Biomedical subjects

U Christensen

Publications and source records attributed to U Christensen.

At least 19 recordsLinked to original sources

PubMed2006

Functional ability at age 75: is there an impact of physical inactivity from middle age to early old age?

The aim of this study is to analyze the impact of physical inactivity from middle age to early old age on functional ability at age 75. Physical activity is measured both as cumulated activity from age 50 to 60 to 70 and at three separate points in time. Three hundred eighty-seven men and women born in 1914 and living in seven municipalities in the western part of the County of Copenhagen were followed for 25 years with examinations in 1964, 1974, 1984 and 1989. Analyses were conducted with physical inactivity as an independent variable (accumulated and separately for each point in time) and smoking, sex, school education, household composition, chronic disease at baseline and functional ability at age 70 as possible confounders. There was a strong association between physical inactivity at age 70 and disability at age 75. However, the analyses showed no effect of cumulated physical inactivity from age 50 to 60 to 70 on disability at age 75 when adjusting for functional ability at age 70. Physical inactivity is a risk factor for disability among old people. Thus, old people should be encouraged to take up and maintain physical training throughout the aging process.

Aged↗

PubMed2005

Communication and coping as predictors of fertility problem stress: cohort study of 816 participants who did not achieve a delivery after 12 months of fertility treatment.

BACKGROUND: We investigated coping strategies and communication strategies as predictors of fertility problem stress 12 months after start of fertility treatment. METHODS: We used a prospective, longitudinal cohort design including 2250 people beginning fertility treatment with a 12-month follow-up. Data were based on self-administered questionnaires measuring communication with partner and with other people, coping strategies: active-avoidance coping, active-confronting coping, passive-avoidance coping, meaning-based coping, and fertility problem stress. The study population included those participants (n = 816, men and women) who had not achieved pregnancy by assisted reproduction or delivery at follow-up. RESULTS: Among both men and women, difficulties in partner communication predicted high fertility problem stress (odds ratio for women, 3.47, 95% confidence interval 2.09-5.76; odds ratio for men, 3.69, 95% confidence interval 2.09-6.43). Active-avoidance coping (e.g. avoiding being with pregnant women or children, turning to work to take their mind off things) was a significant predictor of high fertility problem stress. Among men, high use of active-confronting coping (e.g. letting feelings out, asking other people for advice, seeking social support) predicted low fertility problem stress in the marital domain (odds ratio 0.53, 95% confidence interval 0.28-1.00). Among women, medium or high use of meaning-based coping significantly predicted low fertility problem stress in the personal and marital domain. CONCLUSION: The study provides information about where to intervene with fertility patients in order to reduce their stress after medically unsuccessful treatment.

Adaptation, Psychological↗

PubMed2004

The social epidemiology of coping with infertility.

BACKGROUND: To analyse the cross-sectional association between coping responses with infertility and occupational social class. Infertility is evenly distributed across social classes in Denmark, and there is free access to high-quality assisted reproduction technology. METHODS: Data were based on a questionnaire in a consecutive sample of 1169 women and 1081 Danish men who were about to begin assisted reproduction treatment. The coping measure was developed from an adaptation of Lazarus and Folkman's Ways of Coping Questionnaire and based on results from interviews with infertile people. The measure was developed in four categories: active-avoidance coping; active-confronting coping; passive-avoidance coping; meaning-based coping. These subscales were later confirmed by factor analysis. Occupational social class was measured in a standardized way. RESULTS: Contrary to expectations, the logistic regression analyses showed that women from lower social classes V + VI and men from social classes III + IV used significantly more active-confronting coping. Women from lower social classes V + VI used significantly more meaning-based coping. Both men and women from social classes III - VI used significantly more passive-avoidance coping and significantly less active-avoidance coping. CONCLUSION: Due to the significant social differences in coping with infertility, the study suggested that elements of coping may be learned from one's social network and reference group.

Adaptation, Psychological↗

PubMed2003

High local unemployment and increased mortality in Danish adults; results from a prospective multilevel study.

AIMS: To examine the relation between unemployment rates in area of residence and all-cause mortality, taking the individuals' unemployment experience and a number of social and behavioural factors into account. METHODS: Prospective cohort study with record linkage to mortality and unemployment registers. Data were pooled data from two population studies conducted in Copenhagen, Denmark. The association between unemployment at parish level and mortality was examined in Cox proportional hazard analysis. A total of 15 980 men and women, aged 20-67 years and employed at 1 January 1980, were studied. All-cause mortality was followed from January 1981 to December 1998. RESULTS: The unemployment rate in the area of residence was associated with increased mortality (hazard ratio(highest v lowest quartile) 1.35:0.14-1.60) even after adjustment for individuals' unemployment experience in 1980, which was also a risk factor (hazard ratio(yes/no) 1.38:1.16-1.64). These estimates attenuated somewhat when other social and behavioural covariates were taken into account. The effects were similar in men and women, but the influence of individuals' unemployment experience during one and five years decreased gradually with increasing age. CONCLUSION: This prospective study suggests that high local unemployment and individuals' experience of unemployment increase mortality risk, even after adjustment for other social and behavioural factors.

Adult↗

PubMed2001

Presteady-state kinetics of Bacillus 1,3-1,4-beta-glucanase: binding and hydrolysis of a 4-methylumbelliferyl trisaccharide substrate.

In the present study the first stopped-flow experiments performed on Bacillus 1,3-1,4-beta-glucanases are reported. The presteady-state kinetics of the binding of 4-methylumbelliferyl 3-O-beta-cellobiosyl-beta-D-glucoside to the inactive mutant E134A, and the wild-type-catalysed hydrolysis of the same substrate, were studied by measuring changes in the fluorescence of bound substrate or 4-methylumbelliferone produced. The presteady-state traces all showed an initial lag phase followed by a fast monoexponential phase leading to equilibration (for binding to E134A) or to steady state product formation (for the wild-type reaction). The lag phase, with a rate constant of the order of 100 s(-1), was independent of the substrate concentration; apparently an induced-fit mechanism governs the formation of enzyme-substrate complexes. The concentration dependencies of the observed rate constant of the second presteady-state phase were analysed according to a number of reaction models. For the reaction of the wild-type enzyme, it is shown that the fast product formation observed before steady state is not due to a rate-determining deglycosylation step. A model that can explain the observed results involves, in addition to the induced fit, a conformational change of the productive ES complex into a form that binds a second substrate molecule in a non-productive mode.

Amino Acid Substitution↗

PubMed2001

Stopped-flow kinetics of locked nucleic acid (LNA)-oligonucleotide duplex formation: studies of LNA-DNA and DNA-DNA interactions.

The locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue with an extra 2'-O,4'-C-methylene bridge added to the ribose ring. Oligonucleotides that contain LNA monomers have shown greatly enhanced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given mixed sequence in a nucleic acid duplex and as an antisense molecule. Here the kinetics and thermodynamics of a series of oligonucleotide duplex formations of DNA-DNA and DNA-LNA octamers were studied using stopped-flow absorption measurements at 25 degrees C and melting curves. The reactions of the DNA octamer 5'-CAGGAGCA-3' with its complementary DNA octamer 5'-TGCTCCTG-3', and with the LNA octamers 5'-T(L)GCTCCTG-3' (LNA-1), 5'-T(L)GCT(L)CCTG-3' (LNA-2) and 5'-T(L)GCT(L)CCT(L)G-3'(LNA-3), containing respectively one, two or three thymidine 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotide monomers, designated T(L), were studied. In all cases were seen fast second-order association reactions with k(obs)=2x10(7) M(-1)s(-1). At 25 degrees C the dissociation constants of the duplexes obtained from melting curves were: DNA-DNA, 10 nM; DNA-LNA-1, 20 nM; DNA-LNA-2, 2 nM; and DNA-LNA-3, 0.3 nM; thus the greatly enhanced duplex stability induced by LNA is confirmed. Since the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.

DNA↗

PubMed2001

Phosphinic peptide inhibitors of macrophage metalloelastase (MMP-12). Selectivity and mechanism of binding.

Pseudopeptide inhibitors of MMP-12 with a phosphinic dipeptide G psi[PO(2)H-CH(2)]L covering the P1-P1'- positions originating from a combinatorial solid phase library have been identified and kinetically analysed with respect to binding mechanism and selectivity towards MMP-7, MMP-9, MMP-13 and MMP-14. One compound with a low nanomolar dissociation constant for MMP-12 showed significantly lower affinity towards all other MMPs tested compared to MMP-12. Two compounds showed selectivity against MMP-9, MMP-13 and MMP-14. One additional compound showed selectivity against MMP-7. The selectivity of these compounds could partly be rationalized by analysis of homology models of the enzymes. Truncated versions of one inhibitor spanning P2 to P2', P3 to P2' or P2 to P3' showed that interactions on both the prime and the non-prime side are important for binding. A two-step binding mechanism, with a rate limiting second step, was shown for binding of a tryptophane containing inhibitor to MMP-12 by transient state analysis, using the tryptophane residue of the inhibitor as fluorescent probe.

Animals↗

PubMed2000

Glucoamylase: structure/function relationships, and protein engineering.

Glucoamylases are inverting exo-acting starch hydrolases releasing beta-glucose from the non-reducing ends of starch and related substrates. The majority of glucoamylases are multidomain enzymes consisting of a catalytic domain connected to a starch-binding domain by an O-glycosylated linker region. Three-dimensional structures have been determined of free and inhibitor complexed glucoamylases from Aspergillus awamori var. X100, Aspergillus niger, and Saccharomycopsis fibuligera. The catalytic domain folds as a twisted (alpha/alpha)(6)-barrel with a central funnel-shaped active site, while the starch-binding domain folds as an antiparallel beta-barrel and has two binding sites for starch or beta-cyclodextrin. Certain glucoamylases are widely applied industrially in the manufacture of glucose and fructose syrups. For more than a decade mutational investigations of glucoamylase have addressed fundamental structure/function relationships in the binding and catalytic mechanisms. In parallel, issues of relevance for application have been pursued using protein engineering to improve the industrial properties. The present review focuses on recent findings on the catalytic site, mechanism of action, substrate recognition, the linker region, the multidomain architecture, the engineering of specificity and stability, and roles of individual substrate binding subsites.

Amino Acid Sequence↗

PubMed2000

pH-dependence of the fast step of maltose hydrolysis catalysed by glucoamylase G1 from Aspergillus niger.

The presteady-state kinetic parameters of the interaction ofwild-type glucoamylase from Aspergillus niger (EC 3.2.1.3)with maltose were obtained and analysed in the pH range 3-7 withintervals of 0.25 pH units. In all cases the following three-step reaction scheme was found to apply. [Equation: see text]. The general result of the analysis of the presteady-state kinetics is that glucoamylase G1 is affected by the protonation states of three groups, with pK(a) values of 2.7, 4.5 and 5.7 in the free enzyme and of 2.7, 4.75 and 6.5 in the first enzyme-substrate complex. The protonation of the group in the enzyme-substrate complex with a pK(a) 6.5 hadno effect on k(2) (1640 s(-1)) or k(-2) (20+/-4 s(-1)), but resulted in a stronger enzyme-substrate interaction, due to a decrease of K(1) from 40 to 6.3 mM. In other words,when the substrate is bound, the pK(a) of the acidgroup changes to increase the fraction of reactive enzyme. Since this pK(a) parallels that of the Michaelis complex, known from the pH-dependence of k(cat), the group in question is most probably the catalytic acid Glu-179. Protonation of Glu-179 thus is of no importance in the second step, clearly indicating that this step represents a conformational change and not the actual hydrolysis step of the reaction. Protonation of the pK(a)=4.75 group leads to a small decrease in k(2) to 1090 s(-1), and also to minor changes in K(1). The group with pK(a)=2.7 leads toa major decrease of k(2), of which the limit may bezero, but shows no effect on K(1). Thus no differenceis seen between the pK(a) values of the free enzymeand of the first enzyme-substrate complex at low pH.

Aspergillus niger↗

PubMed1999

[Health, life style and living conditions in Greenland. The 1993-1994 Greenland health survey].

Existing knowledge about the disease pattern in Greenland does not include the Greenlanders' own perception of their health. Consequently, the Greenland Home Rule Government initiated a countrywide health interview survey which was carried out during 1993-1994. A total of 1728 adults were interviewed and filled in a questionnaire. Self-rated health varied with age, gender and socioeconomic conditions. Only few of those bothered by symptoms had contacted the health care services. Musculoskeletal diseases were by far the most frequent cause of symptoms. Suicidal thoughts were frequent; 48% had experienced the suicide of a relative or friend. The life style is characterized by a high consumption of marine mammals and fish, a decreasing but still high alcohol consumption and an 80% prevalence of smoking among both men and women. The health care services are often staffed by Danish physicians and nurses. Occasionally this gives rise to communication problems and dissatisfaction.

Adult↗

PubMed1998

Kinetic analysis of the mechanism and specificity of protein-disulfide isomerase using fluorescence-quenched peptides.

Protein-disulfide isomerase (PDI) is an abundant folding catalyst in the endoplasmic reticulum of eukaryotic cells. PDI introduces disulfide bonds into newly synthesized proteins and catalyzes disulfide bond isomerizations. We have synthesized a library of disulfide-linked fluorescence-quenched peptides, individually linked to resin beads, for two purposes: 1) to probe PDI specificity, and 2) to identify simple, sensitive peptide substrates of PDI. Using this library, beads that became rapidly fluorescent by reduction by human PDI were selected. Amino acid sequencing of the bead-linked peptides revealed substantial similarities. Several of the peptides were synthesized in solution, and a quantitative characterization of pre-steady state kinetics was carried out. Interestingly, a greater than 10-fold difference in affinity toward PDI was seen for various substrates of identical length. As opposed to conventional PDI assays involving larger polypeptides, the starting material for this assay is homogenous. It is furthermore simple and highly sensitive (requires less than 0.5 microgram of PDI/assay) and thus opens the possibility for quantitative determination of PDI activity and specificity.

Amino Acid Sequence↗

PubMed1997

Some details of the reaction mechanism of glucoamylase from Aspergillus niger--kinetic and structural studies on Trp52-->Phe and Trp317-->Phe mutants.

Presteady and steady-state kinetic results on the interactions of a wild-type, and the mutant glucoamylases Trp52-->Phe and Trp317-->Phe, from Aspergillus niger with maltose, maltotriose and maltotetraose have been obtained and analyzed. The results are compared with previous ones on the mutants, Trp120-->Phe and Glu180-->Gln, and with results obtained from structure energy minimization calculations based on known three-dimensional structural data. All results are in accordance with a three-step reaction model involving two steps in the substrate binding and a rate-determining catalytic step. Trp317 and Glu180 belong to different subsites, but are placed on the same flank of the active site (beta-flank). The Trp317-->Phe and the Glu180-->Gln mutants show almost identical kinetic results: weakening of the substrate binding, mainly caused by changes in the second reaction step, and practically no change of the catalytic rate. Structure energy minimization calculations show that the same loss of Arg305 and Glu180 hydrogen bonds to the substrate occurs in the Michaelis complexes of each of these mutants. These results indicate that important interactions of the active site may be better understood from a consideration of its flanks rather than of its subsites. The results further indicate differences in the substrate binding mode of maltose and of longer substrates. Trp52 and Trp120 each interact with the catalytic acid, Glu179, and are placed on the flank (alpha-flank) of the active site opposite to Trp317, Arg305 and Glu180. Also the Trp52-->Phe and Trp120-->Phe mutants show kinetic results similar to each other. The catalytic rates are strongly reduced and the substrates are bound more strongly, mainly as a result of the formation of a more stable complex in the second reaction step. All together, the substrate binding mechanism seems to involve an initial enzyme-substrate complex, in which the beta-flank plays a minor role, except for maltose binding; this is followed by a conformational change, in which hydrogen bonds to Arg305 and Glu180 of the beta-flank are established and the correct alignment on the alpha-flank of Glu179, the general acid catalyst, governed by its flexible interactions with Trp52 and Trp120, occurs.

Aspergillus niger↗

PubMed1997

Glycosylation at Asn-289 facilitates the ligand-induced conformational changes of human Glu-plasminogen.

Glu-plasminogen exists in two major glycoforms (I and II). Glycoform I contains carbohydrate chains linked to Asn-289 and Thr-346, whereas glycoform II is glycosylated only at Thr-346. Disparities in carbohydrate content lead to differences in the important functional properties of the zymogen, e.g. the kinetics of activation. The kinetics of the large ligand-induced conformational changes of each of the Glu-plasminogen glycoforms have been studied using stopped-flow fluorescence. The results are in accordance with a conformational change governed by positive co-operative binding at two weak lysine-binding sites. Additional glycosylation at Asn-289 in Glu-plasminogen I results in a two-fold increase in the overall dissociation constant of a ligand, trans-4-aminomethyl-cyclohexane carboxylic acid. This effect stems directly from the reaction step during which the conformational changes occur. This implies a higher population of Glu-plasminogen I in the open conformation even in the absence of ligands, and thus accounts for a higher rate of activation of Glu-plasminogen I, in comparison with Glu-plasminogen II.

Asparagine↗

PubMed1996

Substrate binding mechanism of Glu180-->Gln, Asp176-->Asn, and wild-type glucoamylases from Aspergillus niger.

Glucoamylase (1,4-alpha-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus, of which the 3D structure is known, releases beta-D-glucose from the non-reducing ends of starch and other related oligo and polysaccharides, cleaving the alpha-1,4-bond positioned between subsites 1 and 2 in the enzyme-substrate complex. The presteady and steady state kinetics of two of the existing mutants, Glu180-->Gln and Asp176-->Asn, are presented here. The kinetic results are analyzed according to two reaction models: One suggested previously [Olsen, K., Svensson, B., & Christensen, U. (1992) Eur. J. Biochem. 209, 777-784], which contains three consecutive steps of the reaction, and one generally accepted and used in calculations of subsite energies [Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1-6], which assumes important non-productive binding and identical values of the intrinsic catalytic constant independent of the chain length of the substrate. It is found that glucoamylase shows kinetics in accordance with a consecutive three-step mechanism, in which the formation of the Michaelis complex occurs in two steps and is followed by a slow catalytic step and fast dissociation of the products with no accumulation of enzyme-product complexes. The kinetics, however, are not in accordance with the model generally used in subsite energy calculations. Thus the kinetic model on which very low values of subsite 1 and high values of subsite 2 interaction energies have been based is not correct. A greater importance of subsite 1 interactions than has hitherto been anticipated is indicated. The results of the Glu180-->Gln mutant show weak overall binding, which stems from large effects on the formation of the Michaelis complex in the second step of the reaction, but no or rather small effects on the initial association of enzyme and substrate, except for maltose. The mutant further shows effective catalysis. A hydrogen bond of the side chain carboxylate of Glu180 with the 2-OH of the sugar ring at subsite 2 is an expected important interaction of the Michaelis complex, as seen from the 3D structures of stabile enzyme-inhibitor complexes. Apparently this bond is established in the second reaction step. It is indicated that subsite 1 and 3 interactions to a great extent govern the initial association. In accordance with a dynamic role of Glu180, structural energy minimization calculations show a flexibility of the gamma-carboxylate of Glu180. The side chain of Asp176 participates in a hydrogen-bonding network also involving the backbone of Glu180 and Glu179, the catalytic acid. Compared with the wild-type enzyme, the Asp176-->Asn mutant shows no significant changes in binding. The catalytic rate is, however, markedly reduced. Apparently changes in the hydrogen bonding network of Asp176 are of importance in the rate-determining catalytic step, but not in the substrate binding steps. Structural energy minimization calculations on the Asp176-->Asn mutant, however, do not confirm this assumption.

Asparagine↗

PubMed1996

Reaction of human alpha2-antiplasmin and plasmin stopped-flow fluorescence kinetics.

The interaction of human plasmin with human alpha2-antiplasmin was measured in the presence and absence of lysine-binding ligands using the corresponding active site fluorescence changes. The stopped-flow method allows for direct determination of reliable values of the second order rate constant for the fast association step of plasmin and alpha2-antiplasmin in the absence of another interacting compound, e.g. a plasmin substrate. At pH 7.4, 25 degrees C, k1 = 2.2 x 10(7) M(-1)s(-1) was obtained. Substantial reductions in k1 were seen in the presence of trans-4-(aminomethyl)cyclohexane-1-carboxylic acid at concentrations corresponding to lysine-binding site interactions at kringle 4 of plasmin; at saturation the rate constant is reduced 20-fold, whereas the effect of saturation of kringle 1 is only a 2-fold reduction. It is thus found that the interaction of alpha2-antiplasmin with the lysine-binding site of kringle 1 is of little importance compared with that of kringle 4 in regulating the inhibition reaction of plasmin with alpha2-antiplasmin. Similar results were recently obtained for the bovine plasmin-bovine alpha2-antiplasmin reaction (Christensen et al. (1995) Biochem. J. 305, 97-102).

Amino Acid Sequence↗

PubMed1995

Stopped-flow kinetic studies of the reaction of barley alpha-amylase/subtilisin inhibitor and the high pI barley alpha-amylase.

The interaction of alpha-amylase/subtilisin inhibitor (BASI) from barley seeds and the high pI barley alpha-amylase (AMY2) de novo synthesized during seed germination, has been studied at pH 8.0, 25 degrees C, using stopped-flow fluorescence spectroscopy, equilibrium fluorescence titration and kinetic analysis of the displacement of BASI from the BASI-AMY2 complex by the substrate blue starch. The results are in accordance with a two-step reaction model: [formula: see text] The resulting values of the kinetic parameters were: k2/K1 = (1.0 +/- 0.2) x 10(6) M-1.s-1, K1 = 0.4 +/- 0.21 mM, k2 = 320 +/- 150 s-1, k-2 = (7.2 +/- 0.6) x 10(-5)s-1, and the overall dissociation constant Kd = (0.7 +/- 0.1) x 10(-10) M. BASI thus is best characterized as a fast reacting, tight-binding inhibitor of AMY2.

Hordeum↗

PubMed1995

Stopped-flow fluorescence kinetics of bovine alpha 2-antiplasmin inhibition of bovine midiplasmin.

In the conversion of bovine plasminogen to bovine plasmin not only the expected urokinase-catalysed cleavage of Arg-557-Val-558, and the following autocatalytic cleavage separating the N-terminal peptide 1-77 from the heavy chain of plasmin, but also a cleavage at Arg-342-Met-343 between kringles 3 and 4 is seen. Here, kinetic studies of the interaction of bovine alpha 2-antiplasmin with bovine plasmin were performed on isolated bovine midiplasmin (lacking kringles 1-3) and on bovine plasmin containing all of the activation products from the bovine plasminogen. A series of experiments using stopped-flow fluorescence fast kinetics as well as conventional techniques suggests a reaction model in accordance with the one known for the human system. First, a tight complex (K1 in the nanomolar range) is formed in a fast reaction step; and second, a tightening of this complex occurs in a slow reaction step. The final complex is indeed so tight (Ki < or = pM), that the reaction for many practical purposes is legitimately considered irreversible. The stopped-flow method allows for the determination of reliable values of the second-order rate constant for the fast association step. At pH 7.4 and 25 degrees C, k+1 = 1.7 x 10(6) M-1 s-1 was obtained in the absence and k+1 = 0.9 x 10(6) M-1.s-1 in the presence of the kringles 1-3 domain of bovine plasmin. In contrast to this, substantial reductions of k+1 were seen in the presence of concentrations of 6-amino-hexanoic acid corresponding to lysine-binding-site interactions and far too low to be attributed to active-site interactions with the bovine plasmins (for each, Ki = 42 mM). All in all, the data indicated that the lysine-binding site(s) not of kringle 1, but of midiplasmin (those of kringles 4 and 5) are regulating the inhibition reaction.

Animals↗

PubMed1994

Involvement of the hydrophobic stack residues 39-44 of factor VIIa in tissue factor interactions.

Des(1-38) factor VIIa and des(1-44) factor VIIa were obtained by limited proteolysis. The binding of tissue factor to these factor VIIa-derivatives was assessed from its stimulation of the proteolytic activity on chromogenic oligopeptide substrates. Compared to native factor VIIa (KTF = 0.6 +/- 0.1 nM), Tissue factor binds to des(1-38) factor VIIa with a lower, but still significant affinity (KTF = 4.8 +/- 0.3 nM). The activity of des(1-44) factor VIIa was only slightly stimulated by TF (KTF approximately 200 nM). Binding of TF depends critically on the presence of Ca2+ ions. Ca2+ ions stimulated the activity of factor VIIa/TF with an apparent KCa = 0.16 +/- 0.02 mM. Factor VIIa in the absence of tissue factor was stimulated by Ca2+ with an apparent KCa = 0.05 +/- 0.01 mM, and similar KCa values were obtained for the truncated derivatives of factor VIIa. Measurements of Ca(2+)-induced changes in intrinsic protein fluorescence suggest a conformational change. The Ca2+ ion concentration at which this change occurred was higher for des(1-44) factor VIIa (apparent KCa = 0.14 mM) than for des(1-38)- and native factor VIIa (apparent KCa = 0.04 mM). The Tb3+ ion luminescence technique was used to further investigate the Ca2+ binding sites. Tb3+ ions bound with a lower affinity to des(1-44) factor VIIa than to des(1-38)-and native factor VIIa. The observed drastic decrease in affinity for tissue factor as a result of truncation of the 'hydrophobic stack' residues 39-44, suggest that this region of factor VIIa provides a structural determinant that together with other regions participates in tissue factor binding.

Amides↗