scholarly journals Kinetic studies of the uptake of aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro

1985 ◽  
Vol 228 (2) ◽  
pp. 493-503 ◽  
Author(s):  
E Marra ◽  
S Passarella ◽  
E Casamassima ◽  
E Perlino ◽  
S Doonan ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Binding Sites ◽  
Aspartate Aminotransferase ◽  
Kinetic Studies ◽  
Membrane System ◽  
Model System ◽  
Kinetic Measurements ◽  
Enzymes Inhibition ◽  
Mitochondrial Aspartate Aminotransferase

Kinetic measurements of the uptake of native mitochondrial aspartate aminotransferase and malate dehydrogenase into mitochondria in vitro were carried out. The uptake of both the enzymes is essentially complete in 1 min and shows saturation characteristics. The rate of uptake of aspartate aminotransferase into mitochondria is decreased by malate dehydrogenase, and vice versa. The inhibition is exerted by isoenzyme remaining outside the mitochondria rather than by isoenzyme that has been imported. The thiol compound beta-mercaptoethanol decreases the rate of uptake of the tested enzymes; inhibition is a result of interaction of beta-mercaptoethanol with the mitochondria and not with the enzymes themselves. The rate of uptake of aspartate aminotransferase is inhibited non-competitively by malate dehydrogenase, but competitively by beta-mercaptoethanol. The rate of uptake of malate dehydrogenase is inhibited non-competitively by aspartate aminotransferase and by beta-mercaptoethanol. beta-Mercaptoethanol prevents the inhibition of the rate of uptake of malate dehydrogenase by aspartate aminotransferase. These results are interpreted in terms of a model system in which the two isoenzymes have separate but interacting binding sites within a receptor in the mitochondrial membrane system.

scholarly journals Removal of an N-terminal peptide from mitochondrial aspartate aminotransferase abolishes its interactions with mitochondria in vitro

1985 ◽  
Vol 228 (3) ◽  
pp. 609-614 ◽  
Author(s):  
K M O'Donovan ◽  
S Doonan ◽  
E Marra ◽  
S Passarella ◽  
E Quagliariello
Keyword(s):  
Catalytic Activity ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Terminal Segment ◽  
Model System ◽  
Native Enzyme ◽  
Cysteine Residues ◽  
Native Form ◽  
Mitochondrial Aspartate Aminotransferase

Treatment of mitochondrial aspartate aminotransferase from rat liver with trypsin leads to specific cleavage of the bonds between residues 26 and 27, and residues 31 and 32. The proteolysed enzyme has only a small residual catalytic activity, but retains a conformation similar to that of the native form as judged by accessibility and reactivity of cysteine residues. Proteolysis abolishes the ability of the enzyme either to bind to mitochondria or to be imported into the organelles. This suggests that the N-terminal segment of the native enzyme is essential for both of these functions, at least in the model system used to study the import process.

Uptake of aspartate aminotransferase into mitochondria in vitro causes efflux of malate dehydrogenase and vice versa

1990 ◽  
Vol 1022 (3) ◽  
pp. 273-282 ◽  
Author(s):  
Salvatore Passarella ◽  
Ersilia Marra ◽  
Anna Atlante ◽  
Maria Barile ◽  
Shawn Doonan ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Aspartate Aminotransferase

Fibrin Binding of Plasminogen Coated Liposomes In Vitro

1996 ◽  
Vol 75 (01) ◽  
pp. 134-139 ◽  
Author(s):  
J L M Heeremans ◽  
P Los ◽  
R Prevost ◽  
D J A Crommelin ◽  
C Kluft
Keyword(s):  
Binding Sites ◽  
In Vitro Model ◽  
Model System ◽  
Binding Properties ◽  
Experimental Conditions ◽  
Binding Rate ◽  
Order Of Magnitude ◽  
Vitro Model

SummaryIn this study, the fibrin binding properties of liposomes containing a number of plasminogen (Pig) molecules on the outside were compared to those of free (non-liposomal) Pig in an in vitro model system. Fibrin monolayer coated 96-wells plates were used, containing fibrin monomer at a density of around 3.4 to 3.9 × 10-4 nmol/cm2. These densities are similar to liposomal Plg-densities, thus allowing multivalent interactions to occur.In the panel of experimental conditions that was chosen, binding of free Pig and liposomes with Pig showed three main differences in characteristics. Firstly, in the fibrin binding of Plg-liposomes not all Pig may be involved, but on the average 40% of the total amount of liposomal Pig. This was shown by lysing the liposomes after binding to the fibrin and estimation of truly bound Pig. With Plg-densities on the liposomes below the fibrin binding sites density, the maximal number of bound Pig molecules remains below the amount of available fibrin binding sites. Secondly, a higher binding rate by at least one order of magnitude was observed for liposomes with Pig compared to free Pig. Thirdly, liposomes with Pig exhibit a fibrin binding affinity which increases with Plg-density, because of the multivalent character of interaction. Liposomal Pig can successfully compete for fibrin binding sites with a 100 fold higher concentration of free Pig.These in vitro findings indicate that in view of avid and rapid fibrin binding, liposomes with attached plasminogen may be suitable for in vivo targeting to fibrin based thrombi.

Assembly of the 30S ribosomal subunit

2006 ◽  
Vol 38 (4) ◽  
pp. 397-403 ◽  
Author(s):  
James R. Williamson
Keyword(s):  
Ribosome Biogenesis ◽  
Energy Landscape ◽  
Ribosomal Subunit ◽  
Kinetic Studies ◽  
Model System ◽  
Quantitative Mass Spectrometry ◽  
Rna Molecules ◽  
Small Proteins ◽  
Assembly Kinetics

The assembly of ribosomes requires a significant fraction of the energy expenditure for rapidly growing bacteria. The ribosome is composed of three large RNA molecules and over 50 small proteins that must be rapidly and efficiently assembled into the molecular machine responsible for protein synthesis. For over 30 years, the 30S ribosome has been a key model system for understanding the process of ribosome biogenesis through in vitro assembly experiments. We have recently developed an isotope pulse-chase experiment using quantitative mass spectrometry that permits assembly kinetics to be measured in real time. Kinetic studies have revealed an assembly energy landscape that ensures efficient assembly by a flexible and robust pathway.

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