Application of A Distributed Nucleus Approximation In Grid Based Minimization of the Kohn-Sham Energy Functional

In the distributed nucleus approximation we represent the singular nucleus as smeared over a smallportion of a Cartesian grid. Delocalizing the nucleus allows us to solve the Poisson equation for theoverall electrostatic potential using a linear scaling multigrid algorithm.This work is done in the context of minimizing the Kohn-Sham energy functionaldirectly in real space with a multiscale approach. The efficacy of the approximation is illustrated bylocating the ground state density of simple one electron atoms and moleculesand more complicated multiorbital systems.Comment: Submitted to JCP (July 1, 1995 Issue), latex, 27pages, 2figure

A Woody Lycopsid Stem From The New Albany Shale (Lower Mississippian) Of Kentucky

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141135/1/ajb208777.pd

Incorporation of a Product of Mevalonic Acid Metabolism Into Proteins of Chinese Hamster Ovary Nuclei

We have examined the nuclear localization of isoprenylated proteins in CHO-K1 cells labeled with [14C]mevalonate. Nuclear proteins of 68, 70, and 74 kD, posttranslationally modified by an isoprenoid, are also components of a nuclear matrix-intermediate filament preparation from CHO cells. Furthermore, the 68-, 70-, and 74-kD isoprenylated polypeptides are immunoprecipitated from cell extracts with two different anti-lamin antisera. Based on exact two-dimensional comigration with lamin B, both from rat liver lamin and CHO nuclear matrix-intermediate filament preparations, and its immunoprecipitation with anti-lamin antisera, we conclude that the 68-kD isoprenylated protein found in nuclei from [14C]mevalonate-labeled CHO cells is lamin B. The more basic 74-kD isoprenylated nuclear protein is similar in molecular mass and isoelectric pH variants to the lamin A precursor polypeptide reported by others. Starving cells for mevalonate results in a dramatic accumulation of a polypeptide that comigrates on two-dimensional, non-equilibrium pH gradient electrophoresis (NEPHGE) gels with the 74-kD isoprenylated protein. The 70-kD isoprenylated protein, which is resolved on NEPHGE gels as being higher in molecular mass and slightly more basic than lamin B, has not yet been identified

What measurable zero point fluctuations can(not) tell us about dark energy

We show that laboratory experiments cannot measure the absolute value of dark energy. All known experiments rely on electromagnetic interactions. They are thus insensitive to particles and fields that interact only weakly with ordinary matter. In addition, Josephson junction experiments only measure differences in vacuum energy similar to Casimir force measurements. Gravity, however, couples to the absolute value. Finally we note that Casimir force measurements have tested zero point fluctuations up to energies of ~10 eV, well above the dark energy scale of ~0.01 eV. Hence, the proposed cut-off in the fluctuation spectrum is ruled out experimentally.Comment: 4 page

When the working day is through: The end of work as identity?

This article seeks to present a counter-case to the ‘end of work thesis’ advocated by writers such as Beck, Sennett and Bauman. It argues that work remains a significant locus of personal identity and that the depiction by these writers of endemic insecurity in the workplace is inaccurate and lacks empirical basis. The article draws upon case study data to illustrate how, across a range of workplaces, work remains an importance source of identity, meaning and social affiliation

Isoprenylation is Required for the Processing of the Lamin A Precursor

The nuclear lamina proteins, prelamin A, lamin B, and a 70-kD lamina-associated protein, are posttranslationally modified by a metabolite derived from mevalonate. This modification can be inhibited by treatment with (3-R,S)-3-fluoromevalonate, demonstrating that it is isoprenoid in nature. We have examined the association between isoprenoid metabolism and processing of the lamin A precursor in human and hamster cells. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase by mevinolin (lovastatin) specifically depletes endogenous isoprenoid pools and inhibits the conversion of prelamin A to lamin A. Prelamin A processing is also blocked by mevalonate starvation of Mev-1, a CHO cell line auxotrophic for mevalonate. Moreover, inhibition of prelamin A processing by mevinolin treatment is rapidly reversed by the addition of exogenous mevalonate. Processing of prelamin A is, therefore, dependent on isoprenoid metabolism. Analysis of the conversion of prelamin A to lamin A by two independent methods, immunoprecipitation and two-dimensional nonequilibrium pH gel electrophoresis, demonstrates that a precursor-product relationship exists between prelamin A and lamin A. Analysis of R,S-[5-3H(N)]mevalonate-labeled cells shows that the rate of turnover of the isoprenoid group from prelamin A is comparable to the rate of conversion of prelamin A to lamin A. These results suggest that during the proteolytic maturation of prelamin A, the isoprenylated moiety is lost. A significant difference between prelamin A processing, and that of p21ras and the B-type lamins that undergo isoprenylation-dependent proteolytic maturation, is that the mature form of lamin A is no longer isoprenylated

Amino Acids Glu323, Tyr324, Glu330, and Val331 of Factor VA Heavy Chain Are Essential for Expression of Cofactor Activity

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4\u27) contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O. (2002) Biochemistry 41, 12715-12728). To ascertain which amino acids within this region are important for the effector and receptor properties of the cofactor with respect to factor Xa, we have synthesized three overlapping peptides (5 amino acids each) spanning the amino acid region 323-331 and tested them for their effect on prothrombinase complex assembly and function. Peptide containing amino acids 323EYFIA327 alone was found to increase the catalytic efficiency of factor Xa but had no effect on the fluorescent anisotropy of active site-labeled factor Xa (human factor Xa labeled in the active site with Oregon Green 488; [OG488]-EGR-hXa). In contrast, peptide containing the sequence 327AAEEV331 was found to interact with [OG488]-EGR-hXa with half-maximal saturation reached at approximately 150 microm, but it was unable to produce a cofactor effect on factor Xa. Peptide 325FIAAE329 inhibited prothrombinase activity and was able to partially decrease the fluorescent anisotropy of [OG488]-EGR-hXa but could not increase the catalytic efficiency of factor Xa with respect to prothrombin. A control peptide with the sequence FFFIA did not increase the catalytic efficiency of factor Xa, whereas a peptide with the sequence AAEMI was impaired in its capability to interact with [OG488]-EGR-hXa. Two mutant recombinant factor Va molecules (Glu323 --\u3e Phe/Tyr324 --\u3e Phe, factor VaFF; Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaMI) showed impaired cofactor activity when used at limiting cofactor concentration, whereas the quadruple mutant (Glu323 --\u3e Phe/Tyr324 --\u3e Phe and Glu330 --\u3e Met/Val331 --\u3e Ile, factor VaFF/MI) had no cofactor activity under similar experimental conditions. Our data demonstrate that amino acid residues Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are critical for expression of factor Va cofactor activity

Contribution of Amino Acid Region 334−335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase†

ABSTRACT: We have demonstrated that amino acids E323, Y324, E330, and V331 from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D334 and Y335 as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D334 f K and Y335 f F (factor VKF) and D334 f A and Y335 f A (factor VAA). Kinetic studies showed that while factor VaKF and factor VaAA had a KD for factor Xa similar to the KD observed for wild-type factor Va (factor VaWT), the clotting activities of the mutant molecules were impaired and the kcat of prothrombinase assembled with factor VaKF and factor VaAA was reduced. The second-order rate constant of prothrombinase assembled with factor VaKF or factor VaAA for prothrombin activation was ∼10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor VaWT. We also created quadruple mutants combining mutations in the amino acid region 334–335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E323Y324 and E330V331). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor VaWT. The dat

The Contribution of Amino Acid Region ASP695-TYR698 of Factor V to Procofactor Activation and Factor VA Function

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively. The two peptides were unable to interfere with the binding of factor Va to active site fluorescently labeled Glu-Gly-Arg human factor Xa, and kinetic analyses showed that HC3 and HC4 are competitive inhibitors of prothrombinase with respect to prothrombin with K(i) values of approximately 6.3 and approximately 5.3 microm, respectively. These data suggest that the peptides inhibit prothrombinase because they interfere with the incorporation of prothrombin into prothrombinase. The shared amino acid motif between HC3 and HC4 is composed of Asp(695)-Tyr-Asp-Tyr-Gln(699) (DYDYQ). A pentapeptide with this sequence inhibited both prothrombinase function with an IC(50) of 1.6 microm (with a K(D) for prothrombin of 850 nm), and activation of factor V by thrombin. Peptides HC3, HC4, and DYDYQ were also found to interact with immobilized thrombin. A recombinant factor V molecule with the mutations Asp(695) --\u3e Lys, Tyr(696) --\u3e Phe, Asp(697) --\u3e Lys, and Tyr(698) --\u3e Phe (factor V(2K2F)) was partially resistant to activation by thrombin but could be readily activated by RVV-V activator (factor Va(RVV)(2K2F)) and factor Xa (factor Va(Xa)(2K2F)). Factor Va(RVV)(2K2F) and factor Va(Xa)(2K2F) had impaired cofactor activity within prothrombinase in a system using purified reagents. Our data demonstrate for the first time that amino acid sequence 695-698 of factor Va heavy chain is important for procofactor activation and is required for optimum prothrombinase function. These data provide functional evidence for an essential and productive contribution of factor Va to the activity of prothrombinase

The Structural Integrity of Anion Binding Exosite I of Thrombin Is Required and Sufficient for Timely Cleavage and Activation of Factor V and Factor VIII

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation