Enhancement of heparin cofactor II anticoagulant activity.
(65/1719)
Heparin cofactor II (HCII) is a serpin whose thrombin inhibition activity is accelerated by glycosaminoglycans. We describe the novel properties of a carboxyl-terminal histidine-tagged recombinant HCII (rHCII-CHis(6)). Thrombin inhibition by rHCII-CHis(6) was increased >2-fold at approximately 5 microgram/ml heparin compared with wild-type recombinant HCII (wt-rHCII) at 50-100 microgram/ml heparin. Enhanced activity of rHCII-CHis(6) was reversed by treatment with carboxypeptidase A. We assessed the role of the HCII acidic domain by constructing amino-terminal deletion mutants (Delta1-52, Delta1-68, and Delta1-75) in wt-rHCII and rHCII-CHis(6). Without glycosaminoglycan, unlike wt-rHCII deletion mutants, the rHCII-CHis(6) deletion mutants were less active compared with full-length rHCII-CHis(6). With glycosaminoglycans, Delta1-68 and Delta1-75 rHCIIs were all less active. We assessed the character of the tag by comparing rHCII-CHis(6), rHCII-CAla(6), and rHCII-CLys(6) to wt-rHCII. Only rHCII-CHis(6) had increased activity with heparin, whereas all three mutants have increased heparin binding. We generated a carboxyl-terminal histidine-tagged recombinant antithrombin III to study the tag on another serpin. Interestingly, this mutant antithrombin III had reduced heparin cofactor activity compared with wild-type protein. In a plasma-based assay, the glycosaminoglycan-dependent inhibition of thrombin by rHCII-CHis(6) was significantly greater compared with wt-rHCII. Thus, HCII variants with increased function, such as rHCII-CHis(6), may offer novel reagents for clinical application. (+info)
Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans.
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Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature. (+info)
Carbohydrate deficient glycoprotein syndrome type IV: deficiency of dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase.
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Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins. Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl-phosphate mannose on to the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-dolichol. The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full-length LLOs. N-glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full-length oligosaccharides and because of the incomplete utilization of N-glycosylation sites. The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene. (+info)
Trypsin-catalysed cleavage of purified ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the resultant irreversible loss of carboxylase activity were prevented by prior incubation with the naturally occurring nocturnal Rubisco inhibitor 2'-carboxy-D-arabitinol 1-phosphate (CA1P), as well as with ribulose 1,5-bisphosphate (RuBP), Mg2+ and CO2. CA1P also protected Rubisco from loss of activity caused by carboxypeptidase A. When similar experiments were carried out using soluble chloroplast proteases, CA1P was again able to protect Rubisco against proteolytic degradation and the consequent irreversible loss of catalytic activity. Thus, CA1P prevents the proteolytic breakdown of Rubisco by endogenous and exogenous proteases. In this way, CA1P may affect the amounts of Rubisco protein available for photosynthetic CO2 assimilation. Rubisco turnover (in the presence of RuBP, Mg2+ and CO2) may confer similar protection against proteases in the light. (+info)
Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.
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D-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which is inhibited by covalent interaction with penicillin G. The penicilloyl enzyme spontaneously reactivates and simultaneously releases a penicillin G degradation product; 0.2 mumol of the latter was isolated after incubation of 4.2 mumol of [8-14C]penicillin G with 10 g of membrane protein. It was identified as phenylacetylglycine by chromatographic techniques, infrared spectroscopy, and mass spectrometry. A mechanism for the degradation is proposed in which the remaining part of penicillin G would be released as 5,5-dimethyl-delta2-thiazoline-4-carboxylic acid. The implications of this finding are discussed. (+info)
Yeast Golgi SNARE interactions are promiscuous.
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The transport of proteins between various compartments of the secretory pathway occurs by the budding of vesicles from one membrane and their fusion with another. A key event in this process is the selective recognition of the target membrane by the vesicle and the current view is that SNARE protein interactions likely play a central role in vesicle-target recognition and or membrane fusion. In yeast, only a single syntaxin (Sed5p) is required for Golgi transport and Sed5p is known to bind to at least 7 SNARE proteins. However, the number of Sed5p-containing SNARE complexes that exist in cells is not known. In this study we examined direct pair-wise interactions between full length soluble recombinant forms of SNAREs (Sed5p, Sft1p, Ykt6p, Vti1p, Gos1p, Sec22p, Bos1p, and Bet1p) involved in ER-Golgi and intra-Golgi membrane trafficking. In the binding assay that we describe here the majority of SNARE-binary interactions tested were positive, indicating that SNARE-SNARE interactions although promiscuous are not entirely non-selective. Interactions between a number of the genes encoding these SNAREs are consistent with our binding data and taken together our results suggest that functionally redundant Golgi SNARE-complexes exist in yeast. In particular, over-expression of Bet1p (a SNARE required for ER-Golgi and Golgi-ER traffic) and can bypass the requirement for the otherwise essential SNARE Sft1p (required for intra-Golgi traffic), suggesting that Bet1p either functions in a parallel pathway with Sft1p or can be incorporated into SNARE-complexes in place of Sftp1. None-the-less this result suggests that Bet1p can participate in two distinct trafficking steps, cycling between the ER and Golgi as well as in retrograde intra-Golgi traffic. In addition, suppressor genetics together with the analysis of the phenotypes of conditional mutations in Sft1p and Ykt6p, are consistent with a role for these SNAREs in more than one trafficking step. We propose that different combinations of SNAREs form complexes with Sed5p and are required for multiple steps in ER-Golgi and intra-Golgi vesicular traffic. And that the apparent promiscuity of SNARE-SNARE binding interactions, together with the requirement for some SNAREs in more than one trafficking step, supports the view that the specificity of vesicle fusion events cannot be explained solely on the basis of SNARE-SNARE interactions. (+info)
Purification and characterization of a cobalt-activated carboxypeptidase from the hyperthermophilic archaeon Pyrococcus furiosus.
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A novel metallocarboxypeptidase (PfuCP) has been purified to homogeneity from the hyperthermophilic archaeon, Pyrococcus furiosus, with its intended use in C-terminal ladder sequencing of proteins and peptides at elevated temperatures. PfuCP was purified in its inactive state by the addition of ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) to purification buffers, and the activity was restored by the addition of divalent cobalt (K, = 24 +/- 4 microM at 80 degrees C). The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) had no effect on the activity. The molecular mass of monomeric PfuCP is 59 kDa as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 58 kDa by SDS-PAGE analysis. In solution, PfuCP exists as a homodimer of approximately 128 kDa as determined by gel filtration chromatography. The activity of PfuCP exhibits a temperature optimum exceeding 90 degrees C under ambient pressure, and a narrow pH optimum of 6.2-6.6. Addition of Co2+ to the apoPfuCP at room temperature does not alter its far-UV circular dichroism (CD) or its intrinsic fluorescence spectrum. Even when the CoPfuCP is heated to 80 degrees C, its far-UV CD shows a minimal change in the global conformation and the intrinsic fluorescence of aromatic residues shows only a partial quenching. Changes in the intrinsic fluorescence appear essentially reversible with temperature. Finally, the far-UV CD and intrinsic fluorescence data suggest that the overall structure of the holoenzyme is extremely thermostable. However, the activities of both the apo and holo enzyme exhibit a similar second-order decay over time, with 50% activity remaining after approximately 40 min at 80 degrees C. The N-blocked synthetic dipeptide, N-carbobenzoxy-Ala-Arg (ZAR), was used in the purification assay. The kinetic parameters at 80 degrees C with 0.4 mM CoCl2 were: Km, 0.9 +/- 0.1 mM; Vmax, 2,300 +/- 70 U mg(-1); and turn over number, 600 +/- 20 s(-1). Activity against other ZAX substrates (X = V, L, I, M, W, Y, F, N, A, S, H, K) revealed a broad specificity for neutral, aromatic, polar, and basic C-terminal residues. This broad specificity was confirmed by the C-terminal ladder sequencing of several synthetic and natural peptides, including porcine N-acetyl-renin substrate, for which we have observed (by MALDI-TOF MS) stepwise hydrolysis by PfuCP of up to seven residues from the C-terminus: Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser. (+info)
Role of penicillin-binding proteins in the initiation of the AmpC beta-lactamase expression in Enterobacter cloacae.
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Penicillin-binding proteins (PBPs) are involved in the regulation of beta-lactamase expression by determining the level of anhydromuramylpeptides in the periplasmatic space. It was hypothesized that one or more PBPs act as a sensor in the beta-lactamase induction pathway. We have performed induction studies with Escherichia coli mutants lacking one to four PBPs with DD-carboxypeptidase activity. Therefore, we conclude that a strong beta-lactamase inducer must inhibit all DD-carboxypeptidases as well as the essential PBPs 1a, 1b, and/or 2. (+info)