Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Partial purification and characterization of phosphotyrosyl-protein phosphatase from Ehrlich ascites tumor cells

We have previously described a phosphotyrosylprotein phosphatase in membrane vesicles from human epidermoid carcinoma A431 cells which is inhibited by micromolar concentration of Zn2+ and is insensitive to ethylenediaminetetraacetic acid (EDTA) and NaF [Brautigan, D. L., Bornstein, P., & Gallis, B. (1981) J. Biol. Chem. 256, 6519-6522]. Here we present the identification and partial purification of a similar enzyme from lysates of Ehrlich ascites tumor cells. the enzyme was purified by using diethylaminoethyl-Sephadex, Zn2+ affinity, and Sephadex G-75 chromatography. During purification, the phosphatase was separated into at least three fractions, all of which exhibited very similar properties and an apparent molecular weight of 40 000 upon gel filtration. The enzyme dephosphorylated phosphotyrosine (P-Tyr)-containing carboxymethylated and succinylated (CM-SC) phosphorylase with an apparent Km of 0.8 microM, as well as P-Tyr containing casein and epidermal growth factor (EGF) receptor kinase, but did not dephosphorylate P-Ser-phosphorylase. The phosphatase was inhibited by Zn2+ at micromolar concentrations (K0.5 with EGF receptor kinase = 5 X 10(-6) M; with CM-SC phosphorylase = 3.3 X 10(-5) M) but not by millimolar concentrations of EDTA and NaF. No inhibition was seen with 1 mM tetramisole, a specific inhibitor of alkaline phosphatases. P-Tyr inhibited the enzyme by 50% at 0.4 X 10(-3) M, while Tyr, Pi, PPi, and p-nitrophenyl phosphate, an excellent substrate for alkaline phosphatases and structurally very similar to P-Tyr, exerted partial inhibition at concentrations above 10(-3) M. The pH optimum was found to be 6.5-7, depending on the substrate used. Very little activity was seen below pH 5 and above pH 8.5. These properties clearly distinguish this enzyme from alkaline phosphatases, as well as the neutral and acidic protein phosphatases so far described, and therefore define it as a new enzyme of the phosphatase family--a phosphotyrosyl-protein phosphatase.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.