scholarly journals Plasma clearance and endocytosis of cytosolic malate dehydrogenase in the rat

1983 ◽  
Vol 210 (2) ◽  
pp. 419-428 ◽  
Author(s):  
M K Bijsterbosch ◽  
A M Duursma ◽  
J M W Bouma ◽  
M Gruber
Keyword(s):  
Bone Marrow ◽  
Malate Dehydrogenase ◽  
Half Life ◽  
Cell Protein ◽  
Net Charge ◽  
Lysosomal Fraction ◽  
First Order Kinetics ◽  
Parenchymal Cells ◽  
Cytosolic Malate Dehydrogenase ◽  
Positively Charged

1. Pig heart cytosolic malate dehydrogenase was radiolabelled with O-(4-diazo-3,5-di-[125I]iodobenzoyl)sucrose and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of about 30 min. 2. The tissue distribution of radioactivity was determined at 2 h after injection. All injected radioactivity was recovered from the tissues. A high percentage of the injected dose was found in liver (37%), spleen (6%) and bone including marrow (19%). 3. Radioactivity in liver and spleen increased up to 2 h after injection and subsequently declined, with a half-life of about 20 h. 4. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. 5. Liver cells were isolated 1 h after injection of labelled enzyme. We found that Kupffer cells, endothelial cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 2725, 94 and 63 ml of plasma/day per g of cell protein respectively. 6. Radioautography indicated that in spleen and bone marrow the enzyme is mainly taken up by macrophages. 7. Internalization of the enzyme by liver, spleen and bone marrow was saturable. This indicates that the enzyme is taken up in these tissues by adsorptive endocytosis. 8. The present results closely resemble those obtained previously for the mitochondrial isoenzyme of malate dehydrogenase and for lactate dehydrogenase M4. Since those enzymes are positively charged at physiological pH, whereas cytosolic malate dehydrogenase is negative, net charge cannot be the major factor determining the rate of uptake of circulating enzymes by reticuloendothelial macrophages, as has been suggested in the literature [Wachsmuth & Klingmüller (1978) J. Reticuloendothel. Soc. 24, 227-241].

scholarly journals Endocytosis and breakdown of mitochondrial malate dehydrogenase in the rat in vivo. Effects of suramin and leupeptin

1982 ◽  
Vol 208 (1) ◽  
pp. 61-67 ◽  
Author(s):  
M K Bijsterbosch ◽  
A M Duursma ◽  
J M W Bouma ◽  
M Gruber
Keyword(s):  
Bone Marrow ◽  
Malate Dehydrogenase ◽  
Kupffer Cells ◽  
Cell Types ◽  
Cell Protein ◽  
Carbon Particles ◽  
Parenchymal Cells ◽  
In Vivo Effects ◽  
Mitochondrial Malate Dehydrogenase

1. The plasma clearance of intravenously injected 125I-labelled mitochondrial malate dehydrogenase (half-life 7 min) was not influenced by previous injection of suramin and/or leupeptin (inhibitors of intralysosomal proteolysis). 2. Pretreatment with both inhibitors considerably delayed degradation of endocytosed enzyme in liver, spleen, bone marrow and kidneys. 3. The tissue distribution of radioactivity was determined at 30 min after injection, when only 3% of the dose was left in plasma. All injected radioactivity was still present in the carcass. The major part of the injected dose was found in liver (49%), spleen (5%), kidneys (13%) and bone, including marrow (11%). 4. Liver cells were isolated 15 min after injection of labelled enzyme. We found that Kupffer cells and parenchymal cells had endocytosed the enzyme at rates corresponding to 9530 and 156 ml of plasma/day per g of cell protein respectively. Endothelial cells do not significantly contribute to uptake of the enzyme. 5. Uptake by Kupffer cells was saturable, whereas uptake by parenchymal cells was not. This suggests that these cell types endocytose the enzyme via different receptors. 6. Previous injection of carbon particles greatly decreased uptake of the enzyme by liver, spleen and bone marrow.

scholarly journals THE ROLE OF THYMOCYTES AND BONE MARROW CELLS IN DEFINING THE RESPONSE TO THE DINITROPHENYL HAPTEN ATTACHED TO POSITIVELY AND NEGATIVELY CHARGED SYNTHETIC POLYPEPTIDE CARRIERS

1973 ◽  
Vol 137 (1) ◽  
pp. 183-195 ◽  
Author(s):  
Yuval Karniely ◽  
Edna Mozes ◽  
G. M. Shearer ◽  
Michael Sela
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Electrical Charge ◽  
Spleen Cells ◽  
Net Charge ◽  
Cellular Basis ◽  
Charged Carrier ◽  
Synthetic Polypeptide ◽  
Positively Charged ◽  
Marrow Cells

An inverse relationship exists between the net electrical charge of immunogens and the antibodies they elicit (1). Results of an earlier study have demonstrated that the net charge phenomenon has a cellular basis, since the immune response potential of murine spleen cells to 2,4-dinitrophenyl (DNP) on a negatively charged synthetic polypeptide carrier was reduced by cell fractionation over negatively charged glass beads, whereas the response to the same hapten on a positively charged carrier was unaffected (14). To verify that the net charge correlation is expressed at the cellular level, spleen cells were fractionated over positively charged poly-L-lysine-coated glass bead columns, and their immunocompetence to DNP on positively and negatively charged carriers was tested by cell transfers in irradiated recipient mice. In this case, the fractionated cells showed reduced response potential to DNP on the positively charged carrier only. Thus, the cellular basis of the net charge phenomenon has been demonstrated for both positively and negatively charged immunogens (for the same specificity) by cell separation techniques over columns of opposite charge. In order to establish whether the cell population relevant for the charge properties of immunogens was of thymus or marrow origin, thymocytes and bone marrow cells were selectively passed over positively or negatively charged columns and mixed with unfractionated cells of the complementary type. Transfers of the filtered and unfiltered cell mixtures in irradiated recipient mice immunized with DNP on either a positive or a negative synthetic polypeptide carrier indicated that fractionation of thymocytes, but not of marrow cells, correlated with the spleen population. Thus, thymocytes fractionated over negatively charged columns and mixed with unfractionated marrow cells exhibited reduced response to DNP on the negative carrier, but normal responses to DNP on the positive carrier. The opposite result was obtained when thymocytes were passed over positively charged columns. No effect on the anti-DNP response was detected by filtration of bone marrow cells over columns of either charge. These findings indicate that it is possible to distinguish between thymocytes on the basis of their capacity to react with more acidic or more basic surfaces and that a population of thymus-derived cells may recognize immunogens on the basis of their overall electrical charge. No evidence was found by these techniques that marrow-derived cells contribute to the net charge phenomenon.

scholarly journals Plasma clearance and endocytosis of mitochondrial malate dehydrogenase in the rat

1981 ◽  
Vol 200 (1) ◽  
pp. 115-121 ◽  
Author(s):  
M K Bijsterbosch ◽  
A M Duursma ◽  
J M W Bouma ◽  
M Gruber ◽  
P Nieuwenhuis
Keyword(s):  
Bone Marrow ◽  
Enzyme Activity ◽  
Malate Dehydrogenase ◽  
Plasma Clearance ◽  
Reticuloendothelial System ◽  
First Order ◽  
First Order Kinetics ◽  
Long Run ◽  
Mitochondrial Malate Dehydrogenase ◽  
Adsorptive Endocytosis

1. Pig mitochondrial malate dehydrogenase was labelled with 125I and intravenously injected into rats. Enzyme activity and radioactivity were cleared from plasma identically, with first-order kinetics, with a half-life of only 7 min. 2. Radioactivity accumulated in liver, spleen, bone (marrow) and kidneys, reaching maxima of 3 1, 4, 6 and 9% of the injected dose respectively, at 10 min after injection. 3. Our data allow us to calculate that in the long run 59, 5, 11 and 13% of the injected dose is taken up and subsequently broken down by liver, spleen, bone and kidneys respectively. 4. Differential fractionation of liver showed that the acid-precipitable radioactivity was mainly present in the lysosomal and microsomal fractions, suggesting that the endocytosed protein is transported via endosomes to lysosomes, where it is degraded. 5. Radioautography of liver and spleen suggested that the labelled protein was taken up by macrophages of the reticuloendothelial system. 6. Mitochondrial malate dehydrogenase is probably internalized in liver, spleen and bone marrow by adsorptive endocytosis, since uptake of the enzyme of these tissues is saturable.

scholarly journals Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose

1982 ◽  
Vol 202 (3) ◽  
pp. 655-660 ◽  
Author(s):  
A S H De Jong ◽  
A M Duursma ◽  
J M W Bouma ◽  
M Gruber ◽  
A Brouwer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Lactate Dehydrogenase ◽  
Kupffer Cells ◽  
Cell Protein ◽  
Dehydrogenase Isoenzyme ◽  
Carbon Particles ◽  
In Vivo Experiments ◽  
Parenchymal Cells

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45-51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.

In-vitro Kinetic Hydrolysis Study of Metronidazole Derivatives with Carvacrol and Eugenol Using Validated RP-HPLC Method

2020 ◽  
Vol 16 ◽  
Author(s):  
M. Alarjah
Keyword(s):  
Half Life ◽  
Hplc Method ◽  
Hydrolysis Rate ◽  
First Order ◽  
First Order Kinetics ◽  
Rp Hplc ◽  
Pharmacokinetic Properties ◽  
Pseudo First Order ◽  
Kinetic Hydrolysis

Background: Prodrugs principle is widely used to improve the pharmacological and pharmacokinetic properties of some active drugs. Much effort was made to develop metronidazole prodrugs to enhance antibacterial activity and or to improve pharmacokinetic properties of the molecule or to lower the adverse effects of metronidazole. Objective: In this work, the pharmacokinetic properties of some of monoterpenes and eugenol pro metronidazole molecules that were developed earlier were evaluated in-vitro. The kinetic hydrolysis rate constants and half-life time estimation of the new metronidazole derivatives were calculated using the validated RP-HPLC method. Method: Chromatographic analysis was done using Zorbbax Eclipse eXtra Dense Bonding (XDB)-C18 column of dimensions (250 mm, 4.6 mm, 5 μm), at ambient column temperature. The mobile phase was a mixture of sodium dihydrogen phosphate buffer of pH 4.5 and methanol in gradient elution, at 1ml/min flow rate. The method was fully validated according to the International Council for Harmonization (ICH) guidelines. The hydrolysis process carried out in an acidic buffer pH 1.2 and in an alkaline buffer pH 7.4 in a thermostatic bath at 37ºC. Results: The results followed pseudo-first-order kinetics. All metronidazole prodrugs were stable in the acidic pH, while they were hydrolysed in the alkaline buffer within a few hours (6-8 hr). The rate constant and half-life values were calculated, and their values were found to be 0.082- 0.117 hr-1 and 5.9- 8.5 hr., respectively. Conclusion: The developed method was accurate, sensitive, and selective for the prodrugs. For most of the prodrugs, the hydrolysis followed pseudo-first-order kinetics; the method might be utilised to conduct an in-vivo study for the metronidazole derivatives with monoterpenes and eugenol.

scholarly journals Contribution of engineered electrostatic interactions to the stability of cytosolic malate dehydrogenase

2001 ◽  
Vol 14 (11) ◽  
pp. 911-917 ◽  
Author(s):  
Francesca Trejo ◽  
Josep Ll. Gelpí ◽  
Albert Ferrer ◽  
Albert Boronat ◽  
Montserrat Busquets ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Electrostatic Interactions ◽  
The Stability ◽  
Cytosolic Malate Dehydrogenase

scholarly journals Effects of bicarbonate on intercompartmental reducing-equivalent translocation in isolated parenchymal cells from rat liver

1974 ◽  
Vol 140 (3) ◽  
pp. 355-361 ◽  
Author(s):  
Michael N. Berry ◽  
Harold V. Werner ◽  
Ernest Kun
Keyword(s):  
Malate Dehydrogenase ◽  
Inhibitory Action ◽  
Ethanol Oxidation ◽  
Specific Inhibitor ◽  
Isolated Liver Cells ◽  
Glycerol Uptake ◽  
Parenchymal Cells ◽  
Rate Limiting ◽  
Isolated Liver ◽  
Phosphate Medium

1. Incubation of isolated liver cells in a medium containing bicarbonate raises malate concentrations almost sixfold compared with values obtained in a bicarbonate-free phosphate medium. The malate concentration of about 0.3mm in bicarbonate medium is of the same order as the Km for malate dehydrogenase. 2. The utilization of ethanol, glyercol and sorbitol was increased by 20–35% in bicarbonate medium. 3. Fluoromalate, a specific inhibitor of malate dehydrogenase and the malate carrier, inhibited or ethanol oxidation by 23%, glycerol uptake by 20% and sorbitol uptake by 42% in bicarbonate medium, but had a much smaller inhibitory action in phosphate medium. In consequence fluoromalate almost abolished the stimulatory effects of bicarbonate on substrate utilization. 4. Difluoro-oxaloacetate, a specific inhibitor of aspartate aminotransferase, had about one-half the inhibitory activity of fluoromalate. The two inhibitors in combination were less effective than fluoromalate by itself. 5. It is concluded that bicarbonate stimulates the utilization of reduced substrates, which are oxidized in the cytoplasmic compartment of the liver cell, by increasing the activity of rate-limiting malate dehydrogenase-dependent intercompartmental hydrogen shuttles. Both malate–oxaloacetate and malate–aspartate systems are involved in these hydrogen-translocation processes.

Use of purified lyophilized human lactate dehydrogenase isoenzyme 5 in a study of measuring lactate dehydrogenase activity.

1989 ◽  
Vol 35 (8) ◽  
pp. 1774-1776 ◽  
Author(s):  
D A Smith ◽  
G C Moses ◽  
A R Henderson
Keyword(s):  
Lactate Dehydrogenase ◽  
Half Life ◽  
Specific Activity ◽  
Lactate Dehydrogenase Activity ◽  
Bovine Albumin ◽  
First Order ◽  
First Order Kinetics ◽  
The Stability ◽  
Lactate Dehydrogenase Isoenzymes ◽  
Excellent Stability

Abstract We examined the stability of human lactate dehydrogenase (EC 1.1.1.27) isoenzyme 5--purified to a specific activity of about 400 kU/g--when lyophilized in a buffered, stabilized matrix of bovine albumin. This isoenzyme was prepared with a final activity of about 500 U/L and stored at -20, 4, 20, 37, and 56 degrees C for as long as six months. This isoenzyme decayed with approximate first-order kinetics, with an estimated half-life at -20 degrees C of about 475 years. Stability of reconstituted samples stored at 20 or 4 degrees C was poor, suggesting that the reconstituted material should be used without delay; material stored at -20 degrees C showed excellent stability for 15 days. We propose that such preparations might be further investigated as standards for use in electrophoresis of lactate dehydrogenase isoenzymes.

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