scholarly journals High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

Plants ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 304 ◽  
Author(s):  
Alexander Hofmann ◽  
Sophia Müller ◽  
Thomas Drechsler ◽  
Mareike Berleth ◽  
Katharina Caesar ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Stress Responses ◽  
Histidine Kinase ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Cd Spectroscopy ◽  
Single Step ◽  
Signaling Mechanism ◽  
Wide Range

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678 ◽  
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

Abstract The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

Different roles for the stress-activated protein kinase pathway in the regulation of trehalose metabolism in Schizosaccharomyces pombe

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1745-1752 ◽  
Author(s):  
V. Paredes ◽  
A. Franco ◽  
T. Soto ◽  
J. Vicente-Soler ◽  
M. Gacto ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Transcription Factors ◽  
Protein Kinase ◽  
Osmotic Stress ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Stress Responses ◽  
Mitogen Activated Protein Kinase ◽  
Wide Range ◽  
Trehalose Metabolism

The Wis1p-Sty1p mitogen-activated protein kinase cascade is a major signalling system in the fission yeast Schizosaccharomyces pombe for a wide range of stress responses. It is known that trehalose functions as a protective metabolite to counteract deleterious effects of environmental stresses. Herein it is reported that the expression of genes related to trehalose metabolism in S. pombe, ntp1 + (neutral trehalase) and tps1 + [trehalose-6-phosphate (T6P) synthase], is partially regulated by the Sty1p kinase under salt-induced osmotic stress and conditions of slight oxidative stress and is fully dependent on this kinase under severe oxidative stress. This control is carried out through transcription factors Atf1p/Pcr1p during osmotic stress and through Pap1p during exposure to low levels of oxidative stress. However, all three transcription factors are needed for gene expression under conditions of extreme oxidative stress. In addition, a role for Sty1p in the modulation of post-transcriptional activation of trehalase mediated by Pka1p/Sck1p kinases, as well as in the activity of T6P synthase under such stressful conditions has been demonstrated. These results reveal a novel dual action of the Wis1p-Sty1p pathway in the regulation of trehalose metabolism in fission yeast.

Fibrin/Fibrinogen Degradation Products in Amniotic Fluid

1975 ◽  
Author(s):  
D. W. Purdie ◽  
P. W. Howie ◽  
W. Edgar ◽  
G. R. M. Prentice
Keyword(s):  
Amniotic Fluid ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Haemagglutination Inhibition ◽  
Gel Filtration ◽  
Normal Group ◽  
Double Immunodiffusion ◽  
Fibrinogen Degradation Products ◽  
The Mean

Fibrin/fibrinogen degradation products (F. D. P.) have been studied in amniotic fluid from normal pregnancies and in pregnancies complicated by hypertension, anencephaly, and hydramnios without foetal anomaly.Using the haemagglutination inhibition immunoassay, F. D. P. were identified in 51 of 53 (mean 3.75 mcg/ml, S.D.±2.80) samples of amniotic fluid from healthy patients with normal pregnancies between 14 and 40 weeks gestation. In the hypertensive group of 16 patients the mean F. D. P. level of 5.00 mcg/ml (S.D.±3.25), was not significantly different from the normal group. In 7 patients with anencephalic foetuses the amniotic fluid F. D. P. level was significantly elevated (mean 17.5 mcg/ml, S.D.±5.75), while in 6 patients with hydramnios without foetal anomaly the F. D. P. level (mean 6.00 mcg/ml ±3.25) was not significantly different from the normal group.Characterization of the amniotic fluid F. D. P. was carried out using double immunodiffusion polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-200. The F. D. P. were present in two distinct components. The major fraction was fragment X (M. W. 240,000) and in a lower concentration, fragment D was identified.The detection of raised amniotic fluid F. D. P. levels in early pregnancy may provide a diagnostic test for open central nervous system foetal abnormalities.

Purification and characterization of the protease inhibitor "monastatin" from a marine Alteromonas sp. with reference to inhibition of the protease produced by a bacterium pathogenic to fish

1985 ◽  
Vol 31 (12) ◽  
pp. 1089-1094 ◽  
Author(s):  
Chiaki Imada ◽  
Masachika Maeda ◽  
Nobuo Taga
Keyword(s):  
Protease Inhibitor ◽  
Inhibitory Activity ◽  
Marine Bacterium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Characterization ◽  
Wide Range ◽  
Thiol Proteases

A novel protease inhibitor, which we named "monastatin," produced by a marine bacterium was purified using ammonium sulfate precipitation, DEAE-cellulofine column chromatography, and gel filtration through Sephadex G-100. Monastatin was purified to homogeneity, as a single staining band in polyacrylamide gel electrophoresis. Monastatin was a glycoprotein with a molecular weight of about 20 000. It was stable up to 100 °C for a 30-min incubation with loss of 20% of inhibitory activity and also showed stability over a wide range of pH from 2 to 12. Metal ions such as Cu2+ and Fe2+ repressed the inhibitory activity to 0%. Monastatin had an inhibitory activity against thiol proteases such as papain and ficin.

Terminal Plasmin Degradation Products Of Duck Fibrinogen

1981 ◽  
Author(s):  
T Krajewski ◽  
P Nowak ◽  
C S Cierniewski
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Amino Acid Content ◽  
Gamma Chain ◽  
Strong Inhibitor ◽  
Peptide Composition ◽  
Proteolytic Action ◽  
Fibrin Monomers

Previously we have found that duck or goose fibrinogen are built up of Aα, Bβ and γ chains like mammalian one. However, they are more sensitive to proteolytic action than pig fibrinogen and rapidly form the D-E complex. The aim of this report is to characterize terminal plasmic products of duck fibrinogen. The protein was digested in the presence of calcium ions with human plasminogen activated by streptokinase. Degradation products were firstly analysed by polyacrylamide gel electrophoresis, secondly, isolated by DEAE-cellulose chromatography and gel filtration on Sephadex G-100, thirdly, characterized in order to establish their peptide composition and amino acid content. Three fragments, namely fg-DH, fg-DM and fg-DL as well as single fragment E were identified among final degradation products. Under conditions used, fg-DH was the major form of fragment D with molecular weight of about 100,000. For fg-DM and fg-DL, fragments Mr 89,000 and Mr 80,000 have been found, respectively. All fragments differed in the length of gamma chain remnants which varied from Mr 42,000 to Mr 24,000. Only a single population of fg-E with Mr 43,000 was identified in plasmic degradation products. The constituent peptide subunits of that fragment were also characterized. Fg-D appeared to be a strong inhibitor of fibrin monomer polymerization both in homologous /duck fibrin monomers/ and heterologous /pig fibrin monomers/ systems.

scholarly journals Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma

Blood ◽  
1990 ◽  
Vol 75 (8) ◽  
pp. 1673-1678
Author(s):  
O Wilhelm ◽  
R Hafter ◽  
A Henschen ◽  
M Schmitt ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
High Performance ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Fibrin Degradation Products ◽  
Sds Page ◽  
Human Ovarian Carcinoma

The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

scholarly journals Purification and Properties of a Polyester Polyurethane-Degrading Enzyme from Comamonas acidovorans TB-35

1998 ◽  
Vol 64 (1) ◽  
pp. 62-67 ◽  
Author(s):  
Yukie Akutsu ◽  
Toshiaki Nakajima-Kambe ◽  
Nobuhiko Nomura ◽  
Tadaatsu Nakahara
Keyword(s):  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Water Soluble ◽  
Degrading Enzyme ◽  
Comamonas Acidovorans ◽  
Water Soluble Compound ◽  
Sds Page ◽  
Hydrophobic Adsorption

ABSTRACT A polyester polyurethane (PUR)-degrading enzyme, PUR esterase, derived from Comamonas acidovorans TB-35, a bacterium that utilizes polyester PUR as the sole carbon source, was purified until it showed a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This enzyme was bound to the cell surface and was extracted by addition of 0.2%N,N-bis(3-d-gluconamidopropyl)deoxycholamide (deoxy-BIGCHAP). The results of gel filtration and SDS-PAGE showed that the PUR esterase was a monomer with a molecular mass of about 62,000 Da. This enzyme, which is a kind of esterase, degraded solid polyester PUR, with diethylene glycol and adipic acid released as the degradation products. The optimum pH for this enzyme was 6.5, and the optimum temperature was 45°C. PUR degradation by the PUR esterase was strongly inhibited by the addition of 0.04% deoxy-BIGCHAP. On the other hand, deoxy-BIGCHAP did not inhibit the activity whenp-nitrophenyl acetate, a water-soluble compound, was used as a substrate. These observations indicated that this enzyme degrades PUR in a two-step reaction: hydrophobic adsorption to the PUR surface and hydrolysis of the ester bond of PUR.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

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