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- Valerio, M, et al.
(författare)
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Tissue-specificity of the regulation of ATP hydrolysis by isolated plant-mitochondria
- 1993
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Ingår i: FEBS Letters. - 0014-5793 .- 1873-3468. ; 318:2, s. 113-117
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Tidskriftsartikel (refereegranskat)abstract
- Pea leaf mitochondria had a high ATP hydrolase activity following the collapse of the membrane potential by addition of valinomycin in state 4. In mitochondria isolated from potato tubers such ATP hydrolase activity was not observed. Pea leaf mitochondria also had a DELTApH, in contrast to what was previously found for potato tuber mitochondria. This DELTApH could, however, not explain the different results on ATP hydrolysis since this activity was also observed in the presence of nigericin. The results suggest a tissue-specific regulation of ATP hydrolysis in resting organs (potato tubers) as compared to active organs (leaves).
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| 2. |
- Domar, Ulla, 1938-
(författare)
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Human intestinal alkaline phosphatase : tissue expression and serum levels
- 1992
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Doktorsavhandling (övrigt vetenskapligt)abstract
- Human alkaline phosphatase (ALP) comprises four isozymes, viz liver/bone/ kidney or tissue unspecific (AP), intestinal (LAP), placental (PLAP) and germ cell or PLAP-like alkaline phosphatase, with their main expression in specific tissues as indicated by their names. The isozymes are coded by different genes, but they are closely related, with more than 50% amino acid sequence homologies. Their biological function is unclear. In certain malignant and benign diseases, serum elevations of one or more of the isozymes occur, which is of diagnostic importance. In this study, the special expression of the intestinal isozyme in human tissues and sera, in normal as well as in pathological conditions, has been investigated by use of isozyme specific monoclonal antibodies.Monoclonal antibodies against the AP, IAP and PLAP isozymes were prepared, and specific assays developed, based on these monoclonal antibodies and the catalytic activity of the isozymes. By use of these assays the basal levels of all three isozymes were examined in selected normal organs. The isozymes were found to be expressed in measurable amounts in all the examined organs.IAP was immunohistochemically localized to the epithelial cells of membranes lining the ducts and tubules of the kidney, liver, pancreas and small intestine.Normal human serum contained all three isozymes. The AP isozyme constituted about 90% of the total ALP activity, the IAP isozyme less than 10% and the PLAP isozyme about 1%. Considerable interindividual variations of the serum IAP activity were observed. The serum activities of the IAP isozyme were related to the individual ABO blood group and secretor status. Non-secretors had low levels of IAP activity amounting to about one tenth of the activity in sera from blood group B or 0 secretors, while blood group A secretors had serum IAP activities in the same order as non-secretors. High individual day to day variations were observed.Fat absorption caused serum IAP to increase significantly for all persons, but it was rapidly cleared from the blood. We found that the release of IAP into the blood was linked to lipid absorption, but removal from the blood was not linked to lipoprotein clearance.Certain tumors of the testis expressed elevated levels of all three ALP isozymes. The highest activitiy of LAP was observed in one yolk sac tumor, in agreement with the endodermal origin of this tumor. In seminoma tissue the AP and PLAP isozymes were significantly, and IAP moderately elevated.Cirrhosis of the liver caused significantly increased serum levels of IAP besides the AP isozyme. In inflammatory diseases of the small intestine, normal serum IAP activities were observed.
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| 3. |
- Hultin, Magnus, 1968-
(författare)
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Turnover of chylomicrons in the rat
- 1995
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Doktorsavhandling (övrigt vetenskapligt)abstract
- Mechanisms involved in the clearance of chylomicrons and aspects of the interactions at the vascular endothelium were studied in the rat.The poly-anion heparin, known to release lipoprotein lipase (LPL) from the vascular endothelium, enhanced the clearance of chylomicrons. Five minutes after heparin injection, the clearance of chylomicron triglycerides and retinyl esters was markedly accelerated. The rapid initial clearance was followed by a slower clearance of heavily lipolyzed chylomicrons. In contrast, one hour after heparin the clearance of both triglycerides and retinyl esters was retarded. This decreased removal of chylomicrons coincided with a decrease in the heparin releasable LPL activity, indicating that the previous release to plasma by heparin had resulted in net loss of functional LPL in the tissues.The poly-cation protamine released hepatic lipase and some LPL from their binding sites to plasma. One hour after protamine, plasma triglyceride levels were increased, indicating that chylomicron removal was impeded. It has been speculated that protamine inactivates LPL in vivo, but this was not the case. Ten minutes after injection of protamine normal amounts of LPL could be released by heparin. Thus, the accumulation of plasma triglycerides was not due to a rapid inactivation of LPL by protamine.LPL has specificity for sn-1,3-ester bonds. To investigate if this specificity is important in vivo, a lipid emulsion containing medium-chain fatty acids (MCFA) in the sn-1,3-position and long-chain fatty acids (LCFA) in the sn-2-position was synthesized, as well as an emulsion containing MCFA-TG mixed with LCFA-TGs (MMM/LLL). In vitro experiments showed large differences in the hydrolysis of the emulsions, but in vivo there were only small differences in the metabolism.To further study if lipid emulsions are cleared by the same mechanisms as chylomicrons, an emulsion was made by the same formulation as Intralipid® with addition of 3H-triolein and ,4C-cholesteryl ester. As measured by the removal of cholesteryl esters, the emulsion was cleared at the same rate as was chylomicrons. The triglyceride label was, however, removed more slowly from the emulsion droplets than from chylomicrons. Together with the lower recirculation of labeled free fatty acids (FFA) in plasma, this suggests that there was less lipolysis of the emulsion. The current view that removal of lipid emulsions in vivo is mainly dependent on LPL-mediated hydrolysis might thus not be correct.To further analyze the metabolism of chylomicrons, a compartmental model was developed. In this process, the distribution volume for chylomicrons was shown to be larger than the blood volume, a model for the metabolism of FFA in the rat was validated, and the full tissue distribution of injected chylomicrons was determined. According to the model, about half of the triglyceride label was removed from the circulation together with the core label while for the emulsion this number was about 80 %. In fasted rats all labeled fatty acids appeared to mix with the plasma FFA pool, while in fed rats about one-fifth of the fatty acids did not mix with the FFA but was apparently channeled directly to tissue metabolism.
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| 6. |
- Davydov, Albert, 1969-
(författare)
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Electron paramagnetic resonance and biochemical studies of red-ox properties of the diferric/radical center in mouse and Mycobacterium tuberculosis ribonucleotide reductase R2 proteins
- 1999
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Doktorsavhandling (övrigt vetenskapligt)abstract
- Ribonucleotide reductase (RNR) catalyses the reduction of all four ribonucleotides to the corresponding deoxyribonucleotides that are used by the cells for DNA synthesis. The active enzyme from mouse, Mycobacterium tuberculosis (M. tuberculosis) and aerobically grown E. coli consists of two nonidentical subunits, proteins R1 and R2, both required for enzymatic activity. The R1 protein contains the regulatory sites and the binding site for the ribonucleotide substrates whereas the R2 subunit contains a stable tyrosyl radical and an antiferromagnetically coupled _-oxo-bridged diferric center. According to the proposed reaction mechanism the radical properties of the R2 protein are transferred to the active site of the R1 protein upon binding of the substrate molecule to the R1. This transfer is proposed to propagate via a conserved chain of hydrogen-bonded amino acids within the R1 and R2 proteins. Once generated the diferric/radical center may exist in different red-ox states: active (with Fe(III)Fe(III) center and tyrosyl radical), met (Fe(III)Fe(III) center without tyrosyl radical), mixed-valent (Fe(II)Fe(III) center) and fully reduced (Fe(II)Fe(II) center). Admission of oxygen to the fully reduced form of an R2 protein (a so-called regeneration reaction) results in a spontaneous formation of the active form. The regeneration reaction may be one of the possible ways employed by the cell for the generation of the active enzyme and therefore the study of this reaction is important for understanding the enzyme functionality.The aim of this thesis is to study the red-ox transitions of the diferric/radical center in mouse and Mycobacterium tuberculosis R2 proteins. Despite the significant similarities in the structure, the red-ox properties of the diferric/radical centers in mouse and M. tuberculosis R2 proteins are significantly different. The diferric/radical center in mouse R2 was found to be much more accessible for the external reductants than the centers of E. coli and M. tuberculosis R2 proteins. A higher accessibility of the diferric/radical center in mouse R2 protein can be explained by the presence of an open channel from the surface of the protein to the diferric/radical center. The tyrosyl radical in mouse R2 protein strongly interacts with the diferric center. Removing the tyrosyl radical in the active mouse R2 protein results in irreversible structural changes of the diferric cluster leading to an inactivation of the protein. Therefore the met form of mouse R2 protein can not be stabilized. Unlike mouse R2, the tyrosyl radical of M. tuberculosis R2 protein exhibits extremely weak magnetic interaction with the diferric center. The met form of M. tuberculosis R2 can be easily obtained and stabilized by the treatment of the active enzyme with hydroxyurea. The results of the chemical reduction of the diferric/radical center in the native mouse R2 protein as well as in two mutants (D266A and Y370W) in a proposed electron transfer pathway indicate that in all cases the second order rate constants in the mutants are comparable or faster than in native protein suggesting that the proposed radical transfer pathway is not important for the chemical reduction to proceed.Studying the oxidation of the fully reduced mouse R2 protein by different non-oxygen oxidants, we have demonstrated that the transitions between Fe(III)Fe(III), Fe(II)Fe(II) and Fe(II)Fe(III) states of this protein are fully reversible. The possibility to form a proper binuclear iron center without using molecular oxygen as an oxidant suggests that the _-oxo-bridge in mouse R2 protein does not necessary need molecular oxygen to be formed. Application of the low temperature reduction to mouse and M. tuberculosis R2 proteins demonstrated a presence of two structurally different diferric clusters giving rise to two distinct mixed-valent EPR signals. Whereas the shape of the mixed-valent EPR signal generated by _-irradiation at 77 K in mouse R2 protein is significantly affected by the presence of the tyrosyl radical, we did not observe any effect of the tyrosyl radical presence on the shape of mixed - valent signal generated in M. tuberculosis R2.
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