scholarly journals H.p.l.c. separation and study of the charge isomers of human placental glutathione transferase

1986 ◽  
Vol 239 (1) ◽  
pp. 53-57 ◽  
Author(s):  
L L Radulovic ◽  
A P Kulkarni
Keyword(s):  
Conformational Change ◽  
Glutathione Transferase ◽  
Tissue Concentration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Studies ◽  
Anionic Form ◽  
Net Charge ◽  
Catalytically Active ◽  
Low Activity

Glutathione transferase (GST) from human placenta was purified by affinity chromatography and anion-exchange h.p.l.c. The enzyme exhibited different chromatographic and electrophoretic behaviours according to the concentration of GSH, suggesting a possible change in the net charge of the molecule and a concomitant conformational change due to ligand binding. Two interconvertible forms were quantitatively separated into distinct catalytically active states by h.p.l.c. Depending upon the GSH concentration, polyacrylamide-gel electrophoresis revealed the presence of one or two bands. A Kd of 0.42 mM for GSH was determined fluorimetrically. The loss in intrinsic fluorescence also suggested a conformational change in the enzyme. Kinetic studies using ethacrynic acid were conducted to determine whether the presumed conformational change could effect the catalytic capability of placental GST. A biphasic response in initial velocities was observed with increasing concentrations of GSH. Two apparent Km values of 0.38 and 50.27 mM were obtained for GSH, whereas Vmax. values showed a 46-fold difference. It was concluded that the enzyme assumes a highly anionic form in the presence of a low GSH concentration, whereas it is converted into relatively weaker anionic form when its immediate environment contains a high GSH concentration. Since the average tissue concentration of total GSH was estimated at 0.11 mM for term placenta, the results suggest that the high-affinity-low-activity conformer would predominate in vivo.

scholarly journals The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in Porphyromonas gingivalis

2006 ◽  
Vol 188 (17) ◽  
pp. 6376-6386 ◽  
Author(s):  
Christine A. Seers ◽  
Nada Slakeski ◽  
Paul D. Veith ◽  
Todd Nikolof ◽  
Yu-Yen Chen ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Proteolytic Activity ◽  
Porphyromonas Gingivalis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Proteolytic Processing ◽  
Terminal Domain ◽  
Catalytically Active ◽  
Membrane Attachment

ABSTRACT Porphyromonas gingivalis produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the P. gingivalis outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain P. gingivalis mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.

scholarly journals A disease-associated G5703A mutation in human mitochondrial DNA causes a conformational change and a marked decrease in steady-state levels of mitochondrial tRNA(Asn).

1997 ◽  
Vol 17 (12) ◽  
pp. 6831-6837 ◽  
Author(s):  
H Hao ◽  
C T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Steady State ◽  
Oxidative Phosphorylation ◽  
Conformational Change ◽  
Tertiary Structure ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Human Osteosarcoma Cells ◽  
Steady State Levels

We introduced mitochondrial DNA (mtDNA) from a patient with a mitochondrial myopathy into established mtDNA-less human osteosarcoma cells. The resulting transmitochondrial cybrid lines, containing either exclusively wild-type or mutated (G5703A transition in the tRNA[Asn] gene) mtDNA, were characterized and analyzed for oxidative phosphorylation function and steady-state levels of different RNA species. Functional studies showed that the G5703A mutation severely impairs oxidative phosphorylation function and mitochondrial protein synthesis. We detected a marked reduction in tRNA(Asn) steady-state levels which was not associated with an accumulation of intermediate transcripts containing tRNA(Asn) sequences or decreased transcription. Native polyacrylamide gel electrophoresis showed that the residual tRNA(Asn) fraction in mutant cybrids had an altered conformation, suggesting that the mutation destabilized the tRNA(Asn) secondary or tertiary structure. Our results suggest that the G5703 mutation causes a conformational change in the tRNA(Asn) which may impair aminoacylation. This alteration leads to a severe reduction in the functional tRNA(Asn) pool by increasing its in vivo degradation by mitochondrial RNases.

scholarly journals Negative Effects of a Nonhost Proteinase Inhibitor of ~19.8 kDa fromMadhuca indicaSeeds on Developmental Physiology ofHelicoverpa armigera(Hübner)

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Farrukh Jamal ◽  
Dushyant Singh ◽  
Prabhash K. Pandey
Keyword(s):  
Artificial Diet ◽  
Insect Pest ◽  
Larval Mortality ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Larval Body ◽  
Seed Flour ◽  
Single Polypeptide Chain

An affinity purified trypsin inhibitor from the seed flour extracts ofMadhuca indica(MiTI) on denaturing polyacrylamide gel electrophoresis showed that MiTI consisted of a single polypeptide chain with molecular mass of ~19.8 kDa. MiTI inhibited the total proteolytic and trypsin-like activities of the midgut proteinases ofHelicoverpa armigeralarvae by 87.51% and 76.12%, respectively, at concentration of 5 µg/mL with an IC50of 1.75 µg/mL against trypsin like midgut proteinases. The enzyme kinetic studies demonstrated that MiTI is a competitive inhibitor with aKivalue of4.1×10−10 M forHelicoverpatrypsin like midgut proteinases.In vivoexperiments with different concentrations of MiTI in artificial diet (0.5, 1.0, and 1.5% w/w) showed an effective downfall in the larval body weight and an increase in larval mortality. The concentration of MiTI in the artificial diet to cause 50% mortality (LD50) of larvae was 1.5% w/w and that to cause reduction in mass of larvae by 50% (ED50) was 1.0% w/w. Nutritional indices observations suggest the toxic and adverse effects of MiTI on the growth and development ofH. armigeralarvae. The results suggest a strong bioinsecticidal potential of affinity purified MiTI which can be exploited in insect pest management of crop plants.

In-vitro conversion of thyroxine to tri-iodothyronine by chicken hepatic 5′-deiodinase: kinetic studies

1986 ◽  
Vol 110 (3) ◽  
pp. 441-446 ◽  
Author(s):  
S. K. Lam ◽  
S. Harvey
Keyword(s):  
Tissue Concentration ◽  
Maximum Velocity ◽  
Kinetic Studies ◽  
Enzymatic System ◽  
Concentration Time ◽  
Liver Homogenates ◽  
Kinetics Of ◽  
Menten Constant

ABSTRACT In-vitro studies with chicken liver homogenates demonstrate that the conversion of thyroxine (T4) to tri-iodothyronine (T3) is dependent upon tissue concentration, time of incubation, pH, temperature, the presence of dithiothreitol (DTT) and the concentration of substrate (T4), and is heat-labile. The generation of T3 is inhibited by iopanoic acid and 6-n-propyl-2-thiouracil. The kinetics of conversion of T4 to T3, determined by Lineweaver–Burke analysis, indicated an apparent Michaelis–Menten constant (Km) of 1·16 μmol/l with a maximum velocity (Vmax) of 44·57 pmol T3 generated/mg protein per min from T4. Dithiothreitol appears to behave as a co-substrate for this system with an apparent Km of 98·5 μmol/l and a Vmax of 1·41 pmol T3 generated/mg protein per min at a T4 concentration of 5 μmol/l. These data suggest that the conversion of T4 to T3 in fowl proceeds by means of an enzymatic system, probably 5′-monodeiodinase, and is responsible for maintaining T3 levels in vivo. J. Endocr. (1986) 110, 441–446.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Preparation of a Viable Population of Indium-111- Labelled Human Blood Platelets

1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Kinetic Studies ◽  
Physiological Saline ◽  
Human Platelets ◽  
Autologous Plasma ◽  
Platelet Suspension ◽  
Viable Population ◽  
Indium 111

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

Insulin secretion in the spiny mouse (Acomys cahirinus). Dose and time kinetic studies with glucose in vivo and in vitro

Diabetes ◽  
1975 ◽  
Vol 24 (12) ◽  
pp. 1094-1100 ◽  
Author(s):  
A. Rabinovitch ◽  
A. Gutzeit ◽  
A. E. Renold ◽  
E. Cerasi
Keyword(s):  
Insulin Secretion ◽  
Kinetic Studies ◽  
Spiny Mouse ◽  
Acomys Cahirinus

Absolute SPECT/CT quantification of cerebral uptake of 99mTc-HMPAO for patients with neurocognitive disorders

2016 ◽  
Vol 55 (04) ◽  
pp. 158-165 ◽  
Author(s):  
Friedrich Welz ◽  
James Sanders ◽  
Torsten Kuwert ◽  
Juan Maler ◽  
Johannes Kornhuber ◽  
...  
Keyword(s):  
Tissue Concentration ◽  
Tracer Uptake ◽  
Planar Imaging ◽  
Quantitative Spect ◽  
Dementia Patients ◽  
Hmpao Spect ◽  
Patient Groups ◽  
The Absolute ◽  
99Mtc Hmpao

SummaryIt was reported from planar imaging studies that the cerebral uptake of injected 99mTc-HMPAO activity is about 4–7% in humans. Recent work has shown that modern SPECT/ CT devices are able to quantify the tissue concentration of radioactivity in vivo in absolute units (Bq/ml), while avoiding the limitations of planar techniques. The aims of this study were (a) to determine the cerebral uptake of 99mTc-HMPAO in absolute units in SPECT/CT, (b) to investigate potential differences in absolute tracer uptake for patients suspected of dementia. Patients, methods: We performed 99mTc-HMPAO SPECT/CT in 65 patients with suspected dementia. 99mTc-HMPAO uptake was determined using a previously published quantitative SPECT/CT protocol. The absolute HMPAO uptake and the results of a regionalized analysis were compared for MMSE and NINCDS-ADRDA based patient groups. Results: The mean absolute uptake of 99mTc-HMPAO for our patient population was 4.3 ± 0.8% of the injected dose. The uptake, as well as the regionalized analysis yielded significantly different results for low ( 23) and high (>23) MMSE groups and also for some of the NINCDS-ADRDA groups. Conclusion: Our results show that the absolute cerebral uptake of 99mTc-HMPAO is in the range of previously reported results, obtained by planar techniques. Absolute uptake is significantly different between the patient groups.

scholarly journals Stu2p binds tubulin and undergoes an open-to-closed conformational change

2006 ◽  
Vol 172 (7) ◽  
pp. 1009-1022 ◽  
Author(s):  
Jawdat Al-Bassam ◽  
Mark van Breugel ◽  
Stephen C. Harrison ◽  
Anthony Hyman
Keyword(s):  
Conformational Change ◽  
Conformational Transition ◽  
Budding Yeast ◽  
Microtubule Dynamics ◽  
Open Structure ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
The Common

Stu2p from budding yeast belongs to the conserved Dis1/XMAP215 family of microtubule-associated proteins (MAPs). The common feature of proteins in this family is the presence of HEAT repeat–containing TOG domains near the NH2 terminus. We have investigated the functions of the two TOG domains of Stu2p in vivo and in vitro. Our data suggest that Stu2p regulates microtubule dynamics through two separate activities. First, Stu2p binds to a single free tubulin heterodimer through its first TOG domain. A large conformational transition in homodimeric Stu2p from an open structure to a closed one accompanies the capture of a single free tubulin heterodimer. Second, Stu2p has the capacity to associate directly with microtubule ends, at least in part, through its second TOG domain. These two properties lead to the stabilization of microtubules in vivo, perhaps by the loading of tubulin dimers at microtubule ends. We suggest that this mechanism of microtubule regulation is a conserved feature of the Dis1/XMAP215 family of MAPs.

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