Comparison of properties of mistletoe lectin I A-chain and ricin B-chain conjugate with native toxins.

Chimeric toxin protein was prepared from the mistletoe lectin I A-chain and ricin B-chain by using the disulfide exchange reaction. Ricin and chimeric protein were indistinguishable in binding to immobilized asialofetuin in ELISA. Chimeric protein was more toxic to Jurkat cells than native mistletoe lectin I, but not so effective as native ricin. In the presence of NH4Cl, which enhances the toxicity of some toxins and immunotoxins, but does not influence ricin toxicity, both ricin and chimeric toxin had equal cytotoxic activity. The possibility is discussed that the ricin B-chain protects the ricin A-chain (RTA) from degradation during delivering RTA from the cell surface to the place where RTA is translocated into the cytosol.

Immunotoxin with mistletoe lectin I A-chain and ricin A-chain directed against CD5 antigen of human T-lymphocytes; comparison of efficiency and specificity.

Monoclonal anti-CD5 antibody was coupled to the enzymatically active subunit of plant toxin [either mistletoe lectin I (ML) or ricin]. The obtained conjugates proved to be selectively toxic to CD5-bearing target cells. The immunotoxin prepared from ML A-chain (MLA) was as toxic as native ML and approximately 80-fold more active than the corresponding conjugate with ricin A-chain (RTA). The comparative studies of the structural properties of isolated MLA and RTA were carried out using intrinsic fluorescence spectroscopy. The results showed similar properties for both proteins. No antigenic cross-reactivity against both toxins was detected when using polyclonal antibodies. The results suggest that MLA-antibody conjugates may be potential candidates for therapeutical use.

[Activity of gluconeogenetic enzymes in swine myocardium and skeletal muscles]. / Aktivität glukoneogenetischer Enzyme in der Herz- und Skelettmuskulatur von Schweinen.

The activity of the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) was determined radiometrically in heart and skeletal muscle (M. semitendinosus) of 21 fetuses of the last third of gestation (80th-112th day), 17 piglets from birth until the 9th day of life and 7 fattening pigs. Simultaneously the activity of the enzymes glucose-6-phosphatase (G6Pase) and fructosebisphosphatase (FDPase) was measured colorimetrically in heart and skeletal muscle of piglets and in skeletal muscles of fattening pigs. Heart and skeletal muscle have only a low PEPCK activity. During the last third of gestation PEPCK in heart remains on a constant level, which can be demonstrated also in fattening pigs, but doubled immediately after birth. During the last two weeks of gestation, at birth and during the first days of life the PEPCK activity in the skeletal muscle is 3-fold higher than at the 80th day of gestation and in fattening pigs. G6Pase and FDPase activity of the heart remains at a constant level during the first days of life. It was impossible to detect G6Pase in the skeletal muscle of piglets. The specific FDPase activity of the skeletal muscle remains constant also postnatally. In fattening pigs skeletal muscles with different types of fibres have the same FDPase and PEPCK activity.

[Postnatal development of gluconeogenic key enzymes, phosphoenolpyruvate carboxykinase and pyruvate carboxylase, in pig liver]. / Postnatale Entwicklung der glukoneogenetischen Schlüsselenzyme Phosphoenolpyruvatkarboxykinase und Pyruvatkarboxylase in der Schweineleber.

Using a radiochemical method the activity of the gluconeogenic key enzymes phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) was determined in liver homogenates of 58 piglets (from birth to 10 days of life), 4 weaners and 18 fattening pigs. Simultaneously, the cytosolic PEPCK activity was estimated. In liver of newborn piglets (immediately after birth without suckling) total PEPCK activity is one half, cytosolic PEPCK activity one sixth and PC activity one half of that occurring in adult pig's liver. During regular suckling within the first 12 h of life, the total PEPCK activity of piglet liver rises slowly, however, cytosolic PEPCK activity increases 3 to 4 times and PC activity in the mitochondria twice. After the 2nd day of life the activity of both enzymes reaches a level which is kept during the whole suckling period: total PEPCK activity is 1.5 to 2-fold, cytosolic PEPCK activity is 2 to 3-fold higher, and PC activity is at the level existing at birth. In weaners cytosolic PEPCK activity further rises to the level existing in the liver of adult pigs. The PEPCK and PC activities in the liver at birth enables the piglet for gluconeogenesis, but due to the postnatal increase of enzyme activities, the prerequisites for an intensive synthesis of glucose are given only after the 2nd day of life.

[Perinatal development of the pyruvate metabolism-regulating enzyme in the pig liver]. / Pränatale Entwicklung der den Pyruvatumsatz regulierenden Enzyme in der Schweineleber.

The gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) and the glycolytic enzyme pyruvate kinase (PK), regulating pyruvate metabolism, were determined in the livers of 71 fetuses, which were developed of 25 dams on the 80th, 100th, 106th, and 112th day of gestation (length: 115 days) by Caesarean section. For comparative purposes the same enzymes were estimated in 12 naturally born piglets immediately after delivery. During the period under examination (the last third of gestation) the total activity of PEPCK has its highest values at the 80th day, the PC and PK activity at the 100th day of gestation. The activities of all 3 enzymes decrease during the last 2 weeks of gestation until birth. The cytosolic part of PEPCK amounts to 10-15 per cent of total activity and develops in a parallel manner. In newborn piglets a further decline of the PC and total PEPCK activity can be observed, but the cytosolic PEPCK activity remains constant, and therefore its relative proportion increases to 25% of the total activity. The PK activity rises distinctly (1.5 times). The role of these enzymes in the carbohydrate metabolism of the fetus, especially in gluconeogenesis, is discussed in detail.

[Activity of gluconeogenetic enzymes in the kidney of pigs during pre- and post-natal development]. / Verhalten von glukoneogenetischen Enzymen in der Niere des Schweines während der prä- und postnatalen Entwicklung.

In pig fetuses (19 of 8 dams) developed by Caesarean section the dry matter and protein content of the kidneys and their PEPCK activity remain constant during the last third (from 80th to 112th day) of gestation. After birth the dry matter content of the kidneys rises slowly, but their protein content remarkably. In the kidneys of suckling piglets (17 animals of 3 offsprings) the FDPase activity remains at the same level from birth to the 9th day of life, while in the same time the G6Pase activity rises 1.5-2 times. In the kidneys of newborn piglets the total PEPCK activity increases 3-4 times and the activity of the cytosolic enzyme 2-3 times during the first 12 hours of life. At the end of the first week of life the total PEPCK activity decreases by one-third, while the activity of the cytosolic enzyme remains stable. In the kidneys of slaughter pigs (n = 7) the dry matter content and the FDPase activity are significantly higher, the protein content and the G6Pase activity are the same as in the kidneys of piglets. The total PEPCK activity is one-half, the activity of the cytosolic enzyme one-third lower than in the kidneys of piglets. In the kidneys of adult pigs the PEPCK activity is localized to equal parts in the cytosol and in the mitochondria, but in some development stages the mitochondrial part exceeds that of the cytosol. In adult pigs the PEPCK activity of the renal cortex is 2.5-3 times higher than that of the renal medulla.

[Behavior of various blood constituents (glucose, fructose, insulin, lactate, pyruvate, free fatty acids, inorganic phosphate) and half-life of monosaccharides in the plasma after i.v. infusion of glucose, fructose, galactose and invert sugar solutions in ruminants. 1. Studies in calves and heifers]. / Untersuchungen über das Verhalten verschiedener Blutbestandteile (Glukose, Fruktose, Insulin, Laktat, Pyruvat, freie Fettsäuren, anorganisches Phosphat) und über die Halbwertszeit der Monosaccharide im Blutplasma nach i.v. Infusion von Glukose-, Fruktose-, Galaktose-und Invertzuckerlösung bei Wiederkäuern.

Studies into the half-life of monosaccharides as well as into the effects of intravenous infusion of sugar solutions (0.5 g/kg body weight of glucose, fructose, galactose, and invert sugar) on the concentration of various blood components were undertaken with six calves and five heads of young cattle. Half-life values of glucose and galactose were of nearly identical magnitude over the first weeks of age, whereas that of fructose was much longer on account of slower conversion. Infusion of invert sugar solution, therefore, cannot be recommended for calves during the first three weeks of age. Infusion of the above monosaccharides led to more or less strongly pronounced rise of insulin levels in the blood serum. More or less strongly marked pyruvate increase took place following infusion, if the pyruvate concentration in the blood plasma had been low before infusion. Lactate levels in the blood usually underwent little change, whereas the levels of free fatty acids and inorganic phosphate in the blood plasma usually were reduced.

[Behavior of various blood constituents (glucose, fructose, insulin, lactate, pyruvate, free fatty acids, inorganic phosphate) and half-life of monosaccharides in the plasma after i.v. infusion of glucose, fructose, galactose and invert sugar solutions in ruminants. 2. Studies in cattle]. / Untersuchungen über das Verhalten verschiedener Blutbestandteile (Glukose, Fruktose, Insulin, Laktat, Pyruvat, freie Fettsäuren, anorganisches Phosphat) und über die Halbwertszeit der Monosaccharide im Blutplasma nach i.v. Infusion von Glukose-, Fruktose-, Galaktose- und Invertzuckerlösung be Wiederkäuern. 2. Mitteilung: Untersuchungen an Rindern.

Monosaccharides were intravenously injected to eight adult heads of cattle, between 380 kg and 670 kg in live weight, to study, in the context of stress endurance, the half-life values of the sugars as well as monosaccharide effects upon concentrations of various blood components. The fructose concentration in the blood plasma went up temporarily following the infusion of glucose solution. Fructose infusion usually caused only little rise of the glucose concentration in blood plasma, with hypoglycaemia occurring quite often towards the end of an experimental period. The half-life values of sugar in blood plasma were between twelve and 29 and those of fructose between ten and 17 minutes. The rate of fructose conversion was higher than that of glucose conversion, but values were identical in some cases. The pyruvate concentration in the blood and the insulin level in blood plasma went up in response to infusion of monosaccharide solutions. Urine excretion of monosaccharides following invert sugar infusion was less than half of that in response to glucose infusion.

[Radiochemical method for the determination of the activity of the gluconeogenetic key enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase in swine livers]. / Radiochemische Methode zur Bestimmung der Aktivität der glukoneogenetischen Schlüsselenzyme Pyruvatkarboxylase und phosphoenolpyruvatkarboxykinase in der Schweineleber.

Described in this paper is a technique for the determination of pyruvate carboxylase and phosphoenolpyruvate carboxykinase, two key enzymes of gluconeogenesis in swine liver. The technique is based on measurement of radioactively labelled carbon incorporated in the common metabolite, oxalacetate. The optimum measuring conditions to establish the enzymes in liver homogenate and supernatant are reported and compared with data given by other authors. The found parameters of kinetic properties were in good agreement with the findings obtained from purified enzymes from swine liver.