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Biomedical subjects

S M Kelly

Publications and source records attributed to S M Kelly.

At least 73 records · Page 4Linked to original sources
PubMed1996

Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's disease amyloid peptide, A beta 25-35.

The abnormal form of the prion protein (PrPSc), a synthetic prion protein peptide fragment (PrP106-126) and fragments of the Alzheimer's protein precursor, APP, have been shown to be cytotoxic in vitro. We have used synchronous, clonal cell models originally developed to study the toxicity of the Alzheimer's disease amyloid peptide, A beta 25-35, to investigate the actions of PrP peptides. We found that the cytotoxicity of the PrP106-126 depends on its state of aggregation and the cellular expression of PrPc, and is independent of a loss of MTT reduction activity in the absence of cell death associated with the cellular effects of A beta 25-35. These factors may play a role in the lesion specificity of different pathological phenotypes of prion-protein related diseases.

Amino Acid Sequence↗
PubMed1996

The effect of a non-absorbable fat substitute, sucrose polyester, on gastrointestinal function.

OBJECTIVES: To assess the effects of a single dose of a non-absorbable fat substitute, sucrose polyester, on gastrointestinal function. METHODS: The effects of 50 g of sucrose polyester taken as a single drink on gastric emptying, small bowel transit time (SBTT), whole gut transit time (WGTT) and faecal weight compared with a control fat were examined in double-blind studies. The effect of sucrose polyester on gallbladder ejection fraction and gastrointestinal hormones was also assessed. RESULTS: Sucrose polyester was found to accelerate gastric emptying significantly (98.33 +/- 71.0 vs. 112.92 +/- 82.0 min, P = 0.042) but to slow SBTT (153.75 +/- 36.25 vs. 128.75 +/- 47.39 min. P = 0.006). A trend to faster WGTT (37.47 +/- 15.61 vs. 46.63 +/- 20.65 h) and increased faecal weight was observed (453.33 +/- 122.05 vs. 395.0 +/- 107.85 g/48 h), but this did not reach statistical significance. There was a striking reduction in gallbladder ejection fraction with sucrose polyester (21.69 +/- 25.32 vs. 45.27 +/- 27.67%), P = 0.039) and a corresponding significant decrease in the release of cholecystokinin. Lower levels of motilin and enteroglucagon were also observed. CONCLUSIONS: Sucrose polyester has significant effects on gastrointestinal transit, gallbladder contraction and gastrointestinal hormones. These effects can be explained on the basis of decreased luminal products of digestion and may have implications for the widespread use of sucrose polyester as a fat substitute.

Administration, Oral↗
PubMed1996

Attenuated Salmonella typhimurium containing interleukin-2 decreases MC-38 hepatic metastases: a novel anti-tumor agent.

Currently, there is no long-term effective treatment for unresectable hepatic malignancies. Salmonella sp. are known to naturally track to the liver during active infection. To develop a biological vector for delivery of Interleukin-2 (IL-2) to the liver for anti-tumor purposes, the avirulent and highly immunogenic chi 4550 strain of Salmonella typhimurium was used as a vector for IL-2. The gene for human IL-2 was cloned into plasmid pYA292 (renamed pIL-2) and inserted into the attenuated Salmonella typhimurium and renamed [chi 4550 (pIL-2)]. This transformant was found to produced biologically active IL-2 demonstrated by NK cell activation in a 4 hour chromium release cytotoxicity assay. To determine anti-tumor potential, MCA-38 murine adenocarcinoma cells were injected intrasplenically into C57BL/6 mice to produce hepatic metastases and metastases were subsequently enumerated after 12 days. Statistical significance was determined by ANOVA with Fisher's test for significance. Hepatic metastases enumerated by blinded observers revealed that the mean number of metastases was 106.4 in control mice, 103.7 in mice gavage fed attenuated salmonella without IL-2 [chi 4550(pYA292)], and 44.3 in mice fed the chi 4550(pIL2); (ANOVA: p < 0.01). Culture of livers and spleens in mice administered a single gavage dose of salmonella demonstrated persistent colonization for up to 4 weeks. No observable toxicity was seen to either IL-2 or salmonella. These studies demonstrate that the chi 4550(pIL2) is a novel form of in vivo biotherapy which produces biologically active IL-2 and employs the oral route of administration to stimulate an immune response against malignancy in the liver.

Animals↗
PubMed1996

Conformational changes associated with activation of bee venom phospholipase A2.

Bee venom PLA2 possesses a binding site for long-chain fatty acids that can be acylated by long-chain fatty acid imidazolides [Drainas, D. and Lawrence, A.J. (1978) Eur. J. Biochem. 91, 131-138]. Occupation of the site either by oleic acid or the oleoyl residue enhances the catalytic activity by 45.7-fold in the presence of 20% 1-propanol and occupation of the site by the oleoyl residue increases the lytic activity against rabbit erythrocytes by 60-fold. Treatment of the enzyme with oleic acid and glutaraldehyde is known to produce irreversible activation [Lawrence, A.J. and Moores, G.R. (1975) FEBS Lett. 49, 287-291]. Here we show that reduction of the glutaraldehyde-treated enzyme with borohydride stabilizes the activated state and enables the fatty acid to be removed, revealing that a large proportion of the induced activation does not require the presence of oleic acid and indicating that activation is due to a change in the conformation rather than the hydrophobicity of the protein. A kinetic study of enzyme activated by oleoyl imidazolide showed that this modification stabilizes the protein against reversible inactivation by 1-propanol. Comparison of the CD spectra of native and oleoyl imidazolide-activated enzyme shows a change in secondary structure with apparent increase in both alpha-helix and beta-sheet content. During reaction of the enzyme with oleoyl imidazolide, the protein fluorescence shows a biphasic response with an initial fall attributed to occupation of the binding site followed by a progressive decrease with a shift of the emission maximum from 341 to 348 nm. The rate of the second phase closely matched the rate of increase in catalytic activity of the enzyme. Free oleic acid caused a rapid fall in fluorescence emission without the subsequent slow change. These results support the proposal that oleic acid or the oleoyl residue occupy a very similar site on the protein and that occupation of this site increases the exposure of one or both of the Trp residues to the aqueous environment. Binding studies show that activation by oleoyl imidazolide does not increase the affinity of the enzyme for the erythrocyte membrane. It is proposed that occupation of a long-chain fatty acid binding site on the enzyme enhances catalytic activity by changing the conformation of the protein rather than acting as a hydrophobic anchor to the substrate surface.

1-Propanol↗
PubMed1996

Ligand-induced conformational changes in wild-type and mutant yeast pyruvate kinase.

A mutant form of pyruvate kinase in which serine 384 has been mutated to proline has been engineered in the yeast Saccharomyces cerevisiae. Residue 384 is located in a helix in a subunit interface of the tetrameric enzyme, and the mutation was anticipated to alter the conformation of the helix and hence destabilize the interface. Previous results indicate that the mutant favours the T quaternary conformation over the R conformation, and this is confirmed by the results presented here. Addition of phosphoenol-pyruvate (PEP), ADP and fructose-1, 6-bisphosphate (Fru-1.6-P2) singly to the wild-type and mutant enzymes results in a significant quenching of tryptophan fluorescence (12-44%), and for Fru-1,6-P2, a red shift of 15 nm in the emission maximum. Fluorescence titration experiments showed that PEP, ADP and Fru-1,6-P2 induce conformations which have similar ligand-binding properties in the wild-type and mutant enzymes. However, the Fru-1,6-P2 induced conformation is demonstrably different from those induced by either ADP or PEP. The enzymes differ in their susceptibility to trypsin digestion and N-ethylmaleimide inhibition. The thermal stability of the enzyme is unaltered by the mutation. Far-UV CD spectra show that both enzymes adopt a similar overall secondary structure in solution. Taken together, the results suggest that the Ser384-Pro mutation causes the enzyme to adopt a different tertiary and/or quaternary structure from the wild-type enzyme and affects the type and extent of the conformational changes induced in the enzyme upon ligand binding. A simplified minimal reaction mechanism is proposed in which the R and T states differ in both affinity and kcat. Thus, in terms of the models of cooperativity and allosteric interaction, pyruvate kinase is both a K and a V system.

Adenosine Diphosphate↗
PubMed1996

The failure of triage criteria to identify geriatric patients with trauma: results from the Florida Trauma Triage Study.

Since 1990, Florida has used a uniform set of eight triage criteria, known as the trauma scorecard, for triaging adult patients with trauma to state-approved trauma centers. If any one of the eight criteria are met, paramedics classify the patient as a "trauma alert" and transport to a state-approved trauma center. Widespread concern within the trauma care community that the scorecard was not providing an effective tool for adult trauma triage, particularly for older adults, was a motivating force for conducting an evaluation of the trauma scorecard's performance. Thus, the Florida Department of Health and Rehabilitative Services, Office of Emergency Medical Services initiated a research effort to assess the effectiveness of the state-adopted trauma triage criteria for adults, giving special attention to geriatric trauma. The results of the Florida Trauma Triage Study indicate that the eight triage criteria comprising the trauma scorecard produce unacceptable levels of undertriage in elderly patients (age 55 years or older) with life-threatening injuries.

Abbreviated Injury Scale↗
PubMed1996

The need for pediatric-specific triage criteria: results from the Florida Trauma Triage Study.

OBJECTIVE: The objective of the Florida Trauma Triage Study was to assess the performance of state-adopted field triage criteria. The study addressed three specific age groups: pediatric (age < 15 years), adult (age 15-54 years), and geriatric (age 55+ years). Since 1990, Florida has used a uniform set of eight triage criteria, known as the trauma scorecard, for triaging adult trauma patients to state-approved trauma centers. However, only five of the criteria are recommended for use with pediatric patients. This article presents the findings regarding the performance of the scorecard when applied to a pediatric population. DESIGN: We used state trauma registry data linked to state hospital discharge data in a retrospective analysis of trauma patients transported by prehospital providers to any acute care hospital within nine selected Florida counties between July 1, 1991, and December 31, 1991. We used cross-table and logistic regression analysis to determine the ability of triage criteria to correctly identify patients who were retrospectively defined as major trauma. We applied the field criteria to physiologic and anatomy/mechanism of injury data contained in the trauma registry to "score" the patient as major or minor trauma. To make our retrospective determination of major or minor trauma we used the protocols developed by an expert medical panel as described by E. J. MacKenzie et al. (1990). MAIN OUTCOME MEASURES: We calculated sensitivity, specificity, and the corresponding over- and undertriage rates by comparing patient classifications (major or minor trauma) produced by the triage criteria and the retrospective algorithm. We used logistic regression to identify which triage criteria were statistically significant in predicting major trauma. RESULTS: Pediatric cases accounted for 9.2% of the total study population, 6.0% of all hospitalized cases, and 6.8% of all trauma deaths. Of the 1505 pediatric cases available for analysis, the triage criteria classified 269 cases as expected major trauma and 1236 cases as expected minor trauma. The retrospective algorithm classified 78 cases as expected major trauma and 1427 cases as expected minor trauma. The resulting specificity is 84.8% (15.2% overtriage), and the sensitivity is 66.7% (33.3% undertriage). Logistic regression indicated that, of the eight state-adopted field triage criteria, only the Glasgow coma score, ejection from vehicle, and penetrating injuries have a statistically significant impact on predicting major trauma in pediatric patients. CONCLUSIONS: Although the state-adopted trauma scorecard, applied to a pediatric population, produced acceptable overtriage, it did not produce acceptable undertriage. However, our undertriage rate is comparable to the results of other published studies on pediatric trauma. As a result of the Florida Trauma Triage Study, a new pediatric triage instrument was developed. It is currently being field-tested.

Adolescent↗
PubMed1996

Airway foreign bodies.

The removal of airway foreign bodies can be challenging even for the most experienced endoscopist. A familiarity with and working knowledge of older time-tested techniques and instrumentation as well as of the newer rigid and flexible fiberoptic equipment is essential for all who desire to accept these challenging situations. Each instrument has inherent advantages, disadvantages, and limitations in certain situations. Occasionally, a foreign body may be removed more safely through open surgical procedures. A knowledge of the lessons learned and techniques developed by pioneering endoscopists coupled with continuing practice of different endoscopy techniques with a variety of instrumentation will prepare the endoscopist to handle unusual foreign body dilemmas with greater skill and safety.

Adolescent↗
PubMed1995

Kinetics of acid-mediated disassembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Molecular basis of pH stability.

The B-subunit pentamer of Escherichia coli heat-labile enterotoxin (EtxB) is highly stable, maintaining its quaternary structure in a range of conditions that would normally be expected to cause protein denaturation. In this paper the structural stability of EtxB has been studied as a function of pH by electrophoretic, immunochemical, and spectroscopic techniques. Disassembly of the cyclic pentameric structure of human EtxB occurs only below pH 2. As determined by changes in intrinsic fluorescence this process follows first-order kinetics, with the rate constant for disassembly being proportional to the square of the H+ ion concentration, and with an activation energy of 155 kJ mol-1. A C-terminal deletion mutant, hEtxB214, similarly shows first-order kinetics for disassembly but with a higher pH threshold, resulting in disassembly being seen at pH 3.4 and below. These findings are consistent with the rate-limiting step for disassembly of human EtxB being the simultaneous disruption of two interfaces by protonation of two C-terminal carboxylates. By comparison, disassembly of the B-subunit of cholera toxin (CtxB), a protein which shows 80% sequence identity with EtxB, exhibits a much lower stability to acid conditions; with disassembly of CtxB occurring below pH 3.9, with an activation energy of 81 kJ mol-1. Reasons for the observed differences in acid stability are discussed, and the implications of these findings to the development of oral vaccines using EtxB and CtxB are considered.

Bacterial Toxins↗
PubMed1995

NADPH oxidase activity of cytochrome P-450 BM3 and its constituent reductase domain.

Cytochrome P-450 BM3 from Bacillus megaterium catalyses NADPH oxidation in the absence of added substrate. This activity is also associated with the independently expressed flavin-containing reductase domain of the protein. The rates of these activities are more than two orders of magnitude lower than those in the presence of fatty acid P-450 substrates or artificial electron acceptors. Electrons derived from NADPH in this fashion are transferred onto oxygen, generating superoxide (O2-) anions. The formation of these active oxygen species is detectable by luminometry and the chemiluminescence can be inhibited through the addition of superoxide dismutase (but not catalase). This activity is reminiscent of the microbicidal NADPH oxidase activity associated with neutrophils and other leukocyte blood cell types. Diphenyliodonium, a potent inhibitor of the neutrophil NADPH oxidase, effectively inhibits fatty acid hydroxylase and electron transferase activities catalysed by P-450 BM3 and its reductase domain. CD studies on the native and NADPH-reduced P-450 BM3 and BM3 reductase indicate that no secondary structural alteration is caused by pre-incubation with the reductant. Therefore, the previously recognised reversible time-dependent inactivation of P-450 BM3 by NADPH may be attributed to the NADPH oxidase activity associated with the reductase domain of the enzyme.

Bacillus megaterium↗
PubMed1995

Conformational changes induced by DNA binding of NF-kappa B.

The transcription factor NF-kappa B makes extensive contacts with its recognition site over one complete turn of the double helix. Structural transitions, in both protein and DNA, that accompany formation of the DNA-protein complex were analysed by proteinase sensitivity and circular dichroism (CD) spectroscopy. In the absence of DNA chymotrypsin cleaved p50 after residues Y60 and N78, while proteinase K cleaved p50 after residues S74 and Q180. Previous experiments had indicated that trypsin cleaved p50 after K77. Cleavages after Y60, S74, K77 and N78 were blocked in the presence of bound DNA, whereas cleavage after Q180 was enhanced. Y60, S74, K77 and N78 are all located in the p50 N-terminal domain AB loop, whereas Q180 is located in the mainly alpha-helical region between p50 N-terminal domain beta-strands G' and H. As only Y60 makes direct contact with the DNA it is likely that the AB loop is highly unstructured in the absence of DNA, but is held in a rigid, proteinase-resistant structure by bound DNA. These conclusions were supported by CD spectroscopic studies of recombinant p50 and p65 homodimers, which indicated that both species changed conformation when binding DNA. Examination of the near UV CD spectra revealed that with some DNA sequences the bound and free forms of the DNA assumed different conformations. While this was evident for a fully symmetrical, high affinity recognition site DNA, it was not apparent with less tightly bound DNA.

Base Sequence↗
PubMed1995

The DvA-1 polyprotein of the parasitic nematode Dictyocaulus viviparus. A small helix-rich lipid-binding protein.

DNA encoding a single unit of the DvA-1 polyprotein of the parasitic nematode Dictyocaulus viviparus was isolated and the polypeptide ("rDvA-1L") expressed in Escherichia coli, to give a protein showing high binding affinity for fatty acids and retinoids. Fluorescent fatty acid probes show substantial changes in emission spectrum in the presence of rDvA-1L, which can be reversed by fatty acids (oleic, palmitic, stearic, arachidonic) and retinoids, but not by tryptophan, squalene, or cholesterol. Moreover, changes in intrinsic fluorescence of retinol or retinoic acid confirm a retinoid binding activity. Fluorescence titration experiments indicate stoichiometric binding to a single protein site per monomer unit with affinities (Kd) in the range 3 x 10(-8) M for 11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, and by competition, 5 x 10(-8) M for oleic acid. The extreme blue shift of bound fluorescent fatty acid suggests an unusually low polarity for the protein binding site. The emission spectrum of the single tryptophan of rDvA-1L indicates that it is deeply buried in a nonpolar environment, and its spectrum is unaffected by ligand binding. Far UV circular dichroism of rDvA-1L reveals a high alpha-helix content (53%). Differential scanning calorimetry studies indicate that rDvA-1L is highly stable (T(m) approximately 98 degrees C), refolding efficiently following thermal denaturation. DvA-1 therefore represents an example of a new class of lipid binding protein, and is the first product of a polyprotein with this activity to be described.

Animals↗
PubMed1995

Dissociation and unfolding of the pyruvate dehydrogenase complex by guanidinium chloride.

The effect of guanidinium chloride (GdnHCl) on the pyruvate dehydrogenase complex (PDC) from bovine heart and its constituent enzymes has been studied. The overall activity of the complex is lost reversibly at low levels of GdnHCl (0.2 M) which cause 40-50% inactivation but no loss of overall secondary or tertiary structures of the individual enzymes; the inactivation of the complex is shown to be caused by dissociation of the E1 and E3 components from the E2/X core assembly. This provides an improved procedure for controlled dissociation of the complex and efficient recovery of its component enzymes in their native states. Higher concentrations of GdnHCl (up to 4 M) lead to the unfolding and irreversible inactivation of the separate enzymes of the complex with the E2/X core proving the most resistant to GdnHCl-induced unfolding. Neither the 60-meric E2/X core assembly nor the dimeric E3 component are dissociated into monomers in the presence of 6 M GdnHCl; the latter enzyme forms higher-M(r) aggregates under these conditions.

Animals↗
PubMed1995

Probing the details of the HIV-1 Rev-Rev-responsive element interaction: effects of modified nucleotides on protein affinity and conformational changes during complex formation.

The solution structure of the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE) has been investigated by enzymic and chemical structural probing of a 71 nt RRE transcript. The minimum sequence information required to maintain recognition by the Rev protein has previously been mapped to a 29 nt stem-loop structure, known as minSLIIB. The key recognition target is a single-stranded RNA bubble at the base of the RNA stem. The fine details of RNA recognition have been probed using chemically synthesized minSLIIBs containing variant base or sugar residues at sites within the bubble. These have been analysed by gel retardation assays and their relative affinities for Rev protein determined. Complex formation between the wild-type minSLIIB RRE and Rev protein was also monitored using CD spectroscopy, which suggests a change in RNA conformation upon Rev binding. The spectral change is consistent with localized melting of RNA, leading to a decrease in the level of base stacking and/or a change in base tilting, during formation of the complex. Deoxynucleotide substitutions on just one side, the 5' side, of the bubble inhibit the conformational change detected by CD. The data are consistent with a dynamic interaction between Rev and its target site. The contact points between Rev and the RRE were probed directly using photo-cross-linking with either ribo-5-bromouridine- or ribo-4-thiouridine-substituted minSLIIBs. The data are consistent with protein-RNA contacts at the bottom of the bubble.

Base Sequence↗
PubMed1995

The ABA-1 allergen of the parasitic nematode Ascaris suum: fatty acid and retinoid binding function and structural characterization.

We report here on the structure and function of the ABA-1 allergen protein of the parasitic nematode Ascaris, the first nematode allergen to be characterized in detail. Using the fluorescent fatty acid analog 11-(((5-(dimethylamino)-1-naphthalenyl)sulfonyl)amino)undecanoic acid (DAUDA), it was demonstrated that ABA-1 is a fatty acid binding protein (FABP) with a high affinity for the fluorescent analog (8.8 x 10(-8) M) and for oleic acid in competition experiments (1.3 x 10(-8) M), with a single binding site for ligand per monomer unit. Blue-shifting of fluorescence emission of DAUDA upon binding was unprecedented in degree among FABPs, being equivalent to that occurring in cyclohexane. A similarly blue-shifted spectrum was obtained with a probe in which the fluorophore was bound to the alpha carbon of a fatty acid, indicating that the carboxylate group of bound fatty acids is probably not exposed to solvent. In competition experiments and by observation of changes in their intrinsic fluorescence, retinol and retinoic acid were also found to bind in the fatty acid binding site. Circular dichroism (CD) of the ABA-1 protein revealed a high alpha-helix content (59%) which was consistent with the four-helix structure for the protein predicted from sequence algorithms. Fluorescence measurements showed that the single, highly conserved tryptophan residue is deeply buried in an unusually apolar environment and that this was unaffected by ligand binding. DSC studies of thermal stability indicate that unfolding of the ABA-1 dimer is cooperative and biphasic (Tm approximately 71 and 89 degrees C), suggesting a two-domain thermal unfolding process, again consistent with the predicted structure. Only the folding of the high-temperature domain is reversible on cooling. DSC confirmed the gel filtration analysis, which indicated that ABA-1 forms a dimer. Aside from being the first nematode allergen for which structure or function has been elucidated, ABA-1 provides a highly manipulable model for investigation of the interaction between hydrophobic ligands and alpha-helical proteins.

Allergens↗
PubMed1995

Purification and characterization of the periplasmic nickel-binding protein NikA of Escherichia coli K12.

The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 microM. Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 microM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23,000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis.

Bacterial Proteins↗
PubMed1995

Conformational studies on plasminogen activator inhibitor (PAI-1) in active, latent, substrate, and cleaved forms.

Plasminogen activator inhibitor 1 (PAI-1), the primary physiological inhibitor of t-PA, is an unusual member of the serpin family of serine protease inhibitors, in that it spontaneously converts to a latent form. Latent PAI-1 has been reported to share characteristics with the cleaved form of other serpins. Here we examine the conformation of four forms of PAI-1, active and latent wild-type, together with a noninhibitory, substrate mutant that is cleavable at P1-P1', and its cleaved product. The circular dichroism spectra of active and latent PAI-1 showed differences consistent with decreased alpha-helix from 26% to 22% and increased beta-sheet from 23% to 34% as active-->latent. Active and substrate PAI-1 were less thermostable than latent PAI-1, which was 50% denatured at 70 degrees C. In contrast, cleaved PAI-1 was very stable, with little loss of structure at 100 degrees C. Cleaved PAI-1 was much more resistant to guanidinium chloride (Gdn-HCl), 50% unfolding requiring 4.5 M Gdn-HCl, while active, latent, and substrate forms of PAI-1 were 50% unfolded in 2-2.5 M Gdn-HCl. The differences in fluorescence emission maxima, latent 339 nm, active 336 nm, substrate 343 nm, and cleaved 333 nm, underline the contrast between latent and cleaved PAI-1. The conformational changes occurring on cleavage are clearly more profound than those seen on transition from active to latent PAI-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗