Disulfide bonds of bovine trypsin, their stability and relation to enzymatic activity

1968 ◽  
Vol 33 (4) ◽  
pp. 1359-1363 ◽  
Author(s):  
B. Mesrob ◽  
V. Holeyšovský
Keyword(s):  
Enzymatic Activity ◽  
Disulfide Bonds ◽  
Bovine Trypsin

Isolation of Antithrombin III from Normal and α1-Antitrypsin-Deficient Human Plasma

1977 ◽  
Vol 38 (02) ◽  
pp. 0475-0485 ◽  
Author(s):  
Anna D. Borsodi ◽  
Ralph A. Bradshaw
Keyword(s):  
Enzymatic Activity ◽  
Isoelectric Focusing ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Antithrombin Activity ◽  
Bovine Trypsin ◽  
Normal Plasma ◽  
Antitrypsin Deficiency ◽  
Simplified Procedure ◽  
Stimulation Of

SummaryThe plasma of individuals, hetero- or homozygous for α1-antitrypsin deficiency, contains greatly decreased amounts of antithrombin activity as assayed against factor Xa. However, heparin stimulation of the residual antithrombin activity is observed, which is comparable to that of normal plasma. Antithrombins isolated from both normal and α1-antitrypsin deficient plasma by a simplified procedure are indistinguishable in both properties and yields. The microheterogeneity observed on isoelectric focusing of both preparations can be eliminated by treatment with neuraminidase. Neither purified human antithrombin nor α1-antitrypsin, when assayed against bovine trypsin, is stimulated by heparin. These results clearly establish the unique natures of antithrombin and α1-antitrypsin and show that about 75% of the antithrombin activity measured in normal plasma is due to α1-antitrypsin. Estimates of anti thrombin III activity in normal plasma by assays dependent on enzymatic activity can probably be obtained only in the presence of heparin.

SK053 An Inhibitor Of Enzymes Involved In Allosteric Disulfide Bonds Formation Induces Differentiation Of Human AML Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4215-4215
Author(s):  
Justyna Chlebowska ◽  
Malgorzata Firczuk ◽  
Karolina Furs ◽  
Angelika Muchowicz ◽  
Radoslaw Sadowski ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Cytotoxic Activity ◽  
Disulfide Bonds ◽  
Thioredoxin Reductase ◽  
Positive Control ◽  
Blue Staining ◽  
Nuclear Fraction ◽  
Hl60 Cells ◽  
Disulfide Reduction ◽  
Turbidimetric Assay

Abstract Introduction of differentiation-inducing agents [all-trans retinoic acid (ATRA) and arsenic trioxide] to the treatment of acute promyelocytic leukemia (APL) was a remarkable therapeutic breakthrough resulting in cure rates exceeding 80%. However, there is no such significant progress in the treatment of other acute myeloid leukemia (AML) types. Thus search for new agents exerting anti-leukemic effects by targeting novel and unique cellular mechanisms is of utmost clinical importance. Numerous human proteins involved in tumor formation, such as C-terminal SRC kinase (CSK) or tissue factor, contain allosteric disulfide bonds that are cleaved by oxidoreductases or by thiol-disulfide exchange. Such disulfide modifications participate in post-translational protein control and affect protein function. Targeting of allosteric disulfide bonds is a novel promising strategy in cancer therapy (Hogg P, Nat Rev Cancer 2013). We have recently developed SK053, a small molecule inhibitor of thioredoxin/thioredoxin reductase system, that showed anti-tumor effects both in vitro and in murine tumor models (Klossowski S, Muchowicz A et al., J Med Chem 2012). Our ongoing studies revealed that SK053 is not a target-specific, but mechanism-selective inhibitor of enzymes involved in allosteric disulfide bonds formation such as protein disulfide isomerase (PDI). Its biotinylated form (SK231) precipitated PDI from human AML NB4 cells, and mass spectrometry analysis revealed that SK053 covalently binds to PDI. In a turbidimetric assay of insulin disulfide reduction SK053 inhibits the enzymatic activity of PDI with IC50 of 10 µM (Fig. 1B). Since PDI blocks translation of CCAAT enhancer binding protein alpha (CEBPA), one of the transcription factors crucial for normal neutrophils maturation (Haefliger S et al., Blood 2011), we set out to evaluate the activity of SK053 in human AML cells to see whether it can induce differentiation and cytostatic/cytotoxic effects against these cells. We observed that SK053 exerts significant cytostatic/cytotoxic activity in various types of human AML cells (HL60, NB4, KG-1 and MOLM14, Fig. 1A), and induces differentiation of AML blasts into more mature myeloid cells as evidenced morphologically in May-Grünwald-Giemsa staining, nitro blue tetrazolium (NBT) reduction assay as well as by increased expression of cell membrane differentiation markers (CD11b, CD14 and CD15), measured with flow cytometry. Moreover, incubation of AML cells with SK053 induces expression of CEBPA and hexokinase 3 (HK3) mRNA in quantitative RT-PCR (Fig. 1C) and increases amount of CEBPA protein in nuclear fraction measured with immunoblotting (Fig. 1D). Finally, SK053 induces differentiation of primary leukemic cells freshly isolated from AML patients. In summary, SK053 targets PDI and thioredoxin/thioredoxin reductase system, has significant anti-leukemic activity and induces differentiation of various types of human AML cells. Thus, targeting of enzymes involved in allosteric disulfide bonds formation with small molecule inhibitors presents a novel and promising therapeutic strategy in acute myeloid leukemia.Figure 1SK053 is a potent anti-leukemic agent that induces differentiation of human AML cells. (A) Cytostatic/cytotoxic activity of SK053 in human AML cell lines evaluated with trypan blue staining, mean percentage of dead cells ± SD, n=6; *P<0.05 vs controls, one-way ANOVA with Dunett’s post test; (B) SK053 inhibits enzymatic activity of recombinant human PDI in a turbidimetric assay of insulin disulfide reduction; 130 µM bacitracin served as a positive control; (C) qPCR results presented as mean target-to-reference ratio ± SD (left) CEBPA expression in HL60 cells incubated for indicated time with 10 µM SK053; (right) HK3 expression in HL60 cells incubated for 48 hours with 10 µM SK053, or 1 µM ATRA (positive control); (D) SK053 increases amount of CEBPA protein in nuclear fraction of HL60 cells incubated for 2 or 5 days with 1 µM ATRA (positive control), 2 mM bacitracin (PDI inhibitor) or 10 µM SK053; the figure presents results of Western blot, HDAC2 served as a marker of nuclear fraction, ß-actin served as a loading control.Figure 1. SK053 is a potent anti-leukemic agent that induces differentiation of human AML cells. (A) Cytostatic/cytotoxic activity of SK053 in human AML cell lines evaluated with trypan blue staining, mean percentage of dead cells ± SD, n=6; *P<0.05 vs controls, one-way ANOVA with Dunett’s post test; (B) SK053 inhibits enzymatic activity of recombinant human PDI in a turbidimetric assay of insulin disulfide reduction; 130 µM bacitracin served as a positive control; (C) qPCR results presented as mean target-to-reference ratio ± SD (left) CEBPA expression in HL60 cells incubated for indicated time with 10 µM SK053; (right) HK3 expression in HL60 cells incubated for 48 hours with 10 µM SK053, or 1 µM ATRA (positive control); (D) SK053 increases amount of CEBPA protein in nuclear fraction of HL60 cells incubated for 2 or 5 days with 1 µM ATRA (positive control), 2 mM bacitracin (PDI inhibitor) or 10 µM SK053; the figure presents results of Western blot, HDAC2 served as a marker of nuclear fraction, ß-actin served as a loading control. The research was supported by the Ministry of Science and Higher Education [IP2011038971 (DN)], National Science Centre Poland [NN405127640 (AM)] and a grant from the European Commission 7th Framework Programme: FP7-REGPOT-2012-CT2012-316254-BASTION. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Globodera pallida Effector GpPDI1 Is a Functional Thioredoxin and Triggers Defense-Related Cell Death Independent of Its Enzymatic Activity

2020 ◽  
Vol 110 (11) ◽  
pp. 1838-1844 ◽  
Author(s):  
Rachel Gross ◽  
Shan Zhang ◽  
Lihui Wei ◽  
Allan Caplan ◽  
Joseph Kuhl ◽  
...  
Keyword(s):  
Cell Death ◽  
Enzymatic Activity ◽  
Disulfide Bonds ◽  
Leaf Tissue ◽  
Globodera Pallida ◽  
Host Immune System ◽  
Plant Host ◽  
Root Cells ◽  
Feeding Site ◽  
Related Cell

The plant-parasitic nematode Globodera pallida is an obligate biotroph that only reproduces on select species in the Solanum family. The establishment of the feeding site, the syncytium, involves secretion of effectors into the plant cell to combat the plant defense response and facilitate transformation of root cells into the syncytium. Despite the important predicted roles of effectors in the plant−pathogen interactions, the functionality of G. pallida effectors is largely unknown. In this study, we identified and characterized a G. pallida effector protein disulfide isomerase (GpPDI1). GpPDI1 contains two thioredoxin domains that function together to reduce disulfide bonds, as manifested by the nullification of enzymatic activity when either domain is absent. The transcript of GpPDI1 is localized in the dorsal gland of the nematode during the J2 stage. In addition, GpPDI1 can trigger defense-related cell death in Nicotiana benthamiana and tomato (Solanum lycopersicum) leaf tissue and localizes in the plant host cell’s cytoplasm and nucleus when transiently expressed in plant cells. Significantly, the ability of elicitation of cell death is not dependent on the enzymatic activity of GpPDI1 or correlated with the subcellular distribution of GpPDI1, suggesting that a nondisulfide reducing function or structural feature of GpPDI1 is responsible for the recognition by the host immune system to elicit cell death.

The Effects of Biguanides on Thrombokinase, Thrombin and Trypsin

1975 ◽  
Vol 34 (01) ◽  
pp. 246-255
Author(s):  
Phyllis S Roberts ◽  
Patricia B Fleming
Keyword(s):  
Methyl Ester ◽  
Enzymatic Activity ◽  
Test Period ◽  
Factor X ◽  
Bovine Thrombin ◽  
Slow Reaction ◽  
Bovine Trypsin ◽  
Rate Of Hydrolysis ◽  
Almost All ◽  
Hydrolysis Of

SummaryA purified preparation of bovine thrombokinase (activated Factor X) loses the ability to hydrolyze TAME (p-toluenesulfonyl-L-arginine methyl ester) when it is incubated at 37° in 0.25 M Tris. HCl buffer, pH 7.4 with lauroxypropyl biguanide, N1, N5-dimethyl, N1-lauroxypropyl biguanide, N1-p-chlorophenethyl, N5-phenethyl biguanide, or N1-methyl, N1-p-chlorobenzyl, N5-o,p-dichlorobenzyl biguanide. Activity is lost much more slowly when 0.15 M NaCl is also present. Lauroxypropyl biguanide is the most potent of the compounds tested, 0.22 mM causing thrombokinase to lose almost all of its activity in about 30 minutes at 37° in pH 7.4 buffered saline.Topical bovine thrombin also loses activity when incubated with either of the lauroxypropyl biguanides but not with the diphenethyl or the dibenzyl compound. Instead, the latter biguanides accelerate thrombin’s hydrolysis of TAME. The percent acceleration is not affected or only slightly decreased by the presence of 0.15 M NaCl or KCl, and it is also unaffected by incubating the enzyme with the compounds in buffered saline for 4 to 120 minutes.Purified bovine trypsin is stabilized by both lauroxypropyl and the diphenethyl biguanide when incubated at 37° in pH 7.4 buffered saline for the 60 minute test period but neither compound has any effect on its rate of hydrolysis of TAME.It is postulated that the enzymes first react rapidly and reversibly with all of the test biguanides and, depending upon the enzyme and the substrate, the rate of hydrolysis of the substrate is unaffected, accelerated or inhibited. The lauroxypropyl biguanides also undergo a second, slower reaction with both thrombokinase and thrombin that produces loss of enzymatic activity. The dibenzyl and diphenethyl biguanides also undergo this second slow reaction with thrombokinase but not with thrombin, and none of the biguanides undergo this second reaction with trypsin.

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

A Method for the Colorimetric Determination of β-Glucuronidase in Urine, Serum, Tissue; Assay of Enzymatic Activity in Health and Disease

1959 ◽  
Vol 36 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Julius A. Goldbarg ◽  
Esteban P. Pineda ◽  
Benjamin M. Banks ◽  
Alexander M. Rutenburg
Keyword(s):  
Enzymatic Activity ◽  
Colorimetric Determination ◽  
Health And Disease ◽  
Urine Serum
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