Preliminary purification and characterization studies of a low molecular weight, high affinity cytosolic lead-binding protein in rat brain

ABSTRACT Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits dd-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k 2/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M−1s−1 for the Actinomadura sp. strain R39 peptidase, 1,400 M−1 s−1 for B. subtilis PBP4a, and 7,000 M−1 s−1 forEscherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k 2/K = 46,000 M−1 s−1). PBP4a is also much more thermostable than the R39 enzyme.

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rab15, a novel low molecular weight GTP-binding protein specifically expressed in rat brain.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)41727-9 ◽

1992 ◽

Vol 267 (31) ◽

pp. 22693

Author(s):

L.A. Elferink ◽

K Anzai ◽

R.H. Scheller

Keyword(s):

Molecular Weight ◽

Rat Brain ◽

Binding Protein ◽

Low Molecular Weight ◽

Gtp Binding Protein ◽

Gtp Binding

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Rap1b Association with the Platelet Cytoskeleton Occurs in the Absence of Glycoproteins IIb/IIIa

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615073 ◽

1998 ◽

Vol 79 (04) ◽

pp. 832-836 ◽

Cited By ~ 3

Author(s):

Thomas Fischer ◽

Christina Duffy ◽

Gilbert White

Keyword(s):

Molecular Weight ◽

Divalent Cation ◽

Binding Proteins ◽

Binding Protein ◽

Platelet Membrane ◽

Low Molecular Weight ◽

Membrane Glycoproteins ◽

Gtp Binding Protein ◽

Gtp Binding Proteins ◽

Gtp Binding

SummaryPlatelet membrane glycoproteins (GP) IIb/IIIa and rap1b, a 21 kDa GTP binding protein, associate with the triton-insoluble, activation-dependent platelet cytoskeleton with similar rates and divalent cation requirement. To examine the possibility that GPIIb/IIIa was required for rap1b association with the cytoskeleton, experiments were performed to determine if the two proteins were linked under various conditions. Chromatography of lysates from resting platelets on Sephacryl S-300 showed that GPIIb/IIIa and rap1b were well separated and distinct proteins. Immunoprecipitation of GPIIb/IIIa from lysates of resting platelets did not produce rap1b or other low molecular weight GTP binding proteins and immunoprecipitation of rap1b from lysates of resting platelets did not produce GPIIb/IIIa. Finally, rap1b was associated with the activation-dependent cytoskeleton of platelets from a patient with Glanzmann’s thrombasthenia who lacks surface expressed glycoproteins IIb and IIIa. Based on these findings, we conclude that no association between GPIIb/IIIa and rap1b is found in resting platelets and that rap1b association with the activation-dependent cytoskeleton is at least partly independent of GPIIb/IIIa.

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Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657333 ◽

1983 ◽

Vol 49 (02) ◽

pp. 109-115 ◽

Cited By ~ 4

Author(s):

M Hoylaerts ◽

E Holmer ◽

M de Mol ◽

D Collen

Keyword(s):

Molecular Weight ◽

Half Life ◽

Low Molecular Weight Heparin ◽

Antithrombin Iii ◽

Factor Xa ◽

Life Time ◽

Low Molecular Weight ◽

High Affinity ◽

Plasma Half Life ◽

Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

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Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽

10.1182/blood-2003-07-2334 ◽

2004 ◽

Vol 103 (4) ◽

pp. 1356-1363 ◽

Cited By ~ 30

Author(s):

Barbara P. Schick ◽

David Maslow ◽

Adrianna Moshinski ◽

James D. San Antonio

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Tandem Repeats ◽

Factor Xa ◽

Low Molecular Weight ◽

Heparin Binding ◽

High Affinity ◽

Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

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