrast, the percentage of LALP in young dogs (22.2%) was much lower than that in middle-aged dogs (ages 1 year to 7 years, 59.3%) and old dogs (ages>7 years, 50.4%). The present results suggested that a commercially available PAG/disk kit is capable of detecting three serum ALP isoenzymes in dogs, and further that it may have clinical applications in the evaluation of ALP isoenzymes in veterinary medicine.     

Effects of physical stress on serum enzymes and other blood constituents in sheep

In connection with transfer of sheep from the lowland near Oslo to mountain pastures at an altitude of 1,200 m above sea level, investigations were carried out in 37 animals to study the effect of physical stress on serum enzymes and other blood constituents. The sheep were adult ewes and lambs. About half of the animals had been accustomed to outdoor life on pasture for more than one month, while the others were moved directly from indoor feeding. Blood was collected before departure, after six hrs. of long-distance transportation by lorry, and after three hrs. of subsequent continuous herding on foot. The following blood components were determined: Aspartate aminotransferase (AspAT = GOT), alanine aminotransferase (A1AT = GPT), α-hydroxybutyrate dehydrogenase (HBD), total lactate dehydrogenase (LDH), LDH isoenzymes, alkaline phosphatase, calcium, inorganic phosphorus, magnesium, blood sugar, total serum proteins, and haemoglobin.In summary, it may be said that the lambs reacted with greater changes of the blood components than adult animals, and that untrained, indoor fed lambs were distinctly more sensitive than those taken from pasture. The “indoor” lambs showed a statistical significant increase from the initial values in AspAT, HBD, total LDH, the isoenzymes LDH₃ and LDH₄, and blood sugar. Significantly decreased values were recorded in Ga, P, Mg, and total serum protein. Some of these changes, as in Mg and P, were most pronounced after transportation, while elevations of serum enzyme levels continued to increase during the subsequent herding.Based upon the shift in LDH isoenzyme distribution towards a more cathodically dominated pattern it is supposed that the main origin of increased serum enzyme activity was skeletal muscle.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum.The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.     

Alkaline phosphatase and its isoenzymes in the tissues and sera of normal dogs

The total alkaline phosphatase (AP) activity and the pattern of its isoenzymes were studied in the tissues and sera of normal adult dogs. Small intestine mucosa showed the greatest total AP activity followed by kidney, bone, pancreas, liver, lung, skeletal muscle and heart muscle. After separation by agarose gel electrophoresis, each tissue showed only one isoenzyme except lung which showed two. The tissue isoenzymes, in decreasing order of migration distance towards the anode, were as follows: fast lung isoenzyme, liver or slow lung isoenzyme, the group consisting of skeletal muscle, bone, small intestine and pancreas isoenzymes and, finally, the kidney isoenzyme. Two isoenzymes occurred in serum. The major band corresponded to liver and the slow lung isoenzyme, while the minor band was considered to be the corticosteroid-induced isoenzyme, previously thought to be absent from normal serum. The AP isoenzyme patterns in lung and skeletal muscle and the presence of an isoenzyme migrating an identical distance to the corticosteroid-induced isoenzyme do not appear to have been reported before in normal dogs.     

Alkaline phosphatase and alkaline phosphatase isoenzymes in the cat

Feline alkaline phosphatase and alkaline phosphatase isoenzymes have been studied in tissue and serum. Alkaline phosphatase from various organs was quantitated and then subjected to cellulose acetate electrophoresis. The effects of bile duct ligation, prednisolone treatment and phenobarbital treatment on serum alkaline phosphatase was measured. The diagnostic importance of feline serum alkaline phosphatase levels is discussed in light of the results of this and other studies.     

Biochemical aspects of the toxic effects of Supermethrin and the histochemical activity of alkaline phosphatase, acid phosphatase and non-specific esterase in subchronic poisoning in sheep]

Fifteen Slovak Merino sheep were included in the experiment. The animals weighing 21-28 kg were divided into three groups per five animals. In a six-week feeding experiment the animals of group I were given 50 mg supermethrin per kg live weight per day while those of group II received 200, and from week four of the experiment 300 mg supermethrin per kg live weight per day. During the experiment changes of aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), acetylcholine esterase (EC 3.1.1.7), urea und creatinine levels in blood serum were observed. Six weeks after supermethrin treatment the sheep were slaughtered and histochemical evaluation of alkaline phosphatase (EC 3.1.3.2), acid phosphatase (EC 3.1.3.1) and non-specific esterase (EC 3.1.1.1) was carried out in liver, kidney, duodenum, jejunum and ileum. In the course of the experiment changes of the enzymatic activities of aspartate aminotransferase observed in both experimental groups of sheep were similar to those seen in the control group of animals (Tab. I). As compared to the starting values, no significant changes in the activity of alanine aminotransferase were observed in group II of the experiment and in the controls. However, a significantly decreased alanine aminotransferase activity could be seen in the blood serum of sheep of group I (Tab. II). In both experimental groups of animals no significant changes in the acetylcholine esterase could be seen (Tab. III). As compared to the starting values, no significant changes were observed in creatinine levels of the control and the 1st experimental group of sheep (Tab. IV). In the sheep of the 2nd group a temporary significant decrease (p < 0.05) in creatinine levels was seen. The dynamics of urea levels was similar to starting values in all animals throughout the experiment Tab. V). In the control group of animals (Fig. 1) the high density of reaction product of alkaline phosphatase was determined in the microvilli of enterocytes of the small intestine. In the small intestine of the animals of both experimental groups, the activity of this enzyme was shown to be located in the same zone (Fig. 2). In all experimental animals in the parenchyma of the liver and kidney no significant changes could be observed. In both experimental and control animals the high activity of acid phosphatase was demonstrated to be located especially in the cytoplasma of enterocytes. The activity of non-specific esterase was located in the cytoplasma of enterocytes of the small intestine, in the intestinal crypts its activity was slight up to high.(ABSTRACT TRUNCATED AT 400 WORDS)     

外源NO及反向调控PEG胁迫下苜蓿萌发种子抗氧化酶及其同工酶动态的研究

以紫花苜蓿为材料,用一氧化氮供体硝普钠(SNP)及一氧化氮清除剂c-PTIO对苜蓿种子进行浸种处理,采用分光光度法和同工酶电泳技术来研究外源NO及反向调控对PEG胁迫下紫花苜蓿抗氧化酶活性及其同工酶的影响,并探讨NO调控苜蓿种子耐旱性的生理机制。结果表明:PEG胁迫下外施0.1 mmol·L^-1 SNP,第6天时较PEG处理POD活性降低了32.72%;第4天时SOD、CAT活性较PEG处理升高了10.48%、23.60%,有效缓解了PEG对紫花苜蓿萌发中种子的氧化损伤,提高其抗氧化能力。PEG胁迫下添加 c-PTIO抑制了苜蓿萌发期的抗氧化系统活性。从同工酶的谱带数量和强弱来看,POD同工酶各区带活力均随PEG胁迫时间的延长而增加,在第2天酶带只有1条,而第4天酶带呈现9条;SOD和CAT同工酶表达量变化不显著,但酶带强弱有一定变化,S3酶带随处理时间的延长表达量逐渐减弱;CAT同工酶谱带则一直保持2条带,无明显强弱变化。因此,外源NO在PEG胁迫下紫花苜蓿萌发中抗氧化酶快速响应并在维护氧自由基代谢平衡中起重要保护作用。     

Isoenzymes of alkaline phosphatase in serum of newly born lambs.

In the serum of lambs at birth most of the circulating alkaline phosphatase, identified by electrophoretic analysis, was of skeletal origin. Suckling was associated with a rapid increase in activity of intestinal alkaline phosphatase isoenzymes in serum. There was a coincidental increase in level of circulating gamma globulin. Activity of the intestinal isoenzymes in serum began to fall between 15 and 21 h after birth and this fall preceded a fall in serum gamma globulin concentration. The electrophoretic characteristics of the intestinal isoenzymes in serum changed as activity of these isoenzymes increased and then decreased.     

红豆草空间诱变突变体叶片同工酶及细胞超微结构分析

经神州4号飞船搭载的红豆草种子,当代出现匍匐型突变体,其叶片同工酶谱及细胞超微结构的分析结果表明:匍匐型突变体、空间诱变直立型植株及对照之间过氧化物酶同工酶酶带数没有明显差异,其中匍匐型突变体脂酶同工酶酶带数与对照相同,空间诱变直立型植株酶带数多于对照;空间诱变的红豆草,叶片细胞壁不规则增厚,细胞质稀薄,液泡大,叶绿体变小,形状多不规则,叶绿体内淀粉粒细小、数量多,基粒片层直径小,但数量明显多于对照,匍匐型突变体表现尤为明显.     

Isoenzymes of equine alkaline phosphatase

Alkaline phosphatase isoenzymes from small intestine, cecum, large colon, small colon, liver, kidney, leukocytes, and serum from ten clinically normal horses were defined by their sensitivities to L-phenylalanine, L-homoarginine, levamisole and heat, and by polyacrylamide gel disc electrophoresis. Readily identifiable isoenzymes occurred in small intestine, granulocytes, kidney, cecum, and large and small colon. By contrast, alkaline phosphatases from liver, lymphocytes, and serum could not be discriminated by this group of tests.     

Lactate dehydrogenase and creatine kinase isoenzyme levels in the tissues and serum of normal lambs

The normal activities of lactate dehydrogenase and creatine kinase isoenzymes in the tissues and serum of clinically normal five-and-a-half-month-old lambs are presented. The percentage distribution of the lactate dehydrogenase isoenzymes in serum, liver, heart, lung and skeletal muscle were in agreement with previous studies but the distribution in ovine abomasum, small intestine, large intestine and red blood cells has not been previously described. Up to five creatine kinase isoenzymes were detected in ovine tissues and four in serum. One creatine kinase isoenzyme present in serum and tissues was thought to represent a mitochondrial isoenzyme which is absent from the normal serum of other species. Estimations of both lactate dehydrogenase and creatine kinase isoenzymes allowed all the tissues examined to be distinguished and were therefore more likely to allow tissue-specific isoenzyme patterns to be detected in serum than the estimation of the isoenzymes of either enzyme alone.     

Evidence for the presence of nematode-derived acetylcholinesterase in sheep infected with Trichostrongylus colubriformis

The distribution of acetylcholinesterase isoenzymes in the ovine intestinal nematode Trichostrongylus colubriformis was compared with that in chronically infected and worm-free lambs. Total acetylcholinesterase activity in homogenates of adult T colubriformis was resolved into five isoenzyme peaks following gel electrophoresis and specific esterase staining. Two isoenzymes with electrophoretic mobilities similar to those present in adult worm homogenates were detected in mucosal homogenates and plasma extracts from all of six sheep chronically infected with T colubriformis, but not in similar preparations from two uninfected animals.     

Diagnostic value of alkaline phosphatase isoenzyme separation by affinity electrophoresis in the dog.

Affinity electrophoresis, using wheat germ lectin, was used to separate the alkaline phosphatase isoenzymes in the sera of 150 dogs with alkaline phosphatase values greater than or equal to 150 IU/L. The method provided clearer separation of the liver, bone and steroid-induced alkaline phosphatase isoenzymes commonly observed in canine serum, compared to conventional cellulose acetate electrophoresis. The dogs were divided into four patient groups determined by previous corticosteroid treatment, evidence of elevated endogenous corticosteroid levels, age and alanine aminotransferase values. The isoenzyme pattern of each patient was qualitatively assessed. The isoenzyme pattern most frequently observed was greater than 50% steroid induced alkaline phosphatase, which was present in 76 of 150 dogs. This pattern was observed in 18 of 22 dogs receiving corticosteroid therapy, two of three dogs with hyperadrenocorticism, and in dogs with a variety of other diagnoses. The majority of immature dogs (12 of 20) had an isoenzyme pattern consisting of greater than 50% bone. The majority of dogs with active hepatocellular injury (16 of 27) had greater than 50% liver isoenzyme. The isoenzyme pattern was not specific for certain diseases, therefore the diagnostic usefulness is limited. However the isoenzyme result is useful in some cases to determine which further diagnostic tests are indicated, and to determine the source of alkaline phosphatase elevation.