Rates of disappearance from plasma of enzymes labeled by coupling with a radioactive lodo-ester.

1976 ◽  
Vol 22 (8) ◽  
pp. 1277-1282
Author(s):  
A R Qureshi ◽  
J H Wilkinson
Keyword(s):  
Creatine Kinase ◽  
Catalytic Activity ◽  
Lactate Dehydrogenase ◽  
Propionic Acid ◽  
Free Acid ◽  
Rabbit Muscle ◽  
Analytical Recovery ◽  
Catalytically Active ◽  
Rapid Hydrolysis

Abstract Lactate dehydrogenase-5 and creatine kinase from rabbit muscle were labeled by coupling with N-hydroxysuccinimidyl 3-(4'-hydroxy-[3',5'-125I]diiodophenyl)propionate. After purification, the analytical recovery of catalytically-active labeled enzyme averaged 90% for lactate dehydrogenase, 81% for creatine kinase. The labeled enzymes were injected intravenously into rabbits and disappearance from plasma of catalytic activity and radioactivity was measured. The disappearance curves for lactate dehydrogenase-5 differed considerably from those observed with the enzyme labeled by direct iodination. The discrepancy was due to rapid hydrolysis in vivo of the labeled amide-enzyme linkage, because about 50% of the injected radioactivity appeared in the urine as 125I-labeled 3-(4'-hydroxy-3',5'-diiodophenyl)propionic acid within 4-8 h of injection. Similar outputs were observed after administration of this acid to rabbits. The free acid was also detected in the urines of rabbits within 4-8 h of the intravenous injection of creatine kinase labeled similarly. We conclude that this method of labeling is unsuitable for preparing radioactive enzymes for study of their catabolism.

scholarly journals In Vivo Dimerization of Types 1, 2, and 3 Iodothyronine Selenodeiodinases

Endocrinology ◽  
2003 ◽  
Vol 144 (3) ◽  
pp. 937-946 ◽  
Author(s):  
Cyntia Curcio-Morelli ◽  
Balazs Gereben ◽  
Ann Marie Zavacki ◽  
Brian W. Kim ◽  
Stephen Huang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Western Blot ◽  
Relative Molecular Mass ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Human Embryonic Kidney ◽  
Cell Lysate ◽  
Sds Page ◽  
Catalytically Active

The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of 75Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2–5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (Mr) of 55,000 for D1, Mr 62,000 for D2, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.

Inactivation in Vitro of Lactate Dehydrogenase-5 in Blood

1977 ◽  
Vol 14 (1-6) ◽  
pp. 48-52 ◽  
Author(s):  
A. R. Qureshi ◽  
J. H. Wilkinson
Keyword(s):  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Whole Blood ◽  
Rabbit Muscle ◽  
Muscle Lactate ◽  
Rabbit Blood

During incubation with rabbit blood in vitro rabbit-muscle lactate dehydrogenase-5 was inactivated at a rate similar to that observed in vivo. By contrast plasma and plasma containing erythrocytes had no effect on the enzyme activity, but plasma containing leucocytes inactivated the enzyme at the same rate as whole blood. The results obtained support the concept that intravascular inactivation accounts for the disappearance of enzymes from the circulation.

IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): Scientific Division, Committee on Reference Systems for Enzymes (C-RSE): Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase: [α-Amylase: 1,4-α-D-glucan 4-glucanohydrolase (AMY), EC 3.2.1.1]

2006 ◽  
Vol 44 (9) ◽  
Author(s):  
G. Schumann ◽  
R. Aoki ◽  
C.A. Ferrero ◽  
G. Ehlers ◽  
G. Férard ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Catalytic Activity ◽  
Lactate Dehydrogenase ◽  
Alanine Aminotransferase ◽  
Clinical Chemistry ◽  
Reference Method ◽  
Reference Procedure ◽  
Activity Concentrations ◽  
Primary Reference

AbstractThis paper is the eighth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37°C. The procedure described here is deduced from the previously described 30°C IFCC reference method. Differences are tabulated and commented on.Clin Chem Lab Med 2006;44:1146–55.

scholarly journals Native ribonucleases process sgRNA transcripts to create catalytic Cas9/sgRNA complexes in planta

2020 ◽  
Author(s):  
Will B Cody ◽  
Herman B. Scholthof
Keyword(s):  
Catalytic Activity ◽  
Gene Editing ◽  
Viral Vector ◽  
Fusion Transcript ◽  
In Vitro Assays ◽  
In Planta ◽  
Guide Rnas ◽  
Catalytically Active

The current CRISPR/Cas9 gene editing dogma for single guide RNAs (sgRNA) delivery is based on the premise that 5′ and 3′ nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides on the 5′ and 3′ termini. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana. This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5′, but not 3′, proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5′ overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript it also produced indels when delivered with Cas9. These results reveal that 5′ "auto-processing" of progenitor sgRNAs occurs natively in plants. Towards a possible mechanism for the perceived "auto-processing", we found, using in vitro generated RNAs and those isolated from plants, that the 5′ to 3′ exoribonuclease XRN1 can degrade elongated progenitor sgRNAs whereas the mature sgRNA end-products are resistant. Comparisons with other studies suggest that sgRNA "auto-processing" may be a phenomenon not unique to plants, but other eukaryotes as well.

EFFECT OF DRAFT LOAD ON LACTATE DEHYDROGENASE AND CREATINE KINASE ACTIVITIES IN MULES.(Equusasinus x Equuscaballus)

2016 ◽  
Vol 12 (1) ◽  
Author(s):  
A. Moolchandani ◽  
M. Sareen
Keyword(s):  
Creatine Kinase ◽  
Lactate Dehydrogenase ◽  
Serum Creatine Kinase

This study was carried out to assess the effects of draft load on serum creatine kinase and lactated ehydorgenase activities in mule. Five adult mules of 5 to 6 years of age were subjected to loading exercise (i.e. 10% draft load for 1 to 5 days and 20% draft load from 6th to 10th day). A highly significant (P<0.01) increase in serum LDH activities in response to 10% and 20% draft load exercise was observed while significant (P<0.05) increase at 10% draft load and highly significant (P<0.01) increase at 20% draft load was observed in CK activities.

Hydrogen peroxide decomposition on a two-component CuO-Cr2O3 catalyst

1988 ◽  
Vol 53 (8) ◽  
pp. 1636-1646 ◽  
Author(s):  
Viliam Múčka ◽  
Kamil Lang
Keyword(s):  
Hydrogen Peroxide ◽  
Catalytic Activity ◽  
Extreme Values ◽  
Catalytic Properties ◽  
Active Components ◽  
Catalytic Systems ◽  
Heterogeneous Catalytic ◽  
Peroxide Decomposition ◽  
Two Component ◽  
Catalytically Active

Some physical and catalytic properties of the two-component copper(II)oxide-chromium(III)oxide catalyst with different content of both components were studied using the decomposition of the aqueous solution of hydrogen peroxide as a testing reaction. It has been found that along to both basic components, the system under study contains also the spinel structure CuCr2O4, chromate washable by water and hexavalent ions of chromium unwashable by water. The soluble chromate is catalytically active. During the first period of the reaction the equilibrium is being established in both homogeneous and heterogeneous catalytic systems. The catalytic activity as well as the specific surface area of the washed solid is a non-monotonous function of its composition. It seems highly probable that the extreme values of both these quantities are not connected with the detected admixtures in the catalytic system. The system under study is very insensitive with regard to the applied doses of gamma radiation. Its catalytic properties are changed rather significantly after the thermal treatment and particularly after the partial reduction to low degree by hydrogen. The observed changes of the catalytic activity of the system under study are very probably in connection with the changes of the valence state of the catalytically active components of the catalyst.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

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