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PubMed1983

Regions specifying transcriptional termination and pausing in the bacteriophage SP01 terminal repeat.

We have determined the nucleotide sequences of four termination sites recognized by Bacillus subtilis RNA polymerase. These sites are located in the terminally repeated segment of the bacteriophage SP01 genome, where most early phage transcription occurs. The SP01 terminators have structures that are similar to those recognized by Escherichia coli RNA polymerase, containing a region of dyad symmetry followed by a stretch of HMU residues in the noncoding DNA strand (HMU is substituted for T in SP01 DNA). We note that in a terminator that is only 60% efficient in vitro, there is a greater distance between these two conserved elements that exists in more efficient terminators. We also find that RNA polymerase molecules which elongate a transcript through a partial terminator often pause at this site.

Bacillus subtilis↗
PubMed1995

The cytoplasmic C-terminal domain but not the N-terminal domain of latent membrane protein 1 of Epstein-Barr virus is essential for B cell activation.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is essential for EBV-induced immortalization of human primary B cells, transforms rodent fibroblasts and induces the up-regulation of several B cell activation markers when transiently expressed in primary B cells. The biochemical function of LMP-1 is unclear and limited information is available on the involvement of different domains of the protein in B cell activation. The present study describes the characterization of LMP-1 N- and C-terminal deletion mutants in terms of their cell surface distribution and ability to induce activation markers in primary human B cells and in the type I Burkitt's lymphoma cell line DG75. The C-terminal deletion mutant was detected by immunofluorescence via antibodies targeted against an eight amino acid FLAG epitope substituted for the entire predicted cytoplasmic C-terminal domain. Our findings show that N-terminal deletion mutants of LMP-1 are unable to attain their usual patched distribution on the plasma membrane but retain the ability to activate B cells. In contrast, the C-terminal deletion mutant shows the same patched cell surface distribution as wild-type LMP-1 but is unable to activate B cells. The patched distribution of LMP-1 in the plasma membrane is therefore not sufficient nor necessary for the induction of B cell activation and the results rule out patching as a direct mechanism in LMP-1-induced activation. This is the first study addressing the role of the LMP-1 C-terminal domain in lymphocytes and our results show that this domain is essential for B cell activation and therefore likely to be important for the early events of B cell immortalization by EBV.

Antigens, CD↗
PubMed2006

Mutagenic definition of a papain-like catalytic triad, sufficiency of the N-terminal domain for single-site core catalytic enzyme acylation, and C-terminal domain for augmentative metal activation of a eukaryotic phytochelatin synthase.

Phytochelatin (PC) synthases are gamma-glutamylcysteine (gamma-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (gamma-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes gamma-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes gamma-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent gamma-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary gamma-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary gamma-Glu-Cys acylation.

Acylation↗
PubMed1995

Low-molecular-weight succinoglycan is predominantly produced by Rhizobium meliloti strains carrying a mutated ExoP protein characterized by a periplasmic N-terminal domain and a missing C-terminal domain.

The membrane topology of the Rhizobium meliloti 2011 ExoP protein involved in polymerization and export of succinoglycan was analysed by translational fusions of lacZ and phoA reporter genes to the exoP gene. Based on this analysis, the ExoP protein could be divided into an N-terminal domain mainly located in the periplasmic space and a C-terminal domain located in the cytoplasm. Whereas the C-terminal domain of ExoP is characterized by a potential nucleotide-binding motif, the N-terminal ExoP domain contains the sequence motif 'PX2PX4SPKX11GXMXG', which is also present in proteins involved in the determination of O-antigen chain length. R. meliloti strains carrying mutated exoP* genes, exclusively encoding the N-terminal ExoP domain, produced a reduced amount of succinoglycan. This reduction could be suppressed by a mutation in the regulatory gene exoR. The ratio of low-molecular-weight to high-molecular-weight succinoglycan was significantly increased in the exoP* mutant strain. In the exoP*/exoR mutant strain only low-molecular-weight succinoglycan could be detected. Based on sequence homologies and similar hydropathic profiles, the N-terminal domain of ExoP was proposed to be a member of a protein family thought to be involved in polysaccharide chain-length determination.

Amino Acid Sequence↗
PubMed1975

Studies on the role and mode of operation of the very-lysine-rich histone H1 (F1) in eukaryote chromatin. The properties of the N-terminal and C-terminal halves of histone H1.

Restricted chymotrypsin digestion of calf thymus H1 histone gives two fragments, residues 1--106 and 107--C-terminal. These were studied by proton magnetic resonance and circular dichroism. The N-terminal fragment exhibited some salt-induced structure in aqueous solution, but this did not parallel the globular structure of the intact H1 molecule. Comparison of circular dichroism results with helix predictions for this portion of the molecule suggests that the secondary structure may be the same in this fragment as it is in the corresponding region of the whole molecule. The C-terminal fragments show very little salt-induced structure. The N-terminal fragments binds to DNA very weakly, but the C-terminal fragment binds as strongly as the whole molecule. In the C-terminal fragment, about one quarter of the lysine residues are not bound to the DNA in water, but initial increase of salt concentration causes them to become bound. This increasing binding occurs under the same ionic conditions that cause chromatin condensation and condensation of H1 - DNA complexes, and it is suggested that there may be a connection between these phenomena.

Amino Acids↗
PubMed1987

Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study.

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121, 213-219]. It is now established that this N-terminal region of the A1-light chain interacts directly with the C-terminal region of actin in the acto-S1(A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+(CH3)3 protons of alpha-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11-15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1(A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3Ala residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (less than 1.5 nm) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head.

Actins↗
PubMed2005

Role of N-terminal amino acids in the absorption-enhancing effects of the c-terminal fragment of Clostridium perfringens enterotoxin.

We recently found that a polypeptide, the C-terminal of Clostridium perfringens enterotoxin (C-CPE), was a novel type of drug absorption enhancer. The C-terminal of C-CPE is thought to play a role in the binding of C-CPE to its receptor, claudin-4; however, the function of the N-terminal of C-CPE is unclear. In the present study, we evaluated the role of the N-terminal domain of C-CPE in jejunal absorption and claudin-4 binding. The treatment of rat jejunum with C-CPE resulted in enhanced absorption of dextran, with a molecular weight of 4000 Da. However, treatment with C-CPE220, which lacks the 36 N-terminal amino acids of C-CPE, did not enhance jejunal absorption. C-CPE had affinity for claudin-4 in rat jejunum lysates and Caco-2 lysates, but C-CPE220 did not. Interaction of C-CPE with the recombinant extracellular domain 2 of human claudin-4 (EC2hCld-4), which is the putative binding site for C-CPE, was observed, but C-CPE220 had no affinity for EC2hCld-4. To investigate the effect of C-CPE220 on the barrier function of tight junctions, we measured transepithelial electric resistance (TER) in C-CPE- or C-CPE220-treated Caco-2 monolayer cells. Although C-CPE decreased TER in Caco-2 monolayer cells, C-CPE220 did not disrupt the barrier function of tight junctions. Together, these results indicate that the 36 N-terminal amino acids of C-CPE may be necessary for the enhanced absorption mediated by C-CPE and play a partial role in binding to claudin-4.

Amino Acid Sequence↗
PubMed1997

Linker mutagenesis of the Caulobacter crescentus S-layer protein: toward a definition of an N-terminal anchoring region and a C-terminal secretion signal and the potential for heterologous protein secretion.

Linker insertion mutagenesis was used to modify the paracrystalline surface layer (S-layer) protein (RsaA) of the gram-negative bacterium Caulobacter crescentus. Eleven unique BamHI linker insertions in the cloned rsaA gene were identified; at the protein level, these linker insertions introduced 4 to 6 amino acids at positions ranging from the extreme N terminus to the extreme C terminus of the 1,026-amino-acid RsaA protein. All linker-peptide insertions in the RsaA N terminus caused the secreted protein to be shed into the growth medium, suggesting that the RsaA N terminus is involved in cell surface anchoring. One linker-peptide insertion in the RsaA C terminus (amino acid 784) had no effect on S-layer biogenesis, while another (amino acid 907) disrupted secretion of the protein, suggesting that RsaA possesses a secretion signal lying C terminal to amino acid 784, near or including amino acid 907. Unlike extreme N- or C-terminal linker-peptide insertions, those more centrally located in the RsaA primary sequence had no apparent effect on S-layer biogenesis. By using a newly introduced linker-encoded restriction site, a 3' fragment of the rsaA gene encoding the last 242 C-terminal amino acids of the S-layer protein was expressed in C. crescentus from heterologous Escherichia coli lacZ transcription and translation initiation information. This C-terminal portion of RsaA was secreted into the growth medium, confirming the presence of a C-terminal secretion signal. The use of the RsaA C terminus for the secretion of heterologous proteins in C. crescentus was explored by fusing 109 amino acids of an envelope glycoprotein from infectious hematopoietic necrosis virus, a pathogen of salmonid fish, to the last 242 amino acids of the RsaA C terminus. The resulting hybrid protein was successfully secreted into the growth medium and accounted for 10% of total protein in a stationary-phase culture. Based on these results and features of the RsaA primary sequence, we propose that the C. crescentus S-layer protein is secreted by a type I secretion system, relying on a stable C-terminal secretion signal in a manner analogous to E. coli alpha-hemolysin, the first example of an S-layer protein secreted by such a pathway.

Bacterial Outer Membrane Proteins↗
PubMed1982

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.

Amino Acid Sequence↗
PubMed2003

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU.

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.

Amino Acid Sequence↗
PubMed1992

Echo-wave termination of ventricular tachycardia. A common mechanism of termination of reentrant arrhythmias by various pharmacological interventions.

BACKGROUND: Based on epicardial mapping, different mechanisms of termination of reentrant ventricular tachycardia by various pharmacological interventions are described. METHODS AND RESULTS: In 40 Langendorff-perfused rabbit hearts, rings of anisotropic left ventricular epicardium were made by a cryoprocedure. Sustained monomorphic ventricular tachycardia based on continuous circus movement of the impulse around the ring was induced by programmed stimulation. Increasing doses of heptanol (n = 10), potassium (n = 10), tetrodotoxin (n = 6), RP62719 (a new class III drug) (n = 4), flecainide (n = 5), and propafenone (n = 5) were administered to terminate ventricular tachycardia. Epicardial mapping (248 points) was used to study the mechanism of termination of ventricular tachycardia. In 28 of 40 hearts, ventricular tachycardia terminated because the drugs produced complete conduction block of the impulse in a segment of the reentrant pathway. In the remaining 12 hearts (heptanol, n = 2; potassium, n = 3; tetrodotoxin, n = 2; RP62719, n = 2; flecainide, n = 1; and propafenone, n = 2), termination of ventricular tachycardia occurred by collision of the circulating impulse with a spontaneous antidromic wave front reflected within the circuit. This phenomenon occurred when the circulating impulse encountered an arc of functional conduction block that did not extend along the whole width of the ring. As a result, the impulse dissociated into a continuing orthodromic circulating wave and a returning antidromic echo-wave caused by microreentry within the ring. CONCLUSIONS: Independent of their mechanisms of action, sodium channel blockers, electrical uncouplers, and class III drugs terminate reentrant ventricular tachycardia either by complete conduction block or by collision of the impulse with an echo-wave.

Animals↗
PubMed1989

Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas.

Truncated glucagon-like peptide-1 (GLP-1) possesses a potent stimulatory activity for insulin secretion and a slight inhibiting activity for glucagon secretion. The aim of this paper is to examine the activities of N- and C-terminal fragments of GLP-1 using a rat pancreas perfusion system. Concerning the N-terminal portion, GLP-1(7-37) amide elicited a clear insulinotropic activity at 0.1 or 1 nM with the perfusate containing 5.5 mM glucose and 5 mM arginine, while 10 nM GLP-1-(1-37) amide, -(6-37) amide, and -(8-37) amide did not. Concerning the C-terminal portion, GLP-1-(7-37) amide, -(7-37), and -(7-36) amide had a similar potency of insulinotropic activity, and GLP-1-(7-35) was less potent; 0.1 nM GLP-1-(7-35) did not stimulate insulin release, nor did 10 nM GLP-1-(7-20). Glucagon release was significantly suppressed by 1 and 10 nM GLP-1-(7-37) amide, 10 nM GLP-1-(7-37), and 1 nM GLP-1-(7-36) amide. Other fragment peptides of GLP-1, including GLP-1-(7-35), had no effect. From these results it is concluded that histidine at position 7 of GLP-1 as a free N-terminal amino acid is very important in GLP-1's insulinotropic activity and probably in glucagon-inhibiting activity, and that C-terminal amidation and three C-terminal amino acids are less important for these activities.

Animals↗
PubMed1994

The extracellular amino-terminal region of the parathyroid hormone (PTH)/PTH-related peptide receptor determines the binding affinity for carboxyl-terminal fragments of PTH-(1-34).

The recombinant human PTH/PTH-related peptide (PTHrP) receptor, when transiently expressed in COS-7 cells, binds [Nle8,18,Tyr34] bovine PTH-(7-34)amide [PTH-(7-34)], human PTH-(10-34)amide [PTH-(10-34)], and bovine PTH-(15-34)amide [PTH-(15-34)] with at least 50-fold higher affinity than does the rat receptor homolog. In contrast, PTH-(1-34) binding affinities are similar for both receptor homologs. To map those areas of the PTH/PTHrP receptors that determine the binding specificity for carboxyl-terminal fragments of PTH-(1-34), we constructed chimeric rat/human PTH/PTHrP receptors. These bound PTH-(1-34) with normal affinity and, therefore, must have an overall conformation that resembles that of native receptors. Chimeras with the amino-terminal extracellular domain of the human PTH/PTHrP receptor have a considerably higher binding affinity for PTH-(7-34), PTH-(10-34), and PTH-(15-34) than do the reciprocal receptor constructs in which the amino-terminal region is from the rat PTH/PTHrP receptor. The opossum PTH/PTHrP receptor homolog also binds PTH-(7-34) with higher affinity than the rat receptor, and studies of rat/opossum chimeras confirm the importance of the amino-terminal extracellular domain in determining the PTH-(7-34) binding specificity. Mutant rat and human PTH/PTHrP receptors in which either residues 61-105 of the extracellular region or most of the intracellular tail were deleted have PTH-(7-34) binding characteristics indistinguishable from those of either wild-type receptor. These findings indicate that the amino-terminal extracellular region of the PTH/PTHrP receptor contains a domain(s) that largely determines the binding affinity of amino-terminally truncated PTH analogs. This region, therefore, is likely to constitute a site for ligand-receptor interaction.

Animals↗
PubMed1989

Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with [32P]ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of [32P]ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identical with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide (Pandey, V. N., and Modak, M. J. (1988a). J. Biol. Chem. 263, 3744-3751). The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.

Adenosine Triphosphate↗
PubMed1983

Interaction of human beta-endorphin with nonopiate binding sites on the terminal SC5b-9 complex of human complement. Significance of COOH-terminal beta H-endorphin fragments.

We have characterized the binding of 125I-labeled human beta-endorphin (125I-beta H-endorphin) to sites present on the terminal fluid-phase complex of human complement, consisting of complement components C5b, C6, C7, C8, C9, and the S-protein (SC5b-9 complex). Specific binding exhibited saturability, reversibility, structural specificity, temperature dependence, and absence of negative cooperative effects. Binding was maximal at 4 degrees C and pH 7.0; it was diminished by monovalent and divalent cations as well as by increasing concentrations of urea and Triton X-100 and apparently required intact disulfide groups. Binding was not inhibited by a number of opioid peptides sharing common sequences with the NH2 terminus of beta H-endorphin. In contrast, binding was inhibited by beta H-endorphin, N-acetyl-beta H-endorphin, and a series of COOH-terminal beta H-endorphin fragments, where of the COOH-terminal dipeptide Gly-Glu represented the minimal effective structure. Stepwise extension towards the NH2 terminus led to an increased binding affinity of the respective fragment. Computer resolution of competition curves yielded one binding component for several shorter COOH-terminal beta H-endorphin fragments and for beta H-endorphin (1-5) + (16-31), whereas two distinct binding components were obtained when beta H-endorphin (27-31), beta H-endorphin (6-31), N-acetyl-beta H-endorphin or beta H-endorphin were used as inhibitors. This study presents detailed data on the binding of COOH-terminal beta H-endorphin fragments to specific nonopiate binding sites present on the terminal SC5b-9 complex of human complement. We suggest that through this interaction, beta H-endorphin may modulate certain functions within the immune system.

Binding Sites↗
PubMed1995

Carboxy-terminal degradation of peptides using perfluoroacyl anhydrides. A C-terminal sequencing method.

An accurate carboxy-terminal sequencing method has long been sought to complement the Edman degradation procedure for amino-terminal amino acid sequence analysis. The method presented here is a unique and simple method to partly fulfill the needs. Exposure of a polypeptide to perfluoroacyl anhydride vapor at -20 degrees C for 0.5-1 h causes sequential chemical degradation of the molecule from the C-terminus. Fast-atom-bombardment mass spectrometric analysis of the resultant mixture of C-terminally truncated molecules permits the determination of the C-terminal sequence by simple calculation of the mass differences in molecular ions. Experiments suggested that this C-terminal degradation proceeds by active intermediates such as oxazolone at the C-terminal carboxyl residues.

Acetic Anhydrides↗
PubMed1994

Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases. C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin.

In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 x 10(-10) M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher M(r) complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 x 10(-9) M for the truncated variant's interaction with TIMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 x 10(-9) M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.

Binding Sites↗