Intravenous magnesium does not influence the activity of the coagulation cascade.

Experimental arterial thrombus formation is reduced during intravenous magnesium infusion. It is well documented that magnesium reduces platelet reactivity, but the antithrombotic effect could also originate from anticoagulant properties or increased fibrinolysis. We therefore evaluated the effect of intravenous magnesium on prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin III complex (TAT) concentrations, and fibrin degradation products (FbDP) in a randomized, cross-over study in 14 healthy volunteers. Citrated blood samples were collected at 0, 30, and 180 min. An additional in vitro study on magnesium's effect on the activity of different coagulation factors was carried out. A transient increase was seen in F1 + 2 and TAT after 30 min but without any significant difference between the placebo and magnesium period. FbDP did not change significantly between the two treatments. Increasing concentrations of magnesium dose-dependently decreased binding of activated factor X to activated factor VII (FVIIa), but the decrease was slight and probably without any significance for coagulation at the concentrations tested. No effect was observed on the activity of FVIIa or activated factor VIII. In conclusion, no significant differences were observed on markers of coagulation or fibrinolytic activity during intravenous magnesium infusion. These results indicate that the observed antithrombotic effect of magnesium is more likely to arise from the previously observed platelet inhibition.

The catalytic properties of murine carbonic anhydrase VII.

Carbonic anhydrase VII (CA VII) appears to be the most highly conserved of the active mammalian carbonic anhydrases. We have characterized the catalytic activity and inhibition properties of a recombinant murine CA VII. CA VII has steady-state constants similar to two of the most active isozymes of carbonic anhydrase, CA II and IV; also, it is very strongly inhibited by the sulfonamides ethoxzolamide and acetazolamide, yielding the lowest Ki values measured by the exchange of 18O between CO2 and water for any of the mammalian isozymes of carbonic anhydrase. The catalytic measurements of the hydration of CO2 and the dehydration of HCO3- were made by stopped-flow spectrophotometry and the exchange of 18O using mass spectrometry. Unlike the other isozymes of this class of CA, for which Kcat/K(m) is described by the single ionization of zinc-bound water, CA VII exhibits a pH profile for Kcat/K(m) for CO2 hydration described by two ionizations at pKa 6.2 and 7.5, with a maximum approaching 8 x 10(7) M-1 s-1. The pH dependence of kcat/K(m) for the hydrolysis of 4-nitrophenyl acetate could also be described by these two ionizations, yielding a maximum of 71 M-1 s-1 at pH > 9. Using a novel method that compares rates of 18O exchange and dehydration of HCO3-, we assigned values for the apparent pKa at 6.2 to the zinc-bound water and the pKa of 7.5 to His 64. The magnitude of Kcat, its pH profile, 18O-exchange data for both wild-type and a H64A mutant, and inhibition by CuSO4 and acrolein suggest that the histidine at position 64 is functioning as a proton-transfer group and is responsible for one of the observed ionizations. A truncation mutant of CA VII, in which 23 residues from the amino-terminal end were deleted, has its rate constant for intramolecular proton transfer decreased by an order of magnitude with no change in Kcat/K(m). This suggests a role for the amino-terminal end in enhancing proton transfer in catalysis by carbonic anhydrase.

Molecular characterization of the human carbonic anhydrase-related protein (HCA-RP VIII).

The very evolutionarily conserved human carbonic anhydrase-related polypeptide (CA-RP VIII) lacks the carbon-dioxide hydration-activity, characteristic of the enzymatically active carbonic anhydrases. We have expressed HCA-RP VIII as a glutathione-S-transferase fusion protein (GST-HCA-RP VIII). The purified HCA-RP VIII showed a substantially higher apparent molecular weight by gel-filtration compared to the molecular weight calculated from the amino acid sequence, indicating a larger than expected Stoke's radius. Like other studied CA's, the protein unfolds through two transitions at increasing concentrations of guanidine hydrochloride. The far-UV CD spectra of HCA-RP VIII indicates a secondary structure similar to that of the catalytically active HCA II. The very high sequence identity between human and mouse CA-RP VIII (98%), might indicate that the function of the protein involves binding of another protein. However, an attempt to use the GST-HCA-RP VIII fusion protein to affinity purify a ligand was unsuccessful.

Signal transduction via the mitogen-activated protein kinase pathway induced by binding of coagulation factor VIIa to tissue factor.

The putative role of tissue factor (TF) as a receptor involved in signal transduction is indicated by its sequence homology to cytokine receptors (Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 6934-6938). Signal transduction induced by binding of FVIIa to cells expressing TF was studied with baby hamster kidney (BHK) cells stably transfected with TF and with a reporter gene construct encoding a luciferase gene under transcriptional control of tandem cassettes of signal transducer and activator of transcription (STAT) elements and one serum response element (SRE). FVIIa induced a significant luciferase response in cells expressing TF, BHK(+TF), but not in cells without TF. The BHK(+TF) cells responded to the addition of FVIIa in a dose-dependent manner, whereas no response was observed with active site-inhibited FVIIa, which also worked as an antagonist to FVIIa-induced signaling. Activation of the p44/42 MAPK pathway upon binding of FVIIa to TF was demonstrated by suppression of signaling with the specific kinase inhibitor PD98059 and demonstration of a transient p44/42 MAPK phosphorylation. No stimulation of p44/42 MAPK phosphorylation was observed with catalytically inactive FVIIa derivatives suggesting that the catalytic activity of FVIIa was obligatory for activation of the MAPK pathway. Signal transduction caused by a putative generation of FXa activity was excluded by experiments showing that FVIIa/TF-induced signaling was not quenched by tick anticoagulant protein, just as addition of FXa could not induce phosphorylation of p44/42 MAPK in BHK(+TF) cells. These results suggest a specific mechanism by which binding of FVIIa to cell surface TF independent of coagulation can modulate cellular functions and possibly play a role in angiogenesis and tumor metastasis as indicated by several recent observations.

The stable, inactive but reactivatable, unfolding intermediate of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase differs from the age-modified form of this protein.

Ca(2+)-ATPase from rat skeletal-muscle sarcoplasmic-reticulum has been reported to be less stable in old animals. These changes were suggested to be due to age-related modifications in the membrane. In the present study, the guanidinium-chloride (GuHCl)-induced inactivation, and reactivation by dilution of the denaturant, of this enzyme have been investigated. The cooperativity of the inactivation-transition was found to increase when the membrane was dissolved in the non-ionic detergent polyoxyethylene 10 laurylether. There is an inactive unfolding intermediate in equilibrium with the native state at low concentrations of GuHCl. This intermediate can be reactivated, even after as long a time as 19 h. The stability of this intermediate is only slightly affected by detergent solubilization of the enzyme. Hence, the membrane probably only plays a minor role in stabilizing the intermediate.

GroEL provides a folding pathway with lower apparent activation energy compared to spontaneous refolding of human carbonic anhydrase II.

The kinetics of the refolding of the enzyme, human carbonic anhydrase II (HCA II), at different temperatures, together with the Escherichia coli chaperonin GroEL, has been studied. The Arrhenius plots for the spontaneous, GroEL-assisted, and GroEL/ES-assisted refolding of HCA II show that the apparent activation energy (E(a)) is lower in the presence of the chaperonin GroEL alone than for the spontaneous reaction, whereas the apparent activation energy for the GroEL/ES-assisted reaction is almost the same as for the spontaneous reaction (85, 46, and 72 kJ/mol, for the spontaneous, GroEL, and GroEL/ES-assisted reactions, respectively).

Expression of the acatalytic carbonic anhydrase VIII gene, Car8, during mouse embryonic development.

The carbonic anhydrase (CA)-like protein, CA VIII, lacks the typical carbon dioxide hydrase activity of the CA isozymes. However, the high degree of amino acid sequence similarity between the products of the mouse and the human CA VIII genes suggests an important biological function. We have attempted to investigate the function of this gene in mammalian development by conducting an in situ hybridization study on sagittal sections of mouse embryos at gestation days of 9.5-16.5 using a 35S-labelled riboprobe. Results indicate that this gene (called Car8 in mice) is expressed as early as day 9.5 in a variety of organs including liver, branchial arches, neuroepithelium and developing myocardium. Between days 10.5 and 12.5, it showed a widespread distribution of mRNA expression that became more restricted as development progressed. The level of expression of Car8 mRNA was relatively high in the brain, liver, lung, heart, gut, thymus and epithelium covering the head and the oronasal cavity.

GroEL reversibly binds to, and causes rapid inactivation of, human carbonic anhydrase II at high temperatures.

The initial yield of reactivation of GuHCl denatured human carbonic anhydrase II does not change with temperature between 3 and 35 degrees C. At temperatures above 35 degrees C, the enzymatic activity is not stable, but decreases over time. If the bacterial chaperonin GroEL is present during reactivation, the initial yield is lower compared to the spontaneous reaction at temperatures of 35-50 degrees C. However, unlike the spontaneous reactivation, the enzymatic activity with time in the presence of GroEL. In the presence of GroEL, native HCA II incubated at elevated temperatures will rapidly loose enzymatic activity to the same value as during reactivation at that particular temperature; most of the activity will recover if the temperature is lowered when GroEL is present. It is evident that there is an equilibrium between an inactive intermediate of HCA II, probably bound to GroEL, and active enzyme. Furthermore, proline isomerization is part of the rate-limiting step of refolding even in the presence of GroEL, and it is very noteworthy that prolyl isomerase will influence the refolding of HCA II in the presence of GroEL.

Expression of mouse carbonic anhydrase VII in E. coli and demonstration of its CO2 hydrase activity.

The alpha-carbonic anhydrase (alpha-CA) gene family in mammals encodes 10 CA or CA-like proteins (CA I-CA X). Although the gene for human CA VII has been cloned and characterized, the corresponding protein has not previously been purified, and hence, the CO2 hydrase activity of its product has not as yet been demonstrated. In this study, we have cloned the mouse CA VII cDNA in an E. coli, glutathione-S-transferase (GST) expression vector. The CO2 hydrase activity of the expressed protein is about 4% that of the high-activity CAII isozyme, demonstrating that this evolutionarily highly conserved protein is a catalytically active member of this CA gene family.

Chromatographic and electrophoretic methods related to the carbonic anhydrase isozymes.

There are three gene families that encode zinc metalloenzymes that catalyze the reversible hydration of CO2. The encoded enzymes are termed carbonic anhydrases (CAs). The CA isozymes have been purified from representatives of all types of organisms. Most CAs are strongly inhibited by aromatic sulfonamides. Several chromatographic and electrophoretic methods have been devised to determine binding constants for sulfonamides to CAs, and these compounds have been extensively used for, often single-step, affinity chromatographic separation of CAs from complex matrixes. The purification of different CA isozymes from different organisms is reviewed, as are methods for detection of CAs during chromatography and electrophoresis.

GroEL/ES-mediated refolding of human carbonic anhydrase II: role of N-terminal helices as recognition motifs for GroEL.

The presence of GroEL/ES during the refolding of human carbonic anhydrase II (pseudo-wild type) was found to increase the yield of active enzyme from 65 to 100%. This chaperone action on the enzyme could be obtained by adding GroEL alone, and the time-course in that case was only moderately slower than the spontaneous process. Truncated forms of carbonic anhydrase, in which N-terminal helices were removed, also served as protein substrates for GroEL/ES. This demonstrates that N-terminally located helices are not obligatory as recognition motifs.

Phosphorescence reveals a continued slow annealing of the protein core following reactivation of Escherichia coli alkaline phosphatase.

When Escherichia coli alkaline phosphatase (AP) is refolded in vitro after extensive denaturation in 6.2 M guanidine hydrochloride, the enzymatic activity reaches its asymptotic value in 1 h at 24 degrees C. In contrast, the structural rigidity of the hydrophobic core of the protein, monitored by the recovery of the tryptophan phosphorescence lifetime, returns to its characteristic native-like value over several days. Moreover, the protein lability, measured by the rate of inactivation in 4.5 M guanidine hydrochloride, also changes on a time scale much longer than the recovery of activity. These results clearly demonstrate that while the return of enzymatic activity, the traditional measure of the attainment of the native state, indicates that AP has refolded to its final, active conformation, the phosphorescence data indicate otherwise. In the context of the rugged energy landscape model [Frauenfelder, H., et al. (1991) Science 254, 1598-1603], the slow annealing of the hydrophobic core is consistent with the presence of high-energy barriers that separate fully active intermediates along the folding pathway. The data suggest that the core of the protein undergoes continued structural rearrangements affecting the rigidity of the protein environment surrounding the emitting tryptophan and the protein lability long after the return of enzyme activity.

Characterization of the gene encoding carbonic anhydrase I from the pigtail macaque.

The structure of the gene encoding carbonic anhydrase I (CA I) was determined for the pigtail macaque Macaca nemestrina. When the deduced amino-acid sequence was compared with those of five other primates, four non-primate mammals and a turtle, seven residues were found to be unique and invariant to all of the CA I sequences. A scheme is presented for the probable evolutionary order of the six polymorphic nucleotide changes found in the coding regions of the CA I locus of pigtail macaques.

Assignment of the gene for human carbonic anhydrase VIII(CA8) to chromosome 8q11-->q12.

The human carbonic anhydrase (CA) VIII gene (CA8) has been mapped to chromosome 8 at q11-->q12 by human-mouse hybrid mapping and by fluorescence in situ hybridization. The closely-linked human CA isozyme genes, CA1, CA2 and CA3, are also located on chromosome 8, but at q22, and therefore not closely linked to the CA8 locus.

Lack of correspondence between the room-temperature phosphorescence decay-components and Trp residues in a series of Trp-->Cys or Trp-->Phe mutants of human carbonic anhydrase II.

The room temperature phosphorescence of native human carbonic anhydrase (CA), and several mutants of this enzyme has been investigated. In these mutants the seven tryptophan residues in the native protein have sequentially been replaced by cysteine or phenylalanine. All of the mutants as well as native CA show room-temperature tryptophan phosphorescence (RTP) spectra. Surprisingly, only small differences in RTP life-times are noticeable among these mutants, indicating that there is more than one tryptophan residue with similar phosphorescence decay kinetics in the protein. The present results illustrate the danger in attributing the room temperature phosphorescence of a multi-tryptophan protein to a particular residue based solely on an analysis of the protein structure.

The deduced amino acid sequence of human carbonic anhydrase-related protein (CARP) is 98% identical to the mouse homologue.

A recently reported mRNA, encoding 'carbonic anhydrase-related polypeptide' (CARP) from the Purkinje cells of mouse cerebellum, was shown to have a 30-40% deduced amino acid sequence identity with the carbonic anhydrases (CA) of mammals. In order to compare the mouse and human CARP sequences, we used the polymerase chain reaction (PCR) to amplify human CARP sequences from several cDNA libraries (salivary gland, testis and placenta). The sequence has an 89.3% sequence identity with mouse CARP at the nucleotide level and 97.9% at the amino acid level. This extremely high evolutionary conservation suggests an important function for the CARP gene product.

Isomerase and chaperone activity of prolyl isomerase in the folding of carbonic anhydrase.

Several proteins have been discovered that either catalyze slow protein-folding reactions or assist folding in the cell. Prolyl isomerase, which has been shown to accelerate rate-limiting cis-trans peptidyl-proline isomerization steps in the folding pathway, can also participate in the protein-folding process as a chaperone. This function is exerted on an early folding intermediate of carbonic anhydrase, which is thereby prevented from aggregating, whereas the isomerase activity is performed later in the folding process.

Mutation creates an open reading frame within the 5' untranslated region of macaque erythrocyte carbonic anhydrase (CA) I mRNA that suppresses CA I expression and supports the scanning model for translation.

A variant allele at the CA I locus that produces a deficiency of erythrocyte-specific CA I occurs as a widespread polymorphism in pigtail macaques from southeast Asia. Sequence analyses revealed a C----G substitution 12 nucleotides downstream of the cap site in the variant erythrocyte CA I mRNA. This mutation forms a new AUG start site and an open reading frame coding for 26 amino acids that terminates 6 nucleotides before the normal AUG initiation codon for CA I. It appears that the presence of this upstream open reading frame greatly diminishes reinitiation of translation from the normal start site, resulting in trace levels of CA I in erythrocytes. Preferential use of the first AUG codon supports the scanning model for translation initiation in eukaryotes.