scholarly journals Conformation, structure and activation of bovine cathepsin D. Unfolding and refolding studies

1984 ◽  
Vol 218 (2) ◽  
pp. 601-608 ◽  
Author(s):  
T Lah ◽  
M Drobniĉ-Koŝorok ◽  
V Turk ◽  
R H Pain
Keyword(s):  
Cathepsin D ◽  
Specific Activity ◽  
Single Chain ◽  
Guanidinium Chloride ◽  
Single Polypeptide Chain ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Kinetics Of ◽  
Chain Form ◽  
Covalent Complex

Cathepsin D is found in the cell in two forms, one a single polypeptide chain (Mr 44 000) and the other a non-covalent complex of two peptides of Mr 14 000 and 30 000. These correspond to the N-terminal and C-terminal regions of the single chain from which they originate. It has been shown that the two forms of the enzyme are closely similar in secondary-structure content, in aromatic amino acid environment and in denaturation behaviour. The two-chain enzyme has half the specific activity of the single-chain form. The denaturation and renaturation of the single-chain cathepsin D has now been studied by c.d., fluorescence and enzyme activity. Activity is lost irreversibly on unfolding, but the loss of backbone ellipticity and of folded aromatic environment is 75% reversible. The enzyme unfolds in two main stages, and the kinetics of these transitions indicate the existence of at least two intermediate forms between the native and the fully unfolded states. A further form of the enzyme exists in 0.5 M-guanidinium chloride. It is characterized by having an activity 40% greater than that of the native state. This increase is not reversed on removing the denaturant. The similarities between cathepsin D and pepsin are discussed.

Conformation and processing of cathepsin D

1985 ◽  
Vol 5 (10-11) ◽  
pp. 957-967 ◽  
Author(s):  
Roger H. Pain ◽  
Tamara Lan ◽  
Vito Turk
Keyword(s):  
Enzyme Activity ◽  
Protein Turnover ◽  
Cathepsin D ◽  
Polypeptide Chain ◽  
Proteolytic Processing ◽  
Single Polypeptide Chain ◽  
Processing Step ◽  
Acid Environment ◽  
Single Polypeptide ◽  
Covalent Complex

Cathepsin D occurs in two forms, a single polypeptide chain (Mr 44000) and a non-covalent complex of two peptides of Mr 14000 and 30000 that is derived by proteolytic processing of the 44000 polypeptide. The two forms from bovine spleen are closely similar in secondary structure content, in aromatic amino acid environment and in the two step denaturation behaviour. Enzyme activity is lost irreversibly on denaturation but conformation can be partially regained. The two separated chains will only refold partially and this is related to their positions in the overall structure of cathepsin D. It is suggested that the processing step is related to protein turnover.

THE INTERACTION OF PURIFIED FACTOR VIII WITH PLATELETS

1987 ◽  
Author(s):  
P R GanZ ◽  
E S Tackberry ◽  
G Rock
Keyword(s):  
Factor Viii ◽  
Specific Activity ◽  
Membrane Surface ◽  
Platelet Membrane ◽  
Factor X ◽  
Single Chain ◽  
Physiological Concentration ◽  
High Molecular Weight Species ◽  
Polyethylene Glycol Precipitation ◽  
Kinetics Of

Factor VIII is known to interact with Factors IXa and X to generate activated Factor X. A requirement for phospholipid in this reaction suggests that this "tenase" protein complex is assembled on a membrane surface. As a first step in studying the involvement of Factor VIII in this process, we wished to determine whether purified Factor VIII could interact directly with platelets. Factor VIII utilized in these experiments was purified from heparinized blood by a six-stage procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single-chain high molecular weight species of approximately 260,000 (specific activity 5,200 units/mg). This homogeneous protein was then radiolabelled with Na125I by a procedure which allowed the retention of approximately 60-80% of the procoagulant activity of Factor VIII. The kinetics of binding of 125I-Factor VIII to washed platelets at physiological concentration (approximately 3xl08/mL) was examined. Our results showed that for Factor VIII concentrations between 0.38 and 3.0 ng/mL there was a linear uptake of radiolabelled Factor VIII, whereas for concentrations above 10ng/mL only a slight increase in uptake occurred. To further define the association of purified Factor VIII with the platelet membrane, we also labelled Factor VIII with a bifunctional, photoactivatable cross-linking reagent, N-[4-(p-azido-m-[125]iodophenylazo)benzoyl]3-aminopropyl-N1 -oxysuccinimide ester. Analysis by PAGE showed thatthis reagent reacts predominantly with residues in the light chain or neahe C-terminal portion of Factor VIII. When mixed with thrombin-stimulated platelets, the cross-linked Factor VIII molecule was shown to transfer greater than 80% of the 125I label to a polypeptide of M.W. 80,000-90,000 isolated from platelet lysates. Autoradiographs of the labelled platelet preparations demonstrated that other minor polypeptides were radiolabelled. These experiments suggest that Factor VIII interacts closely with a platelet membrane protein which could represent a binding site for Factor VIII

scholarly journals Modulation of the alternative complement pathways by beta 1 H globulin.

1976 ◽  
Vol 144 (5) ◽  
pp. 1147-1163 ◽  
Author(s):  
K Whaley ◽  
S Ruddy
Keyword(s):  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Single Polypeptide Chain ◽  
Equilibrium Sedimentation ◽  
Alternative Complement ◽  
Multistep Process ◽  
Single Polypeptide ◽  
Kinetics Of

C3b inactivator accelerator (A-C3bINA) was isolated from human plasma. An antiserum produced against the purified protein gave a reaction of identity with beta 1 H, a well-documented contaminant of C3 preparations. Beta 1 H appears to be composed of a single polypeptide chain containing a significant quantity of carbohydrate, and having a sedimentation coefficient of 5.6 on analytical, and 6.4 on sucrose density gradient ultracentrifugation. Its mol wt based on SDS polyacrylamide gel electrophoresis and equilibrium sedimentation is approximately 150,000, whereas it elutes from Sephadex G200 with an apparent mol wt of 300,000, suggesting that beta 1 H is an asymmetric molecule. Beta 1 H potentiates the inactivation of C3b by C3b inactivator, binds to EAC43 to limit the formation of EAC43bB and EAC43bBP, and in contrast to C3b inactivator, it increases the rate of loss of hemolytic sites from EAC43bB and EAC43bBP. For the C3b inactivator-potentiating effect, beta 1 H and C3b inactivator must necessarily be simultaneously present. The kinetics of inactivation of C3b by C3b inactivator and beta 1 H are first order, suggesting that potentiation is not a multistep process. The mechanisms of binding to C3b and inhibition of the alternative pathway convertases C3bB and C3bBP are currently unknown.

scholarly journals Structural diversity of oligomeric β-propellers with different numbers of identical blades

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Evgenia Afanasieva ◽  
Indronil Chaudhuri ◽  
Jörg Martin ◽  
Eva Hertle ◽  
Astrid Ursinus ◽  
...  
Keyword(s):  
High Resolution ◽  
Building Block ◽  
Polypeptide Chain ◽  
Structural Diversity ◽  
Building Blocks ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Data Support ◽  
Single Polypeptide ◽  
Structural Versatility

β-Propellers arise through the amplification of a supersecondary structure element called a blade. This process produces toroids of between four and twelve repeats, which are almost always arranged sequentially in a single polypeptide chain. We found that new propellers evolve continuously by amplification from single blades. We therefore investigated whether such nascent propellers can fold as homo-oligomers before they have been fully amplified within a single chain. One- to six-bladed building blocks derived from two seven-bladed WD40 propellers yielded stable homo-oligomers with six to nine blades, depending on the size of the building block. High-resolution structures for tetramers of two blades, trimers of three blades, and dimers of four and five blades, respectively, show structurally diverse propellers and include a novel fold, highlighting the inherent flexibility of the WD40 blade. Our data support the hypothesis that subdomain-sized fragments can provide structural versatility in the evolution of new proteins.

scholarly journals Purification and structural studies on the complement-system control protein β1H (Factor H)

1982 ◽  
Vol 205 (2) ◽  
pp. 285-293 ◽  
Author(s):  
R B Sim ◽  
R G DiScipio
Keyword(s):  
Complement System ◽  
Factor H ◽  
System Control ◽  
Amino Acid Residues ◽  
Single Polypeptide Chain ◽  
C3b Receptor ◽  
Single Polypeptide ◽  
The Complement System ◽  
Chain Form ◽  
Control Protein

An efficient procedure for the isolation of the complement-system control protein beta 1H (Factor H) from human plasma was developed. The chemical composition and physical characteristics of the protein were studied, and a sequence of 17 amino acid residues at the N-terminus was determined. Factor H is a single-polypeptide-chain glycoprotein of mol.wt. 155 000 containing 9.3% carbohydrate. Factor H is cleaved by plasma proteinases to a two-chain form. This cleavage can be mimicked by trypsin, and the two-chain form retains fully the C3b-inactivator cofactor activity of Factor H. The proteolytic fragments of Factor H are compared with those of other proteins (C4b-binding protein and erythrocyte C3b-receptor) that act as cofactors for C3b-inactivator.

scholarly journals Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

2021 ◽  
Author(s):  
Jessica Krakow ◽  
Michal Hammel ◽  
Ying Zhu ◽  
Brian J Hillier ◽  
Bryce Paolella ◽  
...  
Keyword(s):  
T Cell ◽  
Single Chain ◽  
Cleavage Rate ◽  
Post Translational Modifications ◽  
Single Polypeptide Chain ◽  
Negative Stain ◽  
Negative Stain Electron Microscopy ◽  
Single Domain Antibodies ◽  
Single Polypeptide ◽  
Structural Architecture

Abstract Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR (SCU-PA) FROM HT-1080 HUMAN FIBROSARCOMA CELLS IS A GENUINE PROENZYME

1987 ◽  
Author(s):  
L Skriver ◽  
L C Petersen ◽  
L R Lund ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Limited Proteolysis ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Single Polypeptide Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pancreatic Trypsin Inhibitor ◽  
Human Fibrosarcoma Cells ◽  
Single Polypeptide ◽  
Hydrolysis Of

U-PA is released from many cells as a single polypeptide chain (scu-PA) that is converted into its active two-chain form (tcu-PA) by limited proteolysis with plasmin. There is general agreement that scu-PA has an extremely low amidolytic activity, but different oppinions exist, as to whether scu-PA itself can activate plasminogen. We have reinvestigated the plasminogen activating activity of our scu-PA preparations by means of a direct [125]I-plasminogen conversion assay and two amidolytic assays for plasmin and u-PA activity. In the [125]I-plasminogen conversion assay in the presence of bovine pancreatic trypsin inhibitor (BPTI) the subsequent plasmin catalyzed conversion of scu-PA is blocked while the plasminogen activation is unaffected. In this assay with 3oo nM Glu-plasminogen and 15 pM BPTI, 4o nM scu-PA caused a low but significant plasminogen conversion, which could be fully inhibited by pretreatment of scu-PA with diisopropylfluorophos-phate (DFP). DFP-treated scu-PA was convertible to fully active tcu-PA. Rates of plasminogen activation in this type of assay for scu-PA activity was at least 4oo fold slower than that measured for tcu-PA. A coupled amidolytic assay with Lys-plasminogen, scuPA or tcu-PA, BPTI, and the high affinity plasmin substrate H-D-Val-Phe-LyspNA (S2390) was performed under conditions that ensures a low steady state concentration of free plasmin. In this assay the initial rate of Lys-plasminogen activation by DFP-treated scu-PA was at least 25o fold slower than that measured for tcu-PA. Finally, u-PA activity was measured in an assay with the chromogenic substrate <Glu-Gly-ArgpNA (S2444) (o.8mM) in the presence of highly purified Glu-plasminogen (3oonM) and DFP-treated scu-PA (2nM) in the absence of BPTI. Within the initial 15 min of incubation no detectable hydrolysis of S2444 occurred. Addition of tcu-PA (2pM) or plasmin (o.lnM) to the scu-PA/Glu-plasminogen mixture caused a significant reduction of the lag period before onset of the cascade reaction leading to scu-PA conversion and subsequent hydrolysis of S2444. We conclude that the low rates of plasminogen activation measured in these assays by scu-PA might be accounted for by the presence of trace amounts of tcu-PA in the scu-PA preparations, and that scu-PA meets the requirements for a genuine proenzyme

Purification Of A New High Mw Single Chain Form Of Urokinase (UK) From Urine

1981 ◽  
Author(s):  
S S Husain ◽  
V Gurewich ◽  
B Lipinski
Keyword(s):  
Disulfide Bonds ◽  
Specific Activity ◽  
Gel Filtration ◽  
Chain Structure ◽  
Isolation Procedure ◽  
Single Chain ◽  
Native Form ◽  
The Uk ◽  
Chain Form ◽  
Fibrin Thrombi

Purified UK exists in 2 forms, a high MW (55,000 daltons) and a low MW (33,000 daltons) enzyme. The former is composed of 2 chains held together by disulfide bonds and is believed to be a precursor of the latter. Little affinity for fibrin has been ascribed to either form. We have purified a third form of UK using affinity chromatography on fibrin-celite, a method which we developed to purify the major plasminogen activator from blood. When freshly voided urine was exposed to fibrin-celite, approximately 20% of the UK present was tightly bound to the fibrin. This high affinity UK (HAUK) was eluted in a sharp peak with arginine (0.2 M). Purification was achieved by gel filtration (Sephadex G-200) of the activator peak. SDS gel electrophoresis showed a single band (56,000 daltons) which remained intact after exposure to reducing agents, indicating that HAUK has a single chain structure and may be the native form of UK. The specific activity of HAUK is relatively low (40,000-50,000 CTA u/mg) suggesting that it may be a proactivator. Freshly voided urine and a rapid isolation procedure are necessary if degradation of HAUK is to be avoided. The unique high fibrin affinity of HAUK, which is not shared by the other 2 forms of UK, may make it a more specific and efficient fibrinolytic agent by confining and concentrating the enzymatic activity to the fibrin surface. The attachment of a suitable radiolabel to HAUK may also provide a useful tool for the detection of intravascular fibrin thrombi.

scholarly journals PHYSIOCHEMICAL AND BIOLOGICAL PROPERTIES OF THE MAJOR BASIC PROTEIN FROM GUINEA PIG EOSINOPHIL GRANULES

1974 ◽  
Vol 140 (2) ◽  
pp. 313-332 ◽  
Author(s):  
Gerald J. Gleich ◽  
David A. Loegering ◽  
Friedrich Kueppers ◽  
Satya P. Bajaj ◽  
Kenneth G. Mann
Keyword(s):  
Guinea Pig ◽  
Disulfide Bonds ◽  
Basic Protein ◽  
Biological Properties ◽  
Low Molecular Weight ◽  
Major Basic Protein ◽  
Guanidinium Chloride ◽  
E Coli ◽  
Single Polypeptide Chain ◽  
Single Polypeptide

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

scholarly journals Rapid inactivation of plant aconitase by hydrogen peroxide

1991 ◽  
Vol 276 (3) ◽  
pp. 643-648 ◽  
Author(s):  
F Verniquet ◽  
J Gaillard ◽  
M Neuburger ◽  
R Douce
Keyword(s):  
Polypeptide Chain ◽  
Specific Activity ◽  
Dose Range ◽  
The State ◽  
Matrix Space ◽  
Single Polypeptide Chain ◽  
Low Field ◽  
The Matrix ◽  
G Value ◽  
Single Polypeptide

Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.

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