Immunoaffinity purification of rat liver transketolase: evidence for multiple forms of the enzyme (original) (raw)
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Journal of Histochemistry and Cytochemistry, 2006
Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver par...
Heterologous expression of human transketolase
The International Journal of Biochemistry & Cell Biology, 1998
Transketolase belongs to the family of thiamin diphosphate dependent enzymes. The aim of this study was to establish a bacterial expression system for human transketolase in order to investigate the functional characteristics of mammalian transketolases. The level of recombinant human enzyme expressed in Escherichia coli was modest. Puri®cation of recombinant transketolase and separation from the E. coli enzyme has been greatly simpli®ed by means of a non-cleavable hexa-histidine tag. The highest speci®c activity was 13.5 U/mg and the K m values were 0.27 20.02 and 0.51 20.05 mM for the substrates D-xylulose 5-phosphate and D-ribose 5-phosphate, respectively. Binding of cofactors to the apoenzyme showed the expected hysteresis. Without preincubation, the K m values for thiamin diphosphate and for Mg 2+ were, respectively, 4.120.8 and 2.5 20.4 mM, but after 1 h of preincubation these values were 85 216 nM and 0.74 20.23 mM. The kinetic constants are similar to those of the native enzyme puri®ed from human erythrocytes. Despite the modest expression level the reported system is well suited to a variety of functional studies.
Enzymes: biochemistry, biotechnology and clinical chemistry
Angewandte …, 2001
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Journal of Biological Chemistry
Methods are described for the purification, close to homogeneity, of rabbit liver glycogen synthase in forms dependent on (D-form) or independent (I-form) of glucose-6-P for activity. In previous studies (Camici, M., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol. Chern. 257,9898-9901), the D-form enzyme was shown to have apparent subunit molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (Mapp) of 90,000 and to be susceptible to partial proteolytic degradation. We report here that the purified I-form consisted of a single polypeptide of Map = 86,000, even when protease inhibitors were present during the purification. However, appropriate phosphorylation of the I-form enzyme led to a decrease in the electrophoretic mobility of the subunit to generate a species of Mapp = 90,000, identical to that of the D-form. Exposure of the I-form enzyme (subunit Mapp = 85,000) to trypsin caused degradation in the sequence 85,000 + 82,000 + 79,000 + 72,000;
Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase
Biochemical and Biophysical Research Communications, 1992
Transketolase is a key enzyme in the pentose-phosphate pathway which has been implicated in the latent human genetic disease, Wemicke-Korsakoff syndrome. Here we report the cloning and partial characterisation of the coding sequences encoding human transketolase from a human brain cDNA library. The library was screened with oligonucleotide probes based on the amino acid sequence of proteolytic fragments of the purified protein. Northern blots showed that the transketolase mRNA is approximately 2.2 kb, close to the minimum expected, of which approximately 60% was represented in the largest cDNA clone. Sequence analysis of the transketolase coding sequences reveals a number of homologies with related enzymes from other species. ® 1992 Academic Press, Inc. Transketolase (EC 2.2.1.1,TK) catalyses the transfer of a two-carbon ketol unit from xylulose 5-phosphate to an aldose acceptor, either ribose 5-phosphate or erythrose 5-phosphate. Along with transaldolase, TK provides the link between the pentose-phosphate pathway and the glycolytic pathway, enabling the recycling of pentose sugars under conditions where NADPH production is required for reductive biosynthesis. TK is also one of the few major enzymes which requires thiamin diphosphate (ThDP) as a cofactor, others being pyruvate dehydrogenase, 2oxoglutarate dehydrogenase and branched chain 2-oxoacid dehydrogenase. TK assays provide a sensitive measure of thiamin deficiency. There is evidence of heterogeneity of human erythrocyte, leucocyte and fibroblast TK by isoelectric focussing [1-4], as well as by variation in its affinity for ThDP [5, 6]. In Alzheimers disease, TK appears to be modified [4, 7] and a variant form of the enzyme, having decreased affinity for ThDP, has been implicated in the specific brain damage characterised in man as the Wernicke-Korsakoff (WK) syndrome [5, 6]. However, others [2, 8] have reported an identical affinity of TK for ThDP in this condition, and constancy of isoelectric focussing patterns [9,10].
Turnover of Several Glycolytic Enzymes in Rabbit Heart, Soleus Muscle and Liver
Biological Chemistry, 1974
Turnover of glycogenolytic and glycolytic enzymes in rabbit liver, heart and soleus muscle was studied by means of single-pulse labelling with [ 14 C]leucine and isolation of the enzymes labelled /'// vivo by the immunochemical method. In liver, glyceraldehyde-phosphate dehydrogenase and lactate dehydrogenase turn over with apparent half-lives of 1.0 and 0.67 days. In heart and soleus muscle, phosphorylase, fructosebisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase, enolase and lactate dehydrogenase reveal apparent half-lives in the range of 0.9 and. 1.2 days. The similarity of the degradation rates of these enzymes suggests that their different tissue levels result primarily from individual rates of synthesis. 6-Phosphofructokinase reveals a significantly higher degradation rate. The apparent half-lives in heart and soleus muscle are 0.43 and 0.67 days. The high turnover of 6-phosphofructokinase is discussed with regard to the regulatory role of this enzyme in glycolysis and possible mechanisms involved in degradation of the enzyme protein. Umsatzraten mehrerer glykolytischer Enzyme in Herz, Soleusmuskel und Leber des Kaninchens Zusammenfassung: Turnoverraten von Enzymen der Glykogenolyse und Glykolyse wurden nach einmaliger Pulsmarkierung mit [ 14 C]Leucin in Leber, Herz und m. soleus des Kaninchens bestimmt. Die in vivo markierten Enzymproteine wurden aus Gewebsextrakten mit Hilfe der Immunpräzipitation isoliert. Die scheinbaren Halbwertszeiten der Glycerinaldehydphosphat-Dehydrogenase und Lactat-Dehydrogenase betragen in der Leber l ,0 bzw. 0,67
Journal of biochemistry, 1989
Aminopeptidase M [EC 3.4.11.2] was purified 772-fold to homogeneity from the microsomal fraction of human liver, with a yield of 18.9%, by a combination of solubilization with 0.5% Triton X-100 and then 1 M urea and chromatography on columns of DEAE-cellulose, hydroxylapatite, Butyl-Toyopearl, and Sephacryl S-300. The purified enzyme had a molecular weight of 140,000 by SDS-polyacrylamide gel electrophoresis and of 280,000 by gel filtration on a column of TSK gel 2000 SW. It was reconstituted into proteoliposomes with asolectin, showing its amphiphilic nature. The aminopeptidase M from liver was found to be efficiently inhibited by bile acids. The enzyme was almost completely inhibited by chenodeoxycholic acid and 70-90% inhibited by cholic acid at a concentration of 6 mM. The extent of inhibition by conjugated and unconjugated bile acids was in the order: unconjugated greater than glycoconjugated greater than tauroconjugated bile acid, independent of the nature of the substrates us...
Histochemical investigations of dehydrogenase reactions in experimental liver necrosis
The Journal of pathology and bacteriology, 1963
THIS paper reports the results of an attempt to determine whether mitochondrial damage can be demonstrated histochemically in rat liver damaged by carbon tetrachloride or thioacetamide. There is a considerable amount of histological and biochemical work on this subject; the stage at which mitochondrial injury occurs is, however, still disputed. Pearse (1958, 1960) has claimed that mitochondrial injury can be detected with facility in tissue sections by use of the diaphorase and dehydrogenase reactions. We found it difficult.