Biomedical subjects
K S Larsen
Publications and source records attributed to K S Larsen.
Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4.
The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties of binding zinc tightly while remaining remarkably reactive as zinc donors. We have used zinc isotope exchange both to probe the coordination dynamics of zinc clusters in metallothionein, the small protein that has the highest known zinc content, and to investigate the potential function of zinc clusters in cellular zinc distribution. When mixed and incubated, metallothionein isoproteins-1 and -2 rapidly exchange zinc, as demonstrated by fast chromatographic separation and radiometric analysis. Exchange kinetics exhibit two distinct phases (k(fast) approximately 5000 min(-1) x M(-1); k(slow) approximately 200 min(-1) x M(-1), pH 8.6, 25 degrees C) that are thought to reflect exchange between the three-zinc clusters and between the four-zinc clusters, respectively. Moreover, we have observed and examined zinc exchange between metallothionein-2 and the Gal4 protein (k approximately 800 min(-1) x M(-1), pH 8.0, 25 degrees C), which is a prototype of transcription factors with a two-zinc cluster. This reaction constitutes the first experimental example of intermolecular zinc exchange between heterologous proteins. Such kinetic reactivity distinguishes zinc in biological clusters from zinc in the coordination environment of zinc enzymes, where the metal does not exchange over several days with free zinc in solution. The molecular organization of these clusters allows zinc exchange to proceed through a ligand exchange mechanism, involving molecular contact between the reactants.
A circadian rhythm of locomotor activity in newly emerged Ceratophyllus sciurorum.
Circadian rhythm in newly emerged individuals of the Red Squirrel (Scuirus vulgaris) flea C.s.sciurorum was studied in a constant environment, using an insect activity monitor. Trials were run over 7 days using two start times (08.00 and 17.00 hours). The results show that, regardless of start time, the fleas display a 24 h activity rhythm. The presence of a rhythm under constant conditions gives a strong indication that C.s.sciurorum has a self-sustaining clock which is started by disturbance and is most likely to be linked to host activity patterns.
D-Phe complexes of zinc and cobalt carboxypeptidase A.
The binding of D-phenylalanine, D-Phe, to both zinc and cobalt carboxypeptidase A, ZnCPD and CoCPD, has been investigated by a combination of kinetic and spectroscopic techniques. Kinetic studies of the ZnCPD catalyzed hydrolysis of dansyl-Gly-Ala-L-Phe indicate that D-Phe inhibition occurs through a two-site sequential competitive inhibition mode with Ki values of 45 microM and 11.6 mM at pH 8.4, 1 M NaCl, 25 degrees C. Spectral titration of CoCPD under the same conditions indicates a very strong binding mode of D-Phe (KD < 100 microM) that only slightly perturbs the visible cobalt electronic transitions. However, the conversion of CoCPD.D-Phe into a CoCPD.D-Phe2 (KD, 1.13 mM) is accompanied by a very strong spectral perturbation resulting in a complex that is characterized by Amax values of 506 nm (epsilon = 27 M-1 cm-1) and 605 nm (epsilon = 17 M-1 cm-1) and a shoulder at 530 nm (epsilon = 23 M-1 cm-1). The spectral properties of this ternary complex differ markedly from that of the CoCPD.L-Phe.N3-ternary complex. X-ray absorption fine structure, XAFS, studies indicate that these differences are likely due to a more regular tetrahedral coordination sphere for the ternary azide complexes compared to an octahedral coordination geometry for the Zn and CoCPD.D-Phe2 complexes.
X-ray absorption fine structure study of the active site of zinc and cobalt carboxypeptidase A in their solution and crystalline forms.
A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of atoms, with four atoms (N or O) located at an average distance of 2.03 +/- 0.01 A and one atom (N or O) located at 2.57 +/- 0.04 A. The four-atom distribution remains the same for ZnCPD in its crystalline state, but the fifth atom is found at 2.36 +/- 0.04 A. Examination of the higher coordination shell, between 2.7 and 4.2 A, reveals the presence of two imidazoles. Combined with X-ray crystallographic results, a structural model is proposed. The four atoms at an average distance of 2.03 A are assigned to the two delta 1 nitrogens of His-69 and His-196, one epsilon 1 oxygen of Glu-72, and the oxygen of a coordinated water molecule. The atom at 2.57 A for ZnCPD in solution is assigned to the epsilon 2 oxygen of Glu-72. The results for CoCPD in solution are similar with the four atoms at an average distance of 2.08 +/- 0.01 A and one atom at 2.50 +/- 0.04 A, which moves to 2.34 +/- 0.04 A in the crystalline enzyme. The intensity of the 3d "pip" peak for CoCPD is consistent with a distorted tetragonal metal geometry for the solution form of the enzyme which is converted to a more pentacoordinated metal site for the crystalline enzyme. The first shell distribution of crystalline CoCPD is quite disordered, which may be largely due to the disorder of His-69 and His-196 as indicated by higher shell analysis. Thus, the XAFS studies show that the metal coordination spheres in the zinc and cobalt enzymes are quite similar in the solution state but differ from their crystalline counterparts. The XAFS studies provide the necessary background for measurement of substrate- and inhibitor-promoted structural changes in the metal coordination sphere of the zinc and other metal-substituted carboxypeptidases in the solution state.
Characterization of an inhibitory metal binding site in carboxypeptidase A.
The specificity of metal ion inhibition of bovine carboxypeptidase A ([(CPD)Zn]) catalysis is examined under stopped-flow conditions with use of the fluorescent peptide substrate Dns-Gly-Ala-Phe. The enzyme is inhibited competitively by Zn(II), Pb(II), and Cd(II) with apparent KI values of 2.4 x 10(-5), 4.8 x 10(-5), and 1.1 x 10(-2) M in 0.5 M NaCl at pH 7.5 and 25 degrees C. The kcat/Km value, 7.3 x 10(6) M-1 s-1, is affected less than 10% at 1 x 10(-4) M Mn(II) or Cu(II) and at 1 x 10(-2) M Co(II), Ni(II), Hg(II), or Pt(IV). Zn(II) and Pb(II) are mutually exclusive inhibitors. Previous studies of the pH dependence of Zn(II) inhibition [Larsen, K. S., & Auld, D. S. (1989) Biochemistry 28, 9620] indicated that [(CPD)Zn] is selectively inhibited by a zinc monohydroxide complex, ZnOH+, and that ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for its binding. The present study allows further definition of this inhibitory zinc site. The ionizable ligand (LH) is assigned to Glu-270, since specific chemical modification of this residue decreases the binding affinity of [(CPD)Zn] for Zn(II) and Pb(II) by more than 60- and 200-fold, respectively. A bridging interaction between the Glu-270-coordinated metal hydroxide and the catalytic metal ion is implicated from the ability of Zn(II) and Pb(II) to induce a perturbation in the electronic absorption spectrum of cobalt carboxypeptidase A ([(CPD)Co]).(ABSTRACT TRUNCATED AT 250 WORDS)
Acquired immune deficiency syndrome: international attitudinal comparisons.
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Endometriosis of the rectum treated with a long term GnRH agonist and surgery.
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Catalytic and conformational changes induced by limited subtilisin cleavage of bovine carboxypeptidase A.
Limited proteolysis of carboxypeptidase A from bovine pancreas with subtilisin Carlsberg generates a stable intermediate, carboxypeptidase S, whose esterase and peptidase activities are increased and decreased, respectively, under standard assay conditions. Carboxypeptidase S was isolated by affinity chromatography. Sequence analysis shows that it is cleaved solely at the Ala154-Gly155 bond. Its enzymatic properties were determined under stopped-flow conditions with Dns-Gly-Ala-Phe and its ester analogue Dns-Gly-Ala-OPhe. For both substrates, the Km values are increased 30-40-fold. The kcat value for peptide hydrolysis is virtually unaffected whereas that for ester hydrolysis is increased 10-fold. The magnitude of the Km effect is equivalent to a loss of 9 kJ/mol of binding energy and likely reflects a disruption of the network of hydrogen bonds that links Tyr-248 and Arg-145 to the backbone carbonyls of Ala-154 and Gly-155. The difference in kcat effects for the two substrate classes is related to differences in the chemical nature of the rate-determining step. Product release is rate determining for catalytic hydrolysis of ester substrates, and hence, the increase in kcat indicates that dissociation of products is facilitated as a result of the Ala154-Gly155 bond scission. The changes in enzymatic activity accompanying limited proteolysis are due to conformational alterations in the vicinity of the active center of the molecule. The affinity of a monoclonal antibody, mAb 100, directed toward the antigenic determinant located between residues 209 and 218 in carboxypeptidase A is diminished considerably for carboxypeptidase S.(ABSTRACT TRUNCATED AT 250 WORDS)
Authoritarianism and attitudes toward AIDS victims.
This study investigated the relationship of authoritarianism and attitudes toward AIDS victims in three samples. One hundred fifty-eight students at Oregon State University participated, including 101 students from the United States, 25 from Japan, and 32 from other Asian societies. The survey instrument included the 20-item Attitudes Toward AIDS Victims (ATAV) Scale, the 18-item F Scale, the Form A, and 14-item Formal Content of Dogmatism Scale. Results showed slight but significant correlations between the ATAV and F (r = .17, p less than .044) and Formal Content of Dogmatism (r = .20, p less than .023) Scales for the United States sample. Highly significant differences were found in the predicted direction among the three samples on authoritarianism, F = 43.94, p less than .001.
AIDS victims and heterosexual attitudes.
This study reports on the development of a Likert scale that measures attitudes toward AIDS victims (ATAV) in five phases. A total of 582 undergraduate (means age = 24.2) completed the survey forms, 249 males and 333 females. The results for Phase 1 yielded a scale with high part-whole correlations (.62-.90, p less than .001), corrected split-half reliability (.87, p less than .001), and alpha coefficients (.91, p less than .001). The following phases yielded significant correlations between the ATAV scale and attitudes toward homosexuals (.60, p less than .001), homosexual parenting (.64, p less than .001), other minority groups (.33, .37, p less than .001), capital punishment (-.27 p less than .001), and sexually liberal attitudes (.22, .37, .23, p, less than .025). Attitudes toward homosexuals are the central component in attitudes toward AIDS victims. A varimax rotated factor analysis of the ATAV yielded one primary factor accounting for 85.9% of the variance.
Carboxypeptidase A: mechanism of zinc inhibition.
Zinc ions competitively inhibit carboxypeptidase A from bovine pancreas. The state(s) of hydroxylation of zinc and their possible site(s) of interaction with the enzyme have been investigated by determining the strength of zinc inhibition over pH range 4.6-10.5. The inhibition kinetics were recorded under stopped-flow conditions using the alpha-Val isozyme and the peptide substrate Dns-Gly-Ala-Phe in 0.5 M NaCl at 25 degrees C. The pH dependence of pKI follows a pattern which indicates that the enzyme is selectively inhibited by zinc monohydroxide, ZnOH+ (KI = 7.1 X 10(-7) M). The formation of the inhibitory ZnOH+ complex from fully hydrated Zn2+ is characterized by an ionization constant of 9.05, and the consecutive conversion of ZnOH+ to Zn(OH)2, Zn(OH)3-, and Zn(OH)4(2-) complexes takes place with ionization constants of 9.75, 10.1, and 10.5, respectively. Ionization of a ligand, LH, in the enzyme's inhibitory site (pKLH 5.8) is obligatory for binding of the ZnOH+ complex. The enzymatic activity (kcat/Km) is influenced by three ionizable groups: pKEH2 5.78, pKEH 8.60, and pKE 10.2. Since the values of pKLH and pKEH2 are virtually identical, it is possible that the inhibitory ZnOH+ complex interacts with the group responsible for pKEH2. Previous studies have suggested that pKEH2 reflects the ionization of Glu-270 and its interaction with a water molecule coordinated to the catalytic zinc ion. It is proposed that the inhibitory zinc ion binds to the carboxylate of Glu-270 and that the inhibition process is specific for zinc monohydroxide because it allows the formation of a stabilizing hydroxide bridge between the inhibitory and catalytic zinc ions.
[Head lice. The club behind my ear].
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[Lumbar disk prolapse in the elderly].
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The Psychological Screening Inventory as a predictor of predisposition to suicide among patients at the Oregon State Hospital.
Examined all closed patient files for the inclusion of scores from the Psychological Screening Inventory (PSI) at the Oregon State Hospital between 1977-1979. Subsequently, the patient files (N = 123) were assessed for suicidal inclination employing five categories, which ranged from "no suicidal ideation" to "serious attempt." t-tests were completed between Ss placed in category 1 (no suicidal ideation) and remaining categories. Results yielded a significant value for "discomfort," with lower discomfort scores related to higher suicide risks. Subsequent extreme group analysis yielded significant but opposite results for males and females, which suggests the importance of evaluating the results of the PSI separately for sex.
Fascism and attitudes toward mandatory sterilization: the development of an attitude scale.
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Approval seeking, situational pressures, and the willingness to administer shock to a victim.
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Emotional responses to frustration of approval seeking and personal identity.
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