Identification of human plasma C1 inhibitor as a target protein for staphylococcal superantigen-like protein 5 (SSL5)

The family of staphylococcal superantigen-like proteins (SSLs) have a structure similar to bacterial superantigens but exhibit no superantigenic activity. These exoproteins have recently been shown to disturb the host immune defense system. One family member, SSL5, was reported to bind to human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and matrix metalloproteinase-9 (MMP-9) and to interfere with leukocyte trafficking. In the present study, we explored human plasma proteins bound by glutathione S-transferase (GST)-tagged recombinant SSL5 (GST-SSL5) and identified plasma protease C1 inhibitor (C1Inh) as a major SSL5-binding protein based on the results of peptide mass fingerprinting analysis with MALDI-TOFMS. GST-SSL5 was found to attenuate the inhibitory activity of recombinant histidine-tagged C1Inh (C1Inh-His) toward complement C1s. We also observed that the treatment of C1Inh-His with neuraminidase markedly decreased its binding to GST-SSL5. Moreover, C1Inh-His produced by Lec2 mutant cells (deficient in sialic acid biosynthesis) showed much lower binding affinity for SSL5 than that produced by the wild-type CHO-K1 cells, as assessed by pull-down assay. These results suggest that SSL5 binds to C1Inh in a sialic acid-dependent fashion and modulates the host immune defense through perturbation of the complement system in association with S. aureus infection.     

Fluid phase activation of proenzymic C1r purified by affinity chromatography

1. Proenzymic C1r was purified from human plasma in a two-step technique involving indirect affinity chromatography on Sepharose Ig anti-C1s. The capacity of C1r to monomerize at pH 5.0 and to redimerize at neutral pH was used for selective elution of C1r. The yield in purified C1r was 39% from plasma; no trace of contaminating serine proteases was detected from [3H]diisopropyl phosphorofluoridate labelling of C1r. 2. C14 was able to undergo a two-way autoactivation: an intramolecular catalytic process catalysed by proenzymic C1r itself and an intermolecular reaction catalysed by activated C1r formed in the process of the reaction. DFP (5mM) and C1 Inh at a C1 Inh/C1r ratio of 1:1 were effective on the solely intermolecular activation, leading to partial inhibition of the autoactivation from proenzymic C1r: C1r formed during the activation was titrated by the inhibitors. Calcium, high ionic strength or acid pH decreased C1r activation. The pH effect was characterized by a slowed-down reaction below pH 6.0 and no net influence at values as high as 10.5. The two types of activation developed similarly as a function of pH. 3. Peripheral iodination of C1r revealed differences in label distribution between proenzymic (A chain moiety 48%, B chain moiety 52%) and activated C1r (A chain 20%, B chain 80%). Two different conformational states of C1r were also suggested by 125I-labelling at different temperatures.     

A novel transmembrane Ser/Thr kinase complexes with protein phosphatase-1 and inhibitor-2

Protein kinases and protein phosphatases exert coordinated control over many essential cellular processes. Here, we describe the cloning and characterization of a novel human transmembrane protein KPI-2 (Kinase/Phosphatase/Inhibitor-2) that was identified by yeast two-hybrid using protein phosphatase inhibitor-2 (Inh2) as bait. KPI-2 mRNA was predominantly expressed in skeletal muscle. KPI-2 is a 1503-residue protein with two predicted transmembrane helices at the N terminus, a kinase domain, followed by a C-terminal domain. The transmembrane helices were sufficient for targeting proteins to the membrane. KPI-2 kinase domain has about 60% identity with its closest relative, a tyrosine kinase. However, it only exhibited serine/threonine kinase activity in autophosphorylation reactions or with added substrates. KPI-2 kinase domain phosphorylated protein phosphatase-1 (PP1C) at Thr(320), which attenuated PP1C activity. KPI-2 C-terminal domain directly associated with PP1C, and this required a VTF motif. Inh2 associated with KPI-2 C-terminal domain with and without PP1C. Thus, KPI-2 is a kinase with sites to associate with PP1C and Inh2 to form a regulatory complex that is localized to membranes.     

Inhibitor-2 regulates protein phosphatase-1 complexed with NimA-related kinase to induce centrosome separation

Centrosome separation is regulated by balance of in situ protein kinase/phosphatase activities during the cell cycle. The mammalian NimA-related kinase Nek2 forms a complex with the catalytic subunit of protein phosphatase-1 (PP1C). This complex is located at centrosomes and has been implicated in regulation of the cycle of duplication and separation. Inhibitor-2 (Inh2) is an inhibitor protein specific for PP1C, and its expression level fluctuates during the cell cycle. Here we report cellular regulation of the Nek2.PP1C complex by Inh2. PP1C-binding segments of Nek2 were isolated by yeast two-hybrid screening using Inh2 bait. Inh2 indirectly associates with Nek2 via PP1C, which binds to both proteins, forming a bridged heterotrimeric complex. Double Ala mutation of the PP1C-binding site (KVHF) in Nek2 eliminated both PP1C and Inh2 interactions in both a yeast conjugation assay and an in vitro binding assay. The kinase activity of Nek2.PP1C was enhanced 2-fold by addition of recombinant Inh2, with EC(50) = 10 nm. Immunofluorescence showed concentration of endogenous Inh2 at centrosomes and in a region surrounding the centrosomes. Transient expression of wild-type Inh2 increased by 5-fold dispersed/split centrosomes in fibroblasts, mimicking the phenotype produced by overexpression of Nek2. Deletion of the Inh2 C-terminal domain yielded Inh2-(1-118), which failed to interact with or activate the Nek2.PP1C complex, suggesting that the C-terminal region of Inh2 is required for regulation of the Nek2.PP1C complex. Thus, Inh2 can enhance the kinase activity of the Nek2.PP1C complex via inhibition of phosphatase activity to initiate centrosome separation.     

Characterization of C1q, C1s and C-1 Inh synthesized by stimulated human monocytes in vitro

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.     

Fluid-phase interaction of C1 inhibitor (C1 Inh) and the subcomponents C1r and C1s of the first component of complement, C1.

Interactions between proenzymic or activated complement subcomponents of C1 and C1 Inh (C1 inhibitor) were analysed by sucrose-density-gradient ultracentrifugation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The interaction of C1 Inh with dimeric C1r in the presence of EDTA resulted into two bimolecular complexes accounting for a disruption of C1r. The interaction of C1 Inh with the Ca2+-dependent C1r2-C1s2 complex (8.8 S) led to an 8.5 S inhibited C1r-C1s-C1 Inh complex (1:1:2), indicating a disruption of C1r2 and of C1s2 on C1 Inh binding. The 8.5 S inhibited complex was stable in the presence of EDTA; it was also formed from a mixture of C1r, C1s and C1 Inh in the presence of EDTA or from bimolecular complexes of C1r-C1 Inh and C1s-C1 Inh. C1r II, a modified C1r molecule, deprived of a Ca2+-binding site after autoproteolysis, did not lead to an inhibited tetrameric complex on incubation with C1s and C1 Inh. These findings suggest that, when C1 Inh binds to C1r2-C1s2 complex, the intermonomer links inside C1r2 or C1s2 are weakened, whereas the non-covalent Ca2+-independent interaction between C1r2 and C1s2 is strengthened. The nature of the proteinase-C1 Inh link was investigated. Hydroxylamine (1M) was able to dissociate the complexes partially (pH 7.5) or totally (pH 9.0) when the incubation was performed in denaturing conditions. An ester link between a serine residue at the active site of C1r or C1s and C1 Inh is postulated.     

Exercise and recovery metabolism in the pacific spiny dogfish (<Emphasis Type="Italic">Squalus acanthias</Emphasis>)

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, -hydroxybutyrate, glucose, and l-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (~12 h post-exercise) in plasma lactate load and a short-lived increase (~4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO₂, PCO₂, or PNH₃ but the metabolic acidosis caused a decrease in arterial HCO₃^– immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53±0.15 nmol g wet tissue^–1 min^–1; n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of -hydroxybutyrate from the plasma.Abbreviations CoA-SH free coenzyme A - CPT-1 carnitine palmitoyltransferase-1 - CrP creatine phosphate - H⁺_m metabolic proton load - Lac lactate load - PDH pyruvate dehydrogenase - PVP polyvinylpyrrolidone - SCFA-carnitine short-chain fatty acyl-carnitine - TAG triacylglycerol - TENS trancutaneous electrical nerve stimulator Communicated by: L.C.-H. Wang     

Proteolysis and deglycosylation of human C1 inhibitor. Effect on functional properties.

The effects of proteolysis and deglycosylation on C1 inhibitor (C1Inh) were tested with respect to both its ability to form complexes with C1s and its capacity to block C1 autoactivation. Limited proteolysis of C1Inh by Staphylococcus aureus V8 proteinase, proline-specific endopeptidase or elastase generated a major high-Mr (approximately 86,000) fragment. In contrast with the fragment produced by elastase, which was inactive, the fragments resulting from V8 proteinase and proline-specific endopeptidase treatment retained activity. Deglycosylation with N-glycanase or O-glycanase, or both, had no major effect on the functional activity of C1Inh.     

The F1-ATPase inhibitor Inh1 (IF1) affects suppression of mtDNA loss-lethality in Kluyveromyces lactis

Loss of mtDNA by the petite-negative yeast Kluyveromyces lactis is lethal (rho(o)-lethality). However, mutations in the alpha, beta and gamma subunits of F(1)-ATPase can suppress lethality by increasing intramitochondrial hydrolysis of ATP. Increased hydrolysis of ATP can also occur on inactivation of Inh1, the natural inhibitor of F(1)-ATPase. However, not all strains of K. lactis show suppression of rho(o)-lethality on inactivation of INH1. Genetic analysis indicates that one or more alleles of modifying factors are required for suppression. Papillae showing enhanced resistance to ethidium bromide (EB) in INH1 disruptants have mutations in the alpha, beta and gamma subunits of F(1)-ATPase. Increased growth of double mutants on EB has been investigated by disruption of INH1 in previously characterized atp suppressor mutants. Inactivation of Inh1, with one exception, results in better growth on EB and increased F(1)-ATPase activity, indicating that suppression of rho(o)-lethality is not due to atp mutations preventing Inh1 from interacting with the F(1)-complex. By contrast, in suppressor mutants altered in Arg435 of the beta subunit, disruption of INH1 did not change the kinetic properties of F(1)-ATPase or alter growth on EB. Consequently, Arg435 appears to be required for interaction of Inh1 with the beta subunit. In a previous study, a mex1-1 allele was found to enhance mgi(atp) expression. In accord with results from double mutants, it has been found that mex1-1 is a frameshift mutation in INH1 causing inactivation of Inh1p.     

Plasma desorption mass spectrometry of haemoglobin tryptic peptides for the characterization of a Hungarian alpha-chain variant.

S-Aminoethylated-alpha A and -beta A globin tryptic peptides separated by reversed-phase high-performance liquid chromatography have been analysed by plasma desorption mass spectrometry. Almost all the expected alpha A and beta A tryptic fragments were tentatively assigned relative to the known globin chain sequences based on the molecular weight obtained by plasma desorption mass spectrometric analysis of the purified peptides. The application of plasma desorption mass spectrometry for structure elucidation of a haemoglobin alpha-chain variant revealed the first case of Hb Hasharon in Hungary.     

Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins

Free-flow electrophoresis (FFE) and rapid (6 min) RP-HPLC was used to fractionate human citrate-treated plasma. Prior to analysis, the six most abundant proteins in plasma were removed by immunoaffinity chromatography; both depleted plasma and the fraction containing the six abundant proteins depleted were taken for MS-based analysis. Fractionated proteins were digested with trypsin and the generated peptides were subjected to MS-based peptide sequencing. To date, 78 plasma proteins have been unambiguously identified by manual validation from 16% (15/96 FFE total fractions) of the collected FFE pools; 55 identifications were based on > or = 2 tryptic peptides and 23 using single peptides. The molecular weight range of proteins and peptides isolated by this method ranged from approximately 190 K (e.g., Complement C3 and C4) to approximately 4-6 K (e.g., CRISPP and Apolipoprotein C1). This FFE/RP-HPLC approach reveals low-abundance proteins and peptides (e.g., L-Selectin approximately 17 ng/mL and the cancer-associated SCM-recognition, immunodefense suppression, and serine protease protection peptide (CRISPP) at approximately 0.5-1 ng/mL), where CRISPP was found in association with alpha-1-antitrypsin as a non-covalent complex, in the fraction containing the depleted high-abundance proteins. In contrast to shotgun proteomic approaches, the FFE/RP-HPLC method described here allows the identification of potentially interesting peptides to be traced back to their protein of origin, and for the first time, has confirmed the "protein sponge" hypothesis where the 35 residue CRISPP polypeptide is non-covalently complexed with the major circulating plasma protein alpha-1-antitrypsin.     

Primary structure of a dimeric haemoglobin from the deep-sea cold-seep clam Calyptogena soyoae.

The heterodont clam Calyptogena soyoae, living in the cold-seep area of the upper bathyal depth of Sagami Bay, Japan, has two homodimeric haemoglobins (Hb I and Hb II) in erythrocytes. The complete amino acid sequence of 136 residues of C. soyoae Hb II was determined. The sequence showed low homology with any other globins (at most 20% identity) and lacked the N-terminal extension of seven to nine amino acid residues characteristic of all the molluscan haemoglobins sequenced hitherto. Although the subunit assembly of molluscan haemoglobin is known to be 'back-to-front' relative to vertebrate haemoglobin, C. soyoae Hb II is unlikely to undergo such a subunit assembly because it lacks homology in the sequence involving subunit interaction. These structural features suggest that C. soyoae haemoglobin may have accomplished a unique molecular evolution. The distal (E7) histidine residue of C. soyoae Hb II is unusually replaced by glutamine. However, the oxyhaemoglobin is stable enough to act as an O2 carrier, since the autoxidation rate at near physiological temperature (3 degrees C) is about 3 times lower than that of human haemoglobin at 37 degrees C. H.p.l.c. patterns of peptides (Figs. 5-7), amino acid compositions of intact protein and peptides (Table 1) and amino acid sequences of intact protein and peptides (Tables 2 and 3) have been deposited as Supplementary Publication SUP 50150 (11 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1989) 257, 5.     

The effect of severe eccentric exercise-induced muscle damage on plasma elastase, glutamine and zinc concentrations

The aim of this study was to determine if severe exercise-induced muscle damage alters the plasma concentrations of glutamine and zinc. Changes in plasma concentrations of glutamine, zinc and polymorphonuclear elastase (an index of phagocytic cell activation) were examined for up to 10 days following eccentric exercise of the knee extensors of one leg in eight untrained subjects. The exercise bout consisted of 20 repetitions of electrically stimulated eccentric muscle actions on an isokinetic dynamometer. Subjects experienced severe muscle soreness and large increases in plasma creatine kinase activity indicative of muscle fibre damage. Peak soreness occurred at 2 days post-exercise and peak creatine kinase activity [21714 (6416) U · l⁻¹, mean (SEM)] occurred at 3 days post-exercise (P < 0.01 compared with pre-exercise). Plasma elastase concentration was increased at 3 days post-exercise compared with pre-exercise (P < 0.05), and is presumably indicative of ongoing phagocytic leucocyte infiltration and activation in the damaged muscles. There were no significant changes in plasma zinc and glutamine concentrations in the days following eccentric exercise. We conclude that exercise-induced muscle damage does not produce changes in plasma glutamine or zinc concentrations despite evidence of phagocytic neutrophil activation. Accepted: 3 November 1997     

Estrogen attenuates HSP 72 expression in acutely exercised male rodents

Estrogen has been shown to reduce post-exercise skeletal muscle damage. Exercise-induced muscle damage may be a factor in the elevated post-exercise expression of heat-shock proteins (HSPs). Thus, the present investigation was conducted in order to examine the influence of estrogen on post-exercise levels of HSP 72 and heat-shock cognate, HSC 73, in male and female rodents. Prior to an acute bout of treadmill running, male and female Sprague-Dawley rats received daily injections of either 40 microg x kg(-1) of beta-estradiol 3-benzoate or olive oil vehicle for 2 weeks. A two- to fourfold reduction in post-exercise HSP 72 content was observed in the heart, liver, lung and red and white vastus muscles of estradiol-treated males compared with their vehicle-injected counterparts (P < 0.05). Compared to the males, the females had significantly lower post-exercise HSP 72 levels which were not affected by estradiol supplementation. Moreover, estradiol administration in male rodents resulted in a HSP response similar to that of females following exercise. Thus, the results of the present investigation suggest that estrogen is the factor responsible for the observed differences in post-exercise HSP 72 levels between males and females.     

Properties of a monoclonal antibody directed to the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase

A synthetic peptide representing the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase (K-R-R-W-K-K-N-F-I-A-V-S-A-A-N-R-F-K-K-I-S-S-S-G-A-L) was used as an antigen to produce a monoclonal antibody. The antibody (designated MAb RSkCBP1, of the IgM class) reacted with similar affinity (KD approximately 20 nM) by competitive enzyme-linked immunoassay (ELISA) with the antigen peptide and intact rabbit skeletal muscle myosin light chain kinase. MAb RSkCBP1 inhibited rabbit skeletal muscle myosin light chain kinase activity competitively with respect to calmodulin (Ki = 20 nM). The antibody also inhibited myosin light chain kinase activity in extracts of skeletal muscle from several mammalian species (rabbit, sheep, and bovine) and an avian species (chicken). The concentration of MAb RSKCBP1 required for 50% inhibition of enzyme activity was similar for the mammalian species (80 nM) but was significantly higher for the avian species (1.2 microM). A competitive ELISA protocol was used to analyze weak cross-reactivity to other calmodulin-binding peptides and proteins. This assay demonstrated no cross-reactivity with the venom peptides melittin or mastoparan; smooth muscle myosin light chain kinases from hog carotid, bovine trachea, or chicken gizzard; bovine brain calmodulin-dependent calcineurin; or rabbit skeletal muscle troponin I. These data support the contention that the synthetic peptide used as the antigen represents the calmodulin-binding domain of rabbit skeletal muscle myosin light chain kinase and that the calmodulin-binding domains of different calmodulin-regulated proteins may have distinct primary and/or higher order structures.     

Free haemoglobin interferes with detection of endothelin peptides.

High levels of endothelin-like immunoreactivity were detected in red blood cells from rat, pig and man. When characterized on HPLC the immunoreactivity coeluted with haemoglobin, however. Thus, the high levels of endothelin-like immunoreactivity did not reflect occurrence of endothelin peptides but rather the interference of haemoglobin in the RIA. Free haemoglobin > 0.8 g/l (which may occur in haemolytic samples) increased measured plasma "endothelin-like immunoreactivity". SepPak extraction of plasma samples markedly reduced this interference, although some effect still remained at high haemoglobin concentrations. The influence of microperoxidase in the RIA suggests that the interference is related to the haeme portion of haemoglobin and thus may be extended to other haeme-containing proteins, e.g. cytochrome c or guanylate cyclase. The present findings emphasize the importance of characterizing endothelin-like immunoreactivity with HPLC, especially in haemolytic samples.     

Membrane association of the Tnp and Inh proteins of IS50R.

Using a radioimmunoassay for the IS50R proteins Tnp and Inh, we found that both proteins were present primarily in the cytoplasm, but 3 to 11% of Tnp and 3 to 5% of Inh were found in association with the inner membrane. The fractions of total Tnp and Inh that became membrane bound were unaffected by the amount of Tnp and Inh synthesized in whole cells, provided that the ratio of total Tnp to total Inh was not changed. In addition, Inh was not found in the membrane fraction in Tnp- IS50R mutants, indicating that Tnp is required for Inh localization.     

The mechanism of aubstrate eecognition of pyroglutamyl-peptidase I from Bacillus amyloliquefaciens as determined by X-ray crystallography and site-directed mutagenesis

Pyroglutamyl-peptidase is able to specifically remove the amino-terminal pyroglutamyl residue protecting proteins or peptides from aminopeptidases. To clarify the mechanism of substrate recognition for the unique structure of the pyrrolidone ring, x-ray crystallography and site-directed mutagenesis were applied. The crystal structure of pyroglutamyl-peptidase bound to a transition state analog inhibitor (Inh), pyroglutaminal, was determined. Two hydrogen bonds were located between the main chain of the enzyme and the inhibitor (71:O.H-N:Inh and Gln71:N-H.OE:Inh), and the pyrrolidone ring of the inhibitor was inserted into the hydrophobic pocket composed of Phe-10, Phe-13, Thr-45, Ile-92, Phe-142, and Val-143. To study in detail the hydrophobic pocket, Phe-10, Phe-13, and Phe-142 were selected for mutation experiments. The k(cat) value of the F10Y mutant decreased, but the two phenylalanine mutants F13Y and F142Y did not exhibit significant changes in kinetic parameters compared with the wild-type enzyme. The catalytic efficiencies (k(cat)/K(m)) for the F13A and F142A mutants were less than 1000-fold that of the wild-type enzyme. The x-ray crystallographic study of the F142A mutant showed no significant change except for a minor one in the hydrophobic pocket compared with the wild type. These findings indicate that the molecular recognition of pyroglutamic acid is achieved through two hydrogen bonds and an insertion in the hydrophobic pocket. In the pocket, Phe-10 is more important to the hydrophobic interaction than is Phe-142, and furthermore Phe-13 serves as an "induced fit" mechanism.     

Characterizing the response of calcium signal transducers to generated calcium transients

Cellular Ca2+ transients and Ca2+-binding proteins regulate physiological phenomena as diverse as muscle contraction, neurosecretion, and cell division. When Ca2+ is rapidly mixed with slow Ca2+ chelators, EGTA, or Mg2+/EDTA, artificial Ca2+ transients (ACTs) of varying duration (0.1-50 ms half-widths (hws)) and amplitude can be generated. We have exposed several Ca2+ indicators, Ca2+-binding proteins, and a Ca2+-dependent enzyme to ACTs of various durations and observed their transient binding of Ca2+, complex formation, and/or activation. A 0.1 ms hw ACT transiently occupied approximately 70% of the N-terminal regulatory sites of troponin C consistent with their rapid Ca2+ on-rate (8.7 +/- 2.0 x 10(7) M-1 s-1). A 1.1 ms hw ACT produced approximately 90% transient binding of the N-terminal of calmodulin (CaM) to the RS-20 peptide, but little binding of CaM's C-terminal to RS-20. A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase. In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided. The use of ACTs has allowed us to visualize how the Ca2+-exchange rates of Ca2+-binding proteins dictate their Ca2+-induced conformational changes, Ca2+-induced protein/peptide and protein/protein interactions, and enzyme activation and inactivation, in response to Ca2+ transients of various amplitude and duration. By characterizing the response of these proteins to ACTs, we can predict with greater certainty how they would respond to natural Ca2+ transients to regulate cellular phenomena.     

Hepatitis C virus NS3 serine protease interacts with the serpin C1 inhibitor

Both NS3 protein (1007-1657) and its protease moiety (NS3p, 1027-1207) were able to interact in vitro with C1 Inhibitor (C1Inh) to give a 95-kDa Mr C1Inh cleavage product similar to that obtained upon proteolysis by complement protease C1s. High-Mr reaction products were also detected after incubation of C1Inh with NS3 but not with NS3p; they correspond to ester-bonded complexes from their hydroxylamine lability. Similar reactivity of NS3 was observed upon incubation with alpha2-antiplasmin. Serpin cleavage was prevented by treatment of NS3 with synthetic serine protease inhibitors. This interaction between viral NS3 and host serpins suggests that NS3 is likely to be controlled by infected cell protease inhibitors.