Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P00750 (PLA)
 16,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The quantification of functional amino (NH2) groups on poly(lactic acid-co-lysine):(poly(L-lactic acid (PLAL:PLA) blends was performed using a colorimetric assay based on the reaction of sulpho-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate (sulpho-SDTB) with primary amino groups. The colorimetric assay was used to assess the available reactive sites for coupling of biologically active species to PLAL. Blends were created that contained from 10 to 70 wt% poly(lactic acid-co-lysine). Bulk lysine contents within the blends were determined by amino acid analysis and ranged from 9.1 micromol g(-1) to 52.9 micromol g(-1) for blends created using PLA of 100000g mol(-1) molecular weight. Surface amino group concentrations on the same set of blends ranged from 0.23 to 1.45 nmol cm(-2). Similar surface amino groups concentrations were measured on blends using 50000, 200000 and 300000g mol(-1) poly(lactic acid). Non-specific interactions of the colorimetric assay reagents with the PLAL-containing blends were measured on blends prepared from epsilon-amino protected PLAL and 100000g mol(-1) PLA. The presence of amino groups within the top 50 angstroms was confirmed by X-ray photoelectron spectroscopy.
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PMID:Colorimetric analysis of surface reactive amino groups on poly(lactic acid-co-lysine):poly(lactic acid) blends. 937 43

Trimeresurus stejnegeri venom which contains TSV-PA (a specific plasminogen activator sharing 60-70% sequence homology with venom fibrinogen-clotting enzymes), also possesses fibrinogen-clotting activity in vitro. A fibrinogen-clotting enzyme (stejnobin) has been purified to homogeneity by gel filtration and ion-exchange chromatography on a Mono-Q column. It is a single-chain glycoprotein with a mol. wt of 44,000. The NH2-terminal amino acid sequence of stejnobin shows great homology with venom fibrinogen-clotting enzymes and TSV-PA. Like TSV-PA, stejnobin was able to hydrolyse several chromogenic substrates. Comparative study of substrate specificities of stejnobin and other venom proteases purified in our laboratory was carried out on five chromogenic substrates. Stejnobin clotted human fibrinogen with a specific activity of 122 NIH thrombin-equivalent units/mg protein. However, stejnobin did not act on other blood coagulation factors, such as factor X, prothrombin and plasminogen. Diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride inhibited its activity, whereas ethylenediamine tetracetic acid had no effect on it, indicating that it is a serine protease. Although stejnobin showed strong immunological cross-reaction with polyclonal antibodies raised against TSV-PA, it was interesting to observe that, unlike the case of TSV-PA, these antibodies did not inhibit the amidolytic and fibrinogen-clotting activities of stejnobin.
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PMID:Characterization of a fibrinogen-clotting enzyme from Trimeresurus stejnegeri venom, and comparative study with other venom proteases. 960 87

The novel mutant streptokinase, SK-K59E, can activate human plasminogen as efficiently as the purified commercially available streptokinase. Several peptide bonds including Lys59-Ser60 in native streptokinase were hydrolyzed in reaction with plasmin and peptides of small molecular masses were generated. The plasminogen activator activity of native streptokinase in reaction with human plasmin declined to 25% of the original activity in a 120-min incubation. On the other hand, the NH2-terminal peptide of SK-K59E remained intact in reaction with plasmin and the activator activity of streptokinase decreased to 75% of the original activity in 120 min. The major degraded peptide fragments of native streptokinase in reaction with plasmin had molecular masses of 36 and 30 kDa. However, two major peptide fragments of 42 and 34 kDa were observed in the reaction of SK-K59E with human plasmin. The 42 kDa peptide fragment, which contained NH2-terminal of streptokinase, could activate human plasminogen as efficiently as the native streptokinase. SK-K59E can induce greater degree of caseinolysis and fibrinolysis than the native streptokinase. In conclusion, the results demonstrate that the prevention of cleavage at Lys59 of streptokinase prolongs the half-life of streptokinase in complex with plasmin and that the NH2-terminal of streptokinase (Ile1-Lys59) plays an important role in maintaining its stability.
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PMID:Preparation of a novel streptokinase mutant with improved stability. 960 35

Staphylokinase (Sak) forms an inactive 1:1 stoichiometric complex with plasminogen which requires both conversion of plasminogen to plasmin and hydrolysis of the Lys10-Lys11 peptide bond of Sak to become a potent plasminogen activator (Schlott, B., Guhrs, K.-H., Hartmann, M., Rocker, A., and Collen, D. (1997) J. Biol. Chem. 272, 6067-6072). Exposure of a positively charged NH2-terminal amino acid after hydrolysis of Sak is a major determinant of the plasminogen-activating potential, but in itself is neither necessary nor sufficient. Here, the structural motifs of the NH2-terminal region Lys11-Gly-Asp-Asp-Ala-Ser16-Tyr-Phe-Glu of processed Sak, required for plasminogen activating potential, were studied by deletion and substitution mutagenesis. Expression in Escherichia coli of variants with deletion of 11, 14, 15, or 16 NH2-terminal amino acids yielded correctly processed but inactive molecules. Expression of their homologues with the NH2-terminal amino acid substituted with Lys-generated derivatives from which the NH2-terminal initiation Met was no longer removed, yielding inactive (</= 10%) Sak42DDeltaN11(M),G12K, active (>50%) Sak42DDeltaN14(M), A15K and Sak42DDeltaN15(M),S16K, and inactive Sak42DDeltaN16(M),Y17K. Lys variants without NH2-terminal Met, generated from fusion proteins in which a His6 tag and a factor Xa recognition sequence were linked to the NH2 terminus of the Sak variants, were indistinguishable from their NH2-terminal Met-containing counterparts. All variants studied had intact affinities for plasminogen as measured by biospecific interaction analysis. The activity of Sak42DDeltaN11(M),G12K could be restored by additional substitution of both Asp13 and Asp14 with Asn, yielding active Sak42DDeltaN11(M),G12K, D13N, D14N, whereas substitution in Sak42DDeltaN16(M),Y17K of Phe18 and Glu19 with Asn yielded inactive Sak42DDeltaN16(M),Y17K,F18N,E19N. These data, in combination with the recent finding that the 20 NH2-terminal amino acids of Sak lack secondary structure, suggest that the NH2-terminal region of Sak is not required for binding to plasmin/plasminogen, but that a positively charged amino acid in the ultimate or penultimate NH2-terminal position corresponding to amino acids 11-16 of this flexible region participates in the reconfiguration of the active site of the plasmin molecule to endow it with plasminogen-activating potential.
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PMID:NH2-terminal structural motifs in staphylokinase required for plasminogen activation. 971 54

Human C4b-binding protein (C4BP) is a regulator of the complement system and plays an important role in the regulation of the anticoagulant protein C pathway. C4BP can bind anticoagulant protein S, resulting in a decreased cofactor function of protein S for activated protein C. C4BP is a multimeric protein containing several identical alpha-chains and a single beta-chain (C4BPbeta), each chain being composed of short consensus repeats (SCRs). Previous studies have localized the protein S binding site to the NH2-terminal SCR (SCR-1) of C4BPbeta. To further localize the protein S binding site, we constructed chimeras containing C4BPbeta SCR-1, SCR-2, SCR-3, SCR-1+2, SCR-1+3, and SCR-2+3 fused to tissue-type plasminogen activator. Binding assays of protein S with these chimeras indicated that SCR-2 contributes to the interaction of protein S with SCR-1, since the affinity of protein S for SCR-1+2 was up to 5-fold higher compared with SCR-1 and SCR-1+3. Using an assay that measures protein S cofactor activity, we showed that cofactor activity was decreased due to binding to constructs that contain SCR-1. SCR-1+2 inhibited more potently than SCR-1 and SCR-1+3. SCR-3 had no additional effect on SCR-1, and therefore the effect of SCR-2 was specific. In conclusion, beta-chain SCR-2 contributes to the interaction of C4BP with protein S.
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PMID:Interaction between protein S and complement C4b-binding protein (C4BP). Affinity studies using chimeras containing c4bp beta-chain short consensus repeats. 1032 21

We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1-44) and COOH-terminal RFYGPV hexapeptide (44-49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OT-mediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OC-OTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.
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PMID:A plasmin-derived hexapeptide from the carboxyl end of osteocalcin counteracts oxytocin-mediated growth inhibition [corrected] of osteosarcoma cells. 1091 58

The blood fibrinolytic system comprises an inactive proenzyme, plasminogen, that can be converted to the active enzyme, plasmin. Plasmin degrades fibrin into soluble fibrin degradation products, by two physiological plasminogen activators (PA), the tissue type PA (t-PA) and the urokinase type PA (u-PA). t-PA mediated plasminogen activation is mainly involved in the dissolution of fibrin in the circulation. u-PA binds to a specific cellular receptor (u-PAR), resulting in enhanced activation of cell bound plasminogen. Inhibition of the fibrinolytic system may occur either at the level of the PA, by specific plasminogen activator inhibitors (PAI), or at the level of plasmin, mainly by alpha 2-antiplasmin. Several molecular interactions have been observed between the fibrinolytic and the matrix metalloproteinase (MMP) system; both systems may cooperate in generating proteolytic activity. Thus, stromelysin-1 (MMP-3) cleaves a 55-kDa kringle 1-4 fragment, containing the lysine binding site(s) involved in cellular binding, from plasminogen and removes a 17-kDa NH2-terminal fragment, containing the cellular receptor binding site, from urokinase (u-PA). Thereby, MMP-3 may downregulate cell associated plasmin activity by decreasing the amount of activatable plasminogen, without affecting cell bound u-PA activity.
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PMID:Elements of the fibrinolytic system. 1146 Apr 80

Matrix metalloproteinase (MMP)-3 inhibited human MDA-MB-231 breast cancer cell invasion through reconstituted basement membrane in vitro. Inhibition of invasion was dependent upon plasminogen and MMP-3 activation, was impaired by the peptide MMP-3 inhibitor Ac-Arg-Cys-Gly-Val-Pro-Asp-NH2 and was associated with: rapid MMP-3-mediated plasminogen degradation to microplasminogen and angiostatin-like fragments; the removal of single-chain urokinase plasminogen activator from MDA-MB-231 cell membranes; impaired membrane plasminogen association; reduced rate of tissue plasminogen activator (t-PA) and membrane-mediated plasminogen activation; and reduced laminin-degrading capacity. Purified human plasminogen lysine binding site-1 (kringles 1-3) exhibited a similar capacity to inhibit MDA-MB-231 invasion, impair t-PA and cell membrane-mediated plasminogen activation and impair laminin degradation by plasmin. Our data provide evidence that MMP-3 can inhibit breast tumour cell invasion in vitro by a mechanism involving plasminogen degradation to fragments that limit plasminogen activation and the degradation of laminin. This supports the hypothesis that MMP-3, under certain conditions, may protect against tumour invasion, which would help to explain why MMP-3 expression, associated with benign and early stage breast tumours, is frequently lost in advanced stage, aggressive, breast disease.
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PMID:Inhibition of human MDA-MB-231 breast cancer cell invasion by matrix metalloproteinase 3 involves degradation of plasminogen. 1223 May 59

This study aimed at immobilizing the antibodies on the surfaces of the solid host membranes in order to improve the property of the biomaterial. The von Willebrand factor (vWf) antibodies were immobilized on the surface of Bombyx mori silk fibroin and PLA (Poly Lactic Acid) membrane by NH3 plasma treatment followed by covalent cross-linking reaction. The immobilization efficiency was evaluated by two methods including the antibody surplus and enzyme-linked immunosorbent assay (ELISA). The in vitro antithrombogenicity representing the activity of immobilized vWf antibody was determined by the method of Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) test. The results demonstrate that the vWf antibodies are immobilized on silk fibroin and PLA membranes in an efficient way with the efficiency of antibody surplus up to 23.88% and ELISA reaction is positive. APTT and TT exceeded the upper limits distinctly, but the value of PT did not change noticeably. The in vitro antithrombogenicity represented the is activity-retaining form of antibodies. These results extend the application of antibody immobilization technique and provide a new idea about the design of biomaterials relating to the coagulation factors.
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PMID:[Immobilization of von Willebrand factor antibody on solid host membranes]. 1629 33

Vascular intimal carcinomatosis refers to a characteristic tumor proliferation on vascular intima that replaces normal endothelium. This pathological event of unknown cause is quite different from tumor thrombotic microangiopathy due to the absence of thrombi on the tumor cell surfaces. We analyzed renal transitional cell carcinoma cases with metastasis to the main pulmonary arteries and marked hyperfibrino(geno)lysis. The fibrinogen-derived products from patients' plasma were identified as D1A/gamma, D1/gamma, and D1/beta by immunoblotting with the NH2-terminus of the fragment D specific antibody JIF-23. In all cases, the neoplastic cells with vascular intimal carcinomatosis were stained positive for anti-human annexin 2, which is a unique cell surface co-receptor for plasminogen and tissue-type plasminogen activator. In contrast, normal renal pelvic mucosa or renal transitional cell carcinoma without vascular intimal carcinomatosis did not express any annexin 2. The isolated transitional cell carcinoma cells contained annexin 2 mRNA and expressed its protein. Anti-annexin 2 antibody and transfection of annexin 2 small interfering RNA into these carcinoma cells significantly inhibited tissue-type plasminogen activator dependent plasmin generation. These findings suggest that annexin 2 mediated fibrinolysis on the transitional cell carcinoma cells may play a role in inducing hemorrhagic disorder in vascular intimal carcinomatosis.
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PMID:Annexin 2 and hemorrhagic disorder in vascular intimal carcinomatosis. 1652 21


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