Characterization of pyruvate kinase of Trypanosoma brucei and its role in the regulation of carbohydrate metabolism.

Pyruvate kinase from Trypanosoma brucei is a labile enzyme, losing its activity within several hours. In mixtures containing 50 mM triethanolamine buffer, pH 7.2, 25% glycerol and 0.

Chemical modification of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

Chemical modifications of Class I aldolases from Trypanosoma brucei, rabbit muscle and Staphylococcus aureus with carboxypeptidase A, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. Aldolase from S. aureus was not affected by any of these reagents.

Kinetic properties of fructose bisphosphate aldolase from Trypanosoma brucei compared to aldolase from rabbit muscle and Staphylococcus aureus.

The kinetic properties of aldolase from Trypanosoma brucei were studied in comparison with aldolase from rabbit muscle and Staphylococcus aureus. The 3 enzymes displayed a similar broad pH optimum for the cleavage of fructose 1,6-bisphosphate (Fru(1,6)P2) and a similar narrow pH optimum for the cleavage of fructose 1-phosphate (Fru-1-P). However, small alterations in the maximal cleavage rate at more extreme pH values yielded disparities between the pH curves.

The cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei. Kinetic properties and comparison with homologous enzymes.

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms.

The cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in Trypanosoma brucei have a distant evolutionary relationship.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde-3-phosphate dehydrogenase: one enzyme resides in a microbody-like organelle, the glycosome; the other is found in the cytosol. Previously we have reported the characterization of the gene for the glycosomal enzyme [Michels, P. A.

Influence of chronic administration of valproate on ultrastructure and enzyme content of peroxisomes in rat liver and kidney. Oxidation of valproate by liver peroxisomes.

Chronic administration to rats of the anticonvulsant drug, valproate, induced proliferation of liver peroxisomes and selectively increased the activity of the enzymes involved in beta-oxidation in these organelles. In kidney cortex, only a moderate increase in enzyme activity could be recorded. Valproate (1% w/w in the diet for 25 to 100 days) caused the appearance on electron micrographs of unusual tubular inclusions in the matrix of liver peroxisomes.

ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions.

The virulence functions of Yersinia enterocolitica include the pYV-encoded Yop proteins and YadA adhesin as well as the chromosome-encoded enterotoxin, Yst. The yop and yadA genes form a temperature-activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes.

Radiochemical assay of adenylosuccinase: demonstration of parallel loss of activity toward both adenylosuccinate and succinylaminoimidazole carboxamide ribotide in liver of patients with the enzyme defect.

A radiochemical assay for adenylosuccinase, an enzyme which intervenes twice in the biosynthesis of adenine nucleotides, has been developed. The two substrates of the enzyme, succinylaminoimidazole carboxamide ribotide (SAICAR) and adenylosuccinate (S-AMP), were synthesized in radioactive form by incubating [2,3-14C]fumarate and, respectively, AICAR and AMP with partially purified adenylosuccinase from yeast. Enzyme activities were determined by measuring the release of labeled fumarate after its separation from the substrate by chromatography on polyethyleneimine thin-layer plates.

Fructose administration stimulates glucose phosphorylation in the livers of anesthetized rats.

A method allowing one to measure the rate of glucose phosphorylation in the livers of anesthetized rats is described. Upon injection of [2-3H]glucose into the portal vein, about 90% of the radioactivity remained in the liver for approximately 30 s. The proportion of radioactivity accounted for by tritiated water increased linearly at a rate of about 5%/min in control animals.

Glucose uptake by Trypanosoma brucei. Rate-limiting steps in glycolysis and regulation of the glycolytic flux.

Glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite Trypanosoma brucei was measured using 14C-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. Glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. Glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glycolytic rate at external glucose concentrations (glcout) below 5 mM.

The evolutionary origin of glycosomes.

The glycolytic pathway of the Kinetoplastida is organized in a unique manner: the majority of its enzymes are contained in organelles called glycosomes. In this article Paul Michels and Fred Opperdoes argue that the glycosomes are equivalent to the microbodies and peroxisomes identified in other eukaryotic cells. They explore the possible evolutionary origin of the glycosome by comparing many of its structural and functional properties with those of other members of the microbody family and with some features of other organelles, the mitochondria and chloroplasts, which have been studied in much more detail.

Thyroid hormone receptors in brain and liver during ageing.

The binding properties--binding capacity (MBC) and affinity (Ka)--of T3 nuclear receptors were analyzed in cortex, cerebellum and liver of rats aged 3, 6, 12 and 24 months. A slight but not significant decrease of Ka was observed in different tissues of normal rats. In hypothyroid animals the Ka in cortex at 24 months was significantly lower than at 3 months.

Purine catabolism in polymorphonuclear neutrophils. Phorbol myristate acetate-induced accumulation of adenosine owing to inactivation of extracellularly released adenosine deaminase.

Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.

The role of the liver in metabolic homeostasis: implications for inborn errors of metabolism.

The mechanisms by which the liver maintains a constant supply of oxidizable substrates, which provide energy to the body as a whole, are reviewed. During feeding, the liver builds up energy stores in the form of glycogen and triglyceride, the latter being exported to adipose tissue. During fasting, it releases glucose and ketone bodies.

Possible role and mechanism of action of dissolved calcium in the degradation of bone collagen by lysosomal cathepsins and collagenase.

Equilibrium experiments with bone powder, at pH values ranging from 6.3 to 3.5, show a linear relation between log([Ca2+]/[Ca2+]0) (where [Ca2+]0 = 1 M-Ca2+) and pH, indicating that [Ca2+] could reach levels of 25 mM at pH 5 and 90 mM at pH 4.

Anti-cholera toxin IgA-, IgG- and IgM-secreting cells in various rat lymphoid tissues after repeated intestinal or parenteral immunizations.

Single antibody-secreting spot-forming cells (SFC) of the 3 main isotypes were counted in lymphoid cells from the gut lamina propria (LP), Peyer's patches (PP), mesenteric nodes (MN) and spleen (SP) of rats immunized 2-6 times intraduodenally (ID) or intraperitoneally (IP) with cholera toxin (CT). Responses for all isotypes peaked in all tissues after 4 ID- or IP-immunizations at much larger values than previously reported, and significantly decreased thereafter, except in LP and PP after IP-injections, where IgA- or IgG-SFC, but not IgM-SFC, only appeared or increased after 6 IP-doses. The highest IgA-SFC numbers (17% of tested cells) were in LP after 4 ID-doses.

Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes.

Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.

A specific defect in CD3 gamma-chain gene transcription results in loss of T-cell receptor/CD3 expression late after human immunodeficiency virus infection of a CD4+ T-cell line.

Sequential effects on cellular protein expression following human immunodeficiency virus (type 1) infection of a CD4+ T-cell line in vitro were investigated. Events in the human interleukin 2-dependent helper T-cell line WE17/10 are similar in several respects to the clinical progression in acquired immunodeficiency syndrome. WE17/10 cell infection is characterized by an extended period during which viral replication occurs without accompanying cytotoxicity and with a maximum 30% decrease in surface CD4.