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M R Witmer

Publications and source records attributed to M R Witmer.

12 recordsLinked to original sources
PubMed1999

Competitive, reversible inhibition of cytosolic phospholipase A2 at the lipid-water interface by choline derivatives that partially partition into the phospholipid bilayer.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 2-(2'-benzyl-4-chlorophenoxy)ethyl-dimethyl-n-octadecyl-ammonium chloride (compound 1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 5 microM). It was over 70 times more selective for the cPLA2 as compared with the human nonpancreatic secreted phospholipase A2, and it did not inhibit other phospholipases. Additionally, it inhibited arachidonate production in N-formyl-methionyl-leucyl-phenylalanine-stimulated U937 cells. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn -glycero-3-phosphomethanol containing 6-10 mol % of 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition could be defined by kinetic equations describing competitive inhibition at the lipid-water interface. The apparent equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.097 +/- 0.032 mol % versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.3 +/- 0.1 mol %. Thus, compound 1 represents a novel structural class of inhibitor of cPLA2 that partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site. Shorter n-alkyl-chained (C-4, C-6, C-8) derivatives of compound 1 were shown to have even smaller KI*app values. However, these short-chained analogs were less potent in terms of bulk inhibitor concentration needed for inhibition when using the [3H]arachidonate-labeled U937 membranes as substrate. This discrepancy was reconciled by showing that these shorter-chained analogs did not partition into the [3H]arachidonate-labeled U937 membranes as effectively as compound 1. The implications for in vivo efficacy that result from these findings are discussed.

Arachidonic Acid↗
PubMed1999

The size and curvature of anionic covesicle substrate affects the catalytic action of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is normally located in the cytosol, but in response to cellular activation the enzyme binds to the membrane at the lipid/water interface where it catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. Synthetic phospholipid vesicle systems have been used in kinetic and mechanistic analyses of cPLA2, but these systems result in a rapid loss of enzyme activity. In the present research, covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing </=10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) as substrate were used to show that this premature cessation of enzyme-catalyzed hydrolysis is dependent on vesicle size with 25-nm-diameter vesicles supporting little activity as compared to 100-, 200-, and 400-nm vesicles. This suggests that the curvature of the vesicle may shift a conformational equilibrium toward an enzyme state which does not support activity. Interestingly, the presence of 30% (v/v) glycerol greatly enhanced the activity of the enzyme, although vesicle size-dependent premature cessation of hydrolysis was still observed. While the premature cessation of hydrolysis in the absence of glycerol is accompanied by enzyme inactivation, little inactivation occured in the presence of glycerol, indicating that premature cessation and inactivation are not absolutely coupled. When using this covesicle substrate system under conditions (6-10 mM CaCl2) where the vesicles are fusing, no premature cessation of hydrolysis has been observed. This is despite a mean vesicle diameter of 400-450 nm under vesicle-fusing conditions, which is comparable to the largest vesicles used under nonfusing conditions (0.5 mM CaCl2) where considerable premature cessation of hydrolysis was observed. Since DMPM has an intrinsic active site dissociation constant at least 330 times larger than that of PAPC, the optimum conditions for conducting kinetic and mechanistic analyses of cPLA2 with this covesicle substrate is one in which cPLA2 is assayed in the presence of glycerol and with fusion-inducing concentrations of calcium. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol (DOPM) instead of DMPM in this system supports much less activity and adds the complication of a strong affinity of DOPM for the active site.

Cell Membrane↗
PubMed1998

A beta-lactam inhibitor of cytosolic phospholipase A2 which acts in a competitive, reversible manner at the lipid/water interface.

Cytosolic phospholipase A2 (cPLA2) catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. When assaying the human recombinant cPLA2 using membranes isolated from [3H]arachidonate-labeled U937 cells as substrate, 3,3-Dimethyl-6-(3-lauroylureido)-7-oxo-4-thia-1-azabicyclo[3,2,0] heptane-2-carboxylic acid (1) was found to inhibit the enzyme in a dose-dependent manner (IC50 = 72 microM). This beta-lactam did not inhibit other phospholipases, including the human nonpancreatic secreted phospholipase A2. The inhibition of cPLA2 was found not to be time-dependent. This, along with the observation that the degradation of the inhibitor was not catalyzed by the enzyme, demonstrates that the inhibition does not result from the formation of an acyl-enzyme intermediate with the active site serine residue. Moreover, the ring-opened form of 1 is also able to inhibit cPLA2 with near-equal potency. To further characterize the mechanism of inhibition, an assay in which the enzyme is bound to vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol containing 6-10 mole percent of 1-palmitoyl-2-[1-14C]-arachidonoyl-sn-glycero-3-phosphocholine was employed. With this substrate system, the dose-dependent inhibition was defined by kinetic equations describing competitive inhibition at the lipid/water interface. The apparent dissociation constant for the inhibitor bound to the enzyme at the interface (KI*app) was determined to be 0.5 +/- 0.1 mole% versus an apparent dissociation constant for the arachidonate-containing phospholipid of 0.4 +/- 0.1 mole%. Thus, 1 represents a novel structural class of inhibitors of cPLA2 which partitions into the phospholipid bilayer and competes with the phospholipid substrate for the active site.

Arachidonic Acid↗
PubMed1997

Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.

Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated. Phosphorylated and dephosphorylated enzyme forms were shown to have identical values of 2.6 microM for KMapp, an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (Ks) and the dissociation constant for phospholipid from the active site (KM*). Moreover, the values of KM* for phosphorylated and dephosphorylated enzyme did not differ significantly (0.4 +/- 0.1 and 0.2 +/- 0.1, respectively). However, dephosphorylation of the enzyme reduced the value of kcat by 39%. The phosphorylation state of the enzyme had no effect on either the cooperativity shown by this enzyme or the thermal stability of the enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on kcat. Instead, glycerol increased the value of kcat by 440% for the phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had only small effects on KMapp. the increase in the kcat upon addition of glycerol results from a substantial decrease in the activation energy from 29.4 to 14.8 kcal. mol-1. To determine whether the effects of phosphorylation of the enzyme or addition of glycerol are unique to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these membranes, the dephosphorylated enzyme was only 21% less active than the phosphorylated enzyme. In the presence of glycerol, there was no detectable difference the two enzyme forms, and the rate of hydrolysis was increased by 300-390% over that measured in the absence of glycerol. These results suggest that the catalytic efficiency of the phosphorylated enzyme is not particularly relevant to its activation in vivo. Moreover, it may be that glycerol is mimicking the effect of some unidentified factor which greatly enhances the catalytic efficiency of the enzyme.

Acid Phosphatase↗
PubMed1995

Cooperativity and binding in the mechanism of cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) hydrolyzes the sn-2 ester of phospholipids and is believed to be responsible for the receptor-regulated release of arachidonic acid from phospholipid pools. The enzyme was assayed using vesicles containing arachidonate-containing phospholipid substrate, such as 1-palmitoyl-2-arachidonoylphosphatidylcholine (PAPC) or 1-stearoyl-2-arachidonoylphosphatidylinositol (SAPI), dispersed within vesicles of 1,2-dimyristoylphosphatidylmethanol (DMPM). We report here that the enzyme shows an apparent cooperative effect with respect to the mole fraction of arachidonate-containing phospholipids within these covesicles. The data can be fit to a modified Hill equation yielding Hill coefficients, n, of 2-3. This effect is unusual in that it is dependent on the nature of the sn-2 ester as opposed to the phosphoglycerol head group. This cooperativity is independent of both the concentration of glycerol, which greatly increases enzyme activity and stability, and the concentration of calcium, which facilitates the fusion of the covesicles. Surprisingly, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine (PAPE) does not show the same cooperative effect, although the rate at which it is hydrolyzed is much greater when PAPC is present. Moreover, PAPE has a dissociation constant from the active site (KD* = 0.7 mol %) which is comparable to that of PAPC and SAPI (KD* values of 0.3 and 0.3 mol %, respectively). These results are consistent with the presence of an allosteric site that, when occupied, induces a change in the enzyme which facilitates enzymatic hydrolysis. If so, PAPC and SAPI, but not PAPE, must be able to bind to this allosteric site. Alternatively, this effect may result from changes in the physical nature of the bilayer which result upon increasing the bilayer concentration of arachidonate-containing phospholipids. This previously unobserved effect may represent another mechanism by which cells can regulate the activity of cPLA2.

Animals↗
PubMed1995

Active recombinant human cytosolic phospholipase A2 is expressed in Escherichia coli.

The cDNA encoding human cytosolic phospholipase A2 (cPLA2) has been subcloned into a prokaryotic pET16b expression vector which also encodes an amino-terminal deca-histidine affinity tag to facilitate purification of the recombinant enzyme. Soluble, active fusion protein, designated His-cPLA2, has been obtained reproducibly from this expression system using the E. coli strain BL21 (DE3). The protein has been purified to homogeneity in four steps and the mass confirmed by electrospray mass spectrometry. His-cPLA2 was characterized by kinetic analysis which demonstrated that the enzyme is similar to native cPLA2 in all respects investigated. Specifically, the enzyme binds to anionic vesicles containing substrate, and acts processively on these vesicles. Enzymatic activity is supported by the presence of Ca2+ and several other divalent metal ions, and is inhibited by several transition metal ions. Finally, the enzyme demonstrates lysophospholipase activity and exhibits a high selectivity for sn-2 arachidonyl esters. This prokaryotic expression system yields moderate amounts of unmodified recombinant His-cPLA2 and is advantageous for rapid production of protein and mutational analyses.

Amino Acid Sequence↗
PubMed1994

Probing the catalytic roles of n2-site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis.

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn(2+)-KD values, UV-cis and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both Mn2+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn(2+)-activated enzymes showed reduced kcat/Km values for ATP (> 7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg(2+)-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate gamma-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.

Adenine↗
PubMed1992

Altered levels of antithrombin III and fibrinogen in the aortic wall of the alloxan-induced diabetic rabbit: evidence of a prothrombotic state.

The distribution and behavior of the rabbit plasma proteins albumin, fibrinogen, and antithrombin III (ATIII) (isoforms alpha and beta), have been examined in groups of alloxan-induced diabetic rabbits and control rabbits. By injecting radiolabeled preparations intravenously, measurements of plasma clearance, rates of catabolism, and compartmental distribution were made for each protein. In addition, after allowing the radiolabeled proteins to circulate for 12 hours, we excised aortas after exsanguination and determined the content of these proteins in the endothelium and subendothelium. The respective fractional catabolic rates of ATIII-alpha and ATIII-beta were similar in the diabetic and control rabbits, but fibrinogen and albumin were catabolized more slowly in the diabetic rabbit than in the control rabbit. The distributions of albumin and the ATIII isoforms between the intravascular, noncirculating vascular, and extravascular compartments in the diabetic rabbit were similar to the respective proteins in the control rabbit, but a smaller proportion of fibrinogen was associated with the vascular compartment of the diabetic rabbit when compared with that in the control rabbit. At 12 hours after injection, the quantities of fibrinogen and albumin associated with the diabetic aorta endothelium and particularly the subendothelium were increased, whereas ATIII-alpha and ATIII-beta were decreased relative to the control aorta. The fibrinogen-to-ATIII ratio in the diabetic aorta was increased twofold to threefold when compared with that in the control aorta. We conclude that the increased ratio of fibrinogen to ATIII in the aorta wall of the diabetic rabbit may be characteristic of the prothrombotic state that is conspicuous in insulin-dependent diabetes.

Albumins↗
PubMed1991

U-3'-BCIP: a chromogenic substrate for the detection of RNase A in recombinant DNA expression systems.

The synthesis of the bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5) chromogenic substrate uridine-3'-(5-bromo-4-chloroindol-3-yl)-phosphate (U-3'-BCIP) is described. RNase A catalyzes the hydrolysis of U-3'-BCIP to release a halogenated indol-3-ol that undergoes rapid aerobic oxidation to the dark blue 5,5'-dibromo-4,4'-dichloroindigo. Preliminary kinetic studies indicate that this compound may have practical use for assaying RNase A activity both in vitro and in vivo, e.g. in screening bacterial colonies for RNase A produced by recombinant DNA methods.

Animals↗
PubMedDate not supplied

Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo.

The properties of two isoforms, alpha and beta, of rabbit antithrombin III (ATIII) were compared in the presence of undamaged or de-endothelialized rabbit aortic wall. Similar quantities of ATIII-alpha and ATIII-beta bound to and rapidly saturated the endothelium in vitro, but the rate of transendothelial passage of ATIII-beta exceeded that of ATIII-alpha by 22%. Furthermore, ATIII-beta was adsorbed approximately twice as rapidly as ATIII-alpha by the subendothelium of the de-endothelialized aorta. Binding of both isoforms was decreased (ATIII-beta more than ATIII-alpha) by pretreating the subendothelial surface with heparitinase. Also, subendothelium-bound ATIII-beta was desorbed more readily than bound ATIII-alpha by thrombin. In vivo, the rate of uptake of iodine-131-labeled ATIII-beta from the circulation by the aortic wall and the major organs was 30-50% faster than that of iodine-125-labeled ATIII-alpha. In contrast, the uptake of 131I-ATIII-beta by the de-endothelialized aorta in vivo was three times faster than that of 125I-ATIII-alpha. By these criteria, ATIII-beta is the more active of the two isoforms. We surmise that plasma and, consequently, vessel wall levels of ATIII-beta may be vital for controlling thrombogenic events caused by injury to the vascular wall.

Adsorption↗
PubMedDate not supplied

[Retinal detachment].

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Cryosurgery↗