Vitamin K-dependent carboxylase: influence of the "propeptide" region on enzyme activity

The liver microsomal vitamin K-dependent carboxylase catalyzes the post-translational conversion of specific glutamyl to gamma-carboxyglutamyl (Gla) residues in precursor forms of a limited number of proteins. These proteins contain an amino-terminal extension (propeptide) that is presumed to serve as an enzyme recognition site to assure their normal processing. The free, noncovalently bound propeptide has also been shown to stimulate the in vitro activity of this enzyme. This peptide has now been shown to lower the app Km of a low-molecular-weight Glu site substrate while having no influence on the app Km of the other substrates, vitamin KH2, O2, and CO2/HCO3-. Propeptide addition was shown to have no influence on the ratio of the two products of the enzyme, Gla and vitamin K-2,3-epoxide. Stimulation of carboxylase activity by the propeptide from human factor X was observed in a number of rat tissues and in the liver of a number of different species. Stability of the enzyme in crude microsomal preparations was greatly enhanced by the presence of propeptide. These observations are consistent with the hypothesis that this region of the protein substrates for the carboxylase not only serves an enzyme recognition or docking function but also modulates the activity of the enzyme by altering the affinity for one of its substrates.     

Augmentation of inorganic pyrophosphate elaboration in cartilage by serum factors

The disordered production of inorganic pyrophosphate (PPi) by articular cartilage is thought to have an important role in the pathogenesis of calcium pyrophosphate dihydrate deposition disease and perhaps osteoarthritis. We have previously shown that fetal calf serum added to the culture media of porcine articular cartilage explants increases the elaboration of PPi into the ambient media. We have examined this PPi stimulatory activity by studying the effects of adult human serum (HS), serum derived from adult human plasma (HP), and an acid-alcohol extract of human platelets (PE) on PPi production in cartilage organ culture. Ten percent HS produces a 1.4-fold increase in PPi production after 48 h of culture, while cartilage incubated in media containing 10% HP produces no more PPi than that incubated in media alone. PE stimulates a mean 2-fold increase in PPi production at 48 h in the presence of low concentrations of HP, and has no effect alone. It does not appear to up-regulate the activity of the ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH), nor does it promote the release of enzyme substrate into the extracellular space. Cartilage exposed to 0.5% HP and PE has 1.51 +/- 0.36 units of NTPPPH activity whereas cartilage exposed to 0.5% HP alone has 1.52 +/- 0.41 units of enzyme activity. PE does not increase the release of [14C]adenine-labeled compounds into the media. Approximately 13% of soluble 14C counts was found in the media of chondrocytes treated with PE while 18% of counts was released in the presence of HP alone. We have demonstrated a factor or factors present in FCS, HS, and an acid-ethanol extract of human platelets which represent(s) the first known physiologic modulators of PPi production in articular cartilage and may increase PPi production without affecting NTPPPH activity.     

NMR studies of differences in the conformations and dynamics of ligand complexes formed with mutant dihydrofolate reductases

Two mutants of Lactobacillus casei dihydrofolate reductase, Trp 21----Leu and Asp 26----Glu, have been prepared by using site-directed mutagenesis methods, and their ligand binding and structural properties have been compared with those of the wild-type enzyme. 1H, 13C, and 31P NMR studies have been carried out to characterize the structural changes in the complexes of the mutant and wild-type enzymes. Replacement of the conserved Trp 21 by a Leu residue causes a decrease in activity of the enzyme and reduces the NADPH binding constant by a factor of 400. The binding of substrates and substrate analogues is only slightly affected. 1H NMR studies of the Trp 21----Leu enzyme complexes have confirmed the original resonance assignments for Trp 21. In complexes formed with methotrexate and the mutant enzyme, the results indicate some small changes in conformation occurring as much as 14 A away from the site of substitution. For the enzyme-NADPH complexes, the chemical shifts of nuclei in the bound coenzyme indicate that the nicotinamide ring binds differently in complexes with the mutant and the wild-type enzyme. There are complexes where the wild-type enzyme has been shown to exist in solution as a mixture of conformations, and studies on the corresponding complexes with the Trp 21----Leu mutant indicate that the delicately poised equilibria can be perturbed. For example, in the case of the ternary complex formed between enzyme, trimethoprim, and NADP+, two almost equally populated conformations (forms I and II) are seen with the wild-type enzyme but only form II (the one in which the nicotinamide ring of the coenzyme is extended away from the enzyme structure and into the solvent) is observed for the mutant enzyme complex. It appears that the Trp 21----Leu substitution has a major effect on the binding of the nicotinamide ring of the coenzyme. For the Asp 26----Glu enzyme there is a change in the bound conformation of the substrate folate. Further indications that some conformational adjustments are required to allow the carboxylate of Glu 26 to bind effectively to the N1 proton of inhibitors such as methotrexate and trimethoprim come from the observation of a change in the dynamics of the bound trimethoprim molecule as seen from the increased rate of the flipping of the 13C-labeled benzyl ring and the increased rate of the N1-H bond breaking.     

The MgADP-induced decrease of the SH1-SH2 fluorescence resonance energy transfer distance of myosin subfragment 1 occurs in two kinetic steps

The fluorescence resonance energy transfer distance between 5-[2-[iodoacetyl)amino)ethyl]aminoaphthalene-1-sulfonic acid covalently attached to the SH1 thiol of myosin subfragment 1 as the energy donor and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide attached to SH2 as the energy acceptor has been found to decrease by about 7 A in the presence of MgADP (Dalby, R. E., Weiel, J., and Yount, R. G. (1983) Biochemistry 22, 4696-4706; Cheung, H. C., Gonsoulin, F., and Garland, F. (1985) Biochim. Biophys. Acta 832, 52-62). Fluorescence stopped-flow experiments on the same system have yielded biphasic traces which are resolvable into a fast and slow component, k1 and k2, respectively. Results of experiments in which k1 and k2 were measured as a function of excess ADP concentration showed: 1) a nonlinear dependence of the apparent rate constants on [ADP]; 2) k1 is a factor of 10 faster than k2. These results are consistent with the 3-step mechanism previously proposed for nucleotide binding to myosin S1 (Garland, F., and Cheung, H. C. (1979) Biochemistry 18, 5281-5289). Kinetic experiments in which the anisotropy of the donor was monitored show this quantity to be unchanged over the course of the reaction. The biphasic decrease of donor intensity is assigned to an increase in energy transfer efficiency which, from the above results, is due to a decrease in donor-acceptor distance, occurring in two steps. The fast step is associated with a 4-5-A decrease of the donor-acceptor separation, while the slow step is associated with a further decrease of approximately 2 A.     

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.     

Differential effect of ultracentrifugation on apolipoprotein A-I-containing lipoprotein subpopulations

Two populations of apolipoprotein (apo) A-I-containing lipoprotein particles are found in high density lipoproteins (HDL): those that also contain apo A-II[Lp(A-I w A-II)] and those that do not [Lp(A-I w/o A-II)]. Lp(A-I w/o A-II) comprised two distinct particle sizes with mean hydrates Stokes diameter of 10.5 nm for Lp(A-I w/o A-II)1 and 8.5 nm for Lp(A-I w/o A-II)2. To study the effect of ultracentrifugation on these particles, Lp(A-I w/o A-II) and Lp(A-I w A-II) were isolated from the plasma and the ultracentrifugal HDL (d 1.063-1.21 g/ml fractions) of five normolipidemic and three hyperlipidemic subjects. The size subpopulations of these particles were studied by gradient polyacrylamide gel electrophoresis. Several consistent differences were detected between plasma Lp(A-I w/o A-II) and HDL Lp(A-I w/o A-II). First, in all subjects, the relative proportion of Lp(A-I w/o A-II)1 to Lp(A-I w/o A-II)2 isolated from HDL was reduced. Second, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 were considerably reduced in HDL. Third, a distinct population of particles with approximate Stokes diameter of 7.1 nm usually absent in plasma was detected in HDL Lp(A-I w/o A-II). Little difference in subpopulation distribution was detected between Lp(A-I w A-II) isolated from the plasma and HDL of the same subject. When plasma Lp(A-I w/o A-II) and Lp(A-I w A-II) were centrifuged, 14% and 4% of A-I were, respectively, recovered in the D greater than 1.21 g/ml fraction. Only 2% A-II was found in this density fraction. These studies show that the Lp(A-I w/o A-II) particles are less stable than Lp(A-I w A-II) particles upon ultracentrifugation. Among the various Lp(A-I w/o A-II) subpopulations, particles larger than Lp(A-I w/o A-II)1 and smaller than Lp(A-I w/o A-II)2 are most labile.     

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices

Summary Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells. This work was supported by an operating grant from the Medical Research Council of Canada.     

Identification of G-proteins in rat parotid gland plasma membranes and granule membranes: presence of distinct components in granule membranes

We have identified by immunoblotting and ADP-ribosylation by cholera toxin and pertussis toxin the presence of Mr 43 and 46 KDa G_s, and 39 and 41 KDa G_i;.. subunits in rat parotid gland plasma membranes but not in granule membranes. A Mr 28 KDa polypeptide that served as substrate for ADP-ribosylation by both cholera toxin and pertussis toxin was present exclusively in granule membranes. Photoaffinity crosslinking of [-³²P]GTP showed the presence of high molecular weight GTP-binding proteins (Mr 160,100 KDa) in granule membranes. Six low molecular weight GTP-binding proteins (Mr 21–28 KDa) were differentially distributed in both plasma membranes and granule membranes. The present study identifies various GTP-binding proteins in rat parotid gland plasma membranes and granule membranes, and demonstrates the presence of distinct molecular weight GTP-binding proteins in granule membranes. These granule-associated GTP-binding proteins may be involved in secretory processes.