Proteolytic activation of calcium-activated, phospholipid-dependent protein kinase by calcium-dependent neutral protease

A Ca2+-dependent protease I), which hydrolyzes casein at Ca2+ concentrations lower than the 10(-5) M range, is purified roughly 4000-fold from the soluble fraction of rat brain. This protease is able to activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) by limited proteolysis analogously to the previously known Ca2+-dependent analogously to the previously known Ca2+-dependent protease (Ca2+ protease II) which is active at the millimolar range of Ca2+ (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616). The protein kinase fragment thus produced shows a molecular weight of about 5.1 X 10(4), and is significantly smaller than native protein kinase C (Mr = 7.7 X 10(4). Although protein kinase C may be normally activated in a reversible manner by the simultaneous presence of phospholipid and diacylglycerol at Ca2+ concentrations less than 10(-6) M, this enzyme fragment is fully active without any lipid fractions and independent of Ca2+. The limited proteolysis of protein kinase C is markedly enhanced in the velocity by the addition of phospholipid and diacylglycerol, which are both required for the reversible activation of the enzyme. However, casein hydrolysis by this protease is not affected by phospholipid and diacylglycerol. Available evidence suggests that, at lower concentrations of this divalent cation, Ca2+ protease I reacts preferentially with the active form of protein kinase C which is associated with membrane, and converts it to the permanently active form. In contrast, the inactive form of protein kinase C, which is free of membrane phospholipid, does not appear to be very susceptible to the proteolytic attack. It remains unknown, however, whether this mechanism of irreversible activation of protein kinase C does operate in physiological processes. It is noted that Ca2+ protease II, which is active at higher concentrations of Ca2+, proteolytically activates protein kinase C irrespective of the presence and absence of phospholipid and diacylglycerol.     

Proteolysis of the calcium-dependent protease inhibitor by myocardial calcium-dependent protease

Bovine heart peak II calcium-dependent protease was capable of hydrolyzing its specific inhibitor protein at high molar ratios of protease to inhibitor. The proteolysis was inhibited by leupeptin and required millimolar calcium. Thus, it appeared to be attributable to the calcium-dependent protease and not to possible contaminating proteases in the purified preparations of inhibitor or calcium-dependent protease. Incubation of the purified inhibitor with the calcium-dependent protease produced a discrete pattern of inhibitor fragments on Western blots developed with an inhibitor-specific monoclonal antibody. Traces of similar or identical lower molecular weight immunoreactive material could be observed in Western blots of bovine heart extracts, and the immunoreactivity present as these lower molecular weight forms could be increased by incubation of the extracts with calcium ion. These results suggest that the inhibitor can be proteolyzed to low molecular weight forms which can be detected in cardiac tissue extracts, and that calcium-dependent protease(s) may be responsible for this phenomenon.     

Proteolytic modification of staphylokinase

There are three types of staphylokinase of different isoelectric points (6.7, 6.1 and 5.7). Staphylokinase of pI 6.7 was converted to that of pI 6.1 and then to that of pI 5.7 by the treatment with trypsin.Heterogeneity of staphylokinase might be the result of post-translational modification by proteolytic enzyme.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.     

Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel inParamecium

Summary The effects of proteolysis on a hyperpolarization- and Ca²⁺-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca²⁺-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca²⁺ concentrations between 10^–4 and 10^–8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca²⁺-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca²⁺ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca²⁺-dependent K channels (BK channels) and the hyperpolarization-, Ca²⁺-dependent K channels inParamecium.     

Proteolytic processing of HIV-1 protease precursor, kinetics and mechanism.

Previously it was demonstrated using a model precursor that processing at the N terminus of the HIV-1 protease (PR) precedes processing at its C terminus. We now show the expression, purification, and kinetics of the autoprocessing reaction of a PR precursor linked to 53 amino acids of the native flanking transframe region (DeltaTFP-p6(pol)) of Gag-Pol and containing its two native cleavage sites. The PR contains the two cysteine residues exchanged to alanines, mutations that do not alter the kinetics or the structural stability of the mature PR. DeltaTFP-p6(pol)-PR, which encompasses the known PR inhibitor sequence Glu-Asp-Leu within DeltaTFP, undergoes cleavage at the DeltaTFP/p6(pol) and p6(pol)/PR sites in two consecutive steps to produce the mature PR. Both DeltaTFP-p6(pol)-PR and p6(pol)-PR exhibit low intrinsic enzymatic activity. The appearance of the mature PR is accompanied by a large increase in catalytic activity. It follows first-order kinetics in protein concentration with a rate constant of 0.13 +/- 0.01 min(-1) in 0.1 M acetate at pH 4.8. The pH-rate profile for the observed first-order rate constant is bell-shaped with two ionizable groups of pK(a) 4.9 and 5.1. The rate constant also exhibits approximately 7-fold higher sensitivity to urea denaturation as compared with that of the mature PR, suggesting that the cleavage at the N terminus of the PR domain from the precursor leads to the stabilization of the dimeric structure.     

Proteolytic modification of staphylokinse.

There are three types of staphylokinase of different isoelectric points (6.7, 6.1 and 5.7). Staphylokinse of pI 6.7 was converted to that of pI 6.1 and then to that of pI 5.7 by the treatment with trypsin. Heterogeneity of staphylokinase might be the result of post-translational modification by proteolytic enzyme.     

Characterization of ionomycin as a calcium ionophore.

The ionophorous properties of a new antibiotic, ionomycin, have been studied. It was found that the antibiotic is capable of extracting calcium ion from the bulk of an aqueous phase into an organic phase. The antibiotic also acts as a mobile ion carrier to transport the cation across a solvent barrier. The divalent cation selectivity order for ionomycin as determined by ion competition experiments was found to be: Ca greater than Mg greater than Sr = Ba, where the binding of strontium and barium by the antibiotic is insignificant. The antibiotic also binds La3+ to some extent, but its complexation with monovalent alkali metal ions is negligible. Measurement of the binding of ionomycin with Ca2+ indicates that ionomycin complexes and transports calcium ion in a one to one stoichiometry.     

Transmembrane calcium movements mediated by ionomycin and phosphatidate in liposomes with Fura 2 entrapped

A novel liposomal method permits studies of Ca movements across the bilayers of multilamellar vesicles (MLV) which had entrapped the Ca-dependent, fluorescent indicator dye Fura 2. Ionomycin-mediated Ca translocation across MLV of phosphatidylcholine (PC)/dicetyl phosphate (DCP), 9:1, obeyed simple first-order kinetics since log-log plots of initial rates versus ionomycin or Ca concentration yielded slopes of approximately 1. Since Ca is translocated in a Ca-dependent fashion in the course of stimulus-response coupling of cells which form diacylglycerol (DAG) and phosphatidate (PA) from polyphosphoinositides, we compared effects of PA with those of DAG. PA and DAG were preincorporated in PC/DCP vesicles, in which trace amounts of ionomycin provided transmembrane potential (due to Ca2+/H+ exchange). Significant increases in Ca movements were observed in the presence of egg lecithin PA, dioleoyl-PA, and dipalmitoyl-PA when compared with DCP- or DAG-containing MLV. DAGs such as 1-oleoyl-2-acetoylglycerol or 1,2-dioleoylglycerol in liposomes decreased rates of Ca translocation. Ca influx into PA-containing MLV was dependent on the mole percent of the PA in bilayers; the complex kinetics of Ca influx were compatible with the formation of nonbilayer states. Incorporation of cholesterol into the liposomes inhibited initial rates of Ca uptake by MLV presumably by condensing the bilayers. Ca influx increased with increasing pH of the external medium from 6.9 to 7.9 in liposomes with an internal pH of 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic activity in brains of rabbits treated with aluminum

Aluminum injection in rabbits leads to neurofibrillary changes which are at light microscopic level similar to those found in Alzheimer's disease. We used this animal model to see whether changes in proteolytic activity occur that may affect protein degradation in the altered neurofibrillary structure. Rabbits were injected via the cisterna magna with aluminum chloride, and after ten days tissue was excised from the spinal cord, hippocampus, occipital lobe, and cerebellum. Sections from the hippocampus and spinal cord were examined for neurofibrillary changes; enzyme activity was measured in all four areas. The enzymes studied were cathepsins A, B, and D, and the angiotensin-converting enzyme. No significant differences could be established in enzymatic activity in aluminum-injected animals compared to controls. However, a significant decrease in Triton-soluble proteins was observed in the treated animals, which correlated with changes in neurofibrillary structure. This decrease was most noticeable in the spinal cord (from 16.6 to 12.5 mg/g).     

Proteolytic modification of mouse liver catalase

When catalase was immunoprecipitated from different subfractions of mouse liver homogenates, the enzyme which was obtained from extracts of the large granular fraction exhibited a lower molecular weight than that from either the cytosol or purified peroxisomal fractions, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This modification of the enzyme could be prevented by the addition of proteolytic inhibitors to extraction buffers; and consequently, unmodified catalase was able to be purified in the presence of 5 mM iodoacetamide. Electrophoretic comparison of the catalases against standards of known molecular sizes indicated that the unmodified enzyme had a subunit mass approximately 2,000 daltons larger than the modified enzyme. The significance of these proteolytic modifications has been discussed in relation to the involvements of catalase and peroxisome turnover.     

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.     

Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity

Calcium-dependent protease II (CDP-II) from bovine heart is a heterodimer with subunit molecular weights of 80,000 and 26,000. Previous studies have demonstrated that the protease requires 350 microM Ca2+ for half-maximal activity and that the large subunit contains both the catalytic and Ca2+ binding functions of the enzyme. The function of the small subunit has been unclear. We have examined the effect of Ca2+ on structural and catalytic properties of CDP-II in the presence and absence of substrate proteins. When incubated with Ca2+ in the absence of substrate, CDP-II undergoes a series of autoproteolytic cleavages that sequentially reduce the small subunit's molecular weight from 26,000 to 24,000 to 22,000 to 17,000. During this time there is no detectable change in the 80-kDa subunit, which remains associated with the autolyzed small subunit. The rate of autoproteolysis is dependent on temperature and on the concentration of Ca2+ (half-maximal rate at approximately 600 microM Ca2+). The first cleavage appears to be unimolecular because its rate is unaffected by CDP-II concentration or by the presence of exogenous protein substrates. Subsequent cleavages result in the formation of the 80-kDa/17-kDa heterodimer and appear to occur by bimolecular reactions; rates of these reactions were slowed by decreasing CDP-II concentrations and by the presence of protein substrates. Autoproteolysis of the small subunit has two distinct functional consequences, each of which is associated with different forms of the autolyzed protease. Our results indicate that the 80-kDa/26-kDa form of CDP-II represents an inactive proenzyme and that the initial Ca2+-dependent cleavage of the 26-kDa subunit results in activation of the protease. The activated enzyme hydrolyzes protein substrates with a Ca2+ concentration requirement of 350 microM for half-maximal rates. The further autoproteolysis, which results in the formation of the 80-kDa/17-kDa heterodimer, serves to reduce the Ca2+ concentration requirement for protease activity by 25-fold. Thus, these results provide evidence for specific roles of the small subunit in the regulation of CDP-II activity.     

Proteolytic regulation of neurite outgrowth from neuroblastoma cells by thrombin and protease nexin-1

This review summarizes studies on the reciprocal regulation of neuroblastoma neurite outgrowth by thrombin and protease nexin-1 (PN-1). PN-1 recently was shown to possess the same deduced amino acid sequence as the glial-derived neurite-promoting factor. The neurite outgrowth activity of PN-1 depends on its ability to inhibit thrombin. Thrombin not only blocks the neurite outgrowth activity of PN-1, but it also brings about neurite retraction in the presence of PN-1. Thrombin also produces neurite retraction in the absence of PN-1 and other regulatory factors. This suggests that its activity is due to a direct action on cells. The neurite retraction by thrombin depends on its proteolytic activity. It does not occur with the other serine proteases that have been tested, indicating that it is a specific effect and is not due to a general proteolytic effect that could detach neurites from the culture dish. Serum brings about neurite retraction in certain neuroblastoma cells and primary neuronal cultures; most of this activity is due to residual thrombin in the serum. Together, these results suggest that PN-1 and thrombin (or a thrombin-like protease) play a role in regulation of neurite outgrowth.     

Proteolytic modification of Escherichia coli alkaline phosphatase

Proteolytic modification of the native alkaline phosphatase dimer is restricted to sites in the amino-terminal portion of the sequence. Complementing previous studies of the product of trypsin cleavage at the R-11, A-12 bond (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733; Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 260, 7557-7561) circular dichroic spectroscopy indicates that cleavage at this site results in a rearrangement of secondary structure and change in tertiary structure as monitored in the far and near UV regions, respectively. Under more vigorous reaction conditions, trypsin cleaves at the R-35, D-36 bond. The deletion of an additional 24 residues yields a species whose functional and structural properties are similar to the initial product of trypsin cleavage. Treatment of the enzyme with Protease V-8 results in cleavage at the E-9, N-10 bond. In contrast to the products of trypsin treatment, this truncated enzyme is similar to the native enzyme. These results indicate that the residues at the N-10 and R-11 positions play a unique role in maintaining the structural integrity and catalytic potency of the enzyme although this locus is distant from the enzyme active centers. These observations are discussed in terms of the three-dimensional structure of the enzyme.     

Purification and characterization of a calcium-dependent protease from rat liver

A calcium-dependent protease, previously identified in rat liver and designated peak II [DeMartino, G. N. (1981) Arch. Biochem. Biophys. 211, 253-257], was purified and characterized. The calcium-dependent proteolytic activity was accounted for by an 80 000-dalton protein. Depending on the method of purification, we found that this protease could be associated with a 28 000-dalton subunit, which was devoid of protease activity. The catalytic characteristics of the two different forms of the protease were indistinguishable. Each was half-maximally activated by approximately 250 microM calcium.     

A calcium-dependent protease associated with the neural cytoskeleton. Purification and partial characterisation

Calcium-dependent protease activity was found associated with a neurofilament-enriched cytoskeleton isolated from the bovine spinal cord. The protease was extracted from the cytoskeleton by 0.6 M KCl, and purified to apparent homogeneity (3300-fold) by chromatography on organomercurial-Sepharose 4B, casein-Sepharose 4B, and Sepharose CL-6B. A cytosolic calcium-dependent protease was similarly purified from the bovine spinal cord, after the cytosol was fractionated on DEAE-cellulose. Both cytoskeleton-bound and cytosolic enzymes had an apparent molecular mass of 100 kDa as judged by gel filtration, and consisted of two subunits (79 kDa and 20 kDa) upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Both enzymes exhibited caseinolytic activity with 0.5 mM Ca2+ and above, and the activity was strongly inhibited by various thiol protease inhibitors. In the presence of 0.1-0.2 mM Ca2+, the 68-kDa and 160-kDa components, and to a lesser extent the 200-kDa component, of the neurofilament triplet polypeptides were degraded by the cytosolic protease, whereas the cytoskeleton-bound protease needed two-fold higher concentration of Ca2+ to degrade the neurofilaments. Nevertheless, the cytoskeleton-bound protease in situ, i.e. before its extraction form the cytoskeleton by 0.6 M KCl, preferentially degraded the 160-kDa component in the presence of 0.1-0.2 mM Ca2+, suggesting that a proper locational relation of this enzyme to the neurofilament structure is a prerequisite to its preference for the 160-kDa component. It appears that a factor or factors involved in such an interaction between the protease and the neurofilament were eliminated during the course of enzyme purification. The glial fibrillary acidic protein was almost insensitive to the proteases purified in the present study.     

The action of calcium-dependent protease on platelet surface glycoproteins

The action of exogenous calcium-dependent protease (CDP) on tritium-labeled surface glycoproteins was analyzed by incubation of labeled, washed human platelets with CDP partially purified from human platelets. Labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography. Incubation of the labeled platelets with the protease led to a loss (calcium-dependent) from the platelets of glycoproteins Ib and V and concomitant appearance in the supernatant solution of glycocalicin (a proteolytic fragment of glycoprotein Ib), glycoprotein V, and other, unidentified glycoproteins. These changes in surface label were accompanied by alterations in three parameters of platelet function. Compared to control platelets, the CDP-treated platelets were activated by thrombin more slowly and showed less saturable and nonsaturable binding of thrombin. The CDP-treated platelets, but not the controls, aggregated on addition of fibrinogen, indicating that treatment with CDP had exposed fibrinogen receptors. The alterations in surface glycoproteins and functional parameters were compared over a 1000-fold range of CDP treatment. The decreased binding of thrombin and the exposure of fibrinogen receptors were correlated with the release of surface glycoproteins to the supernatant solution, but the slow activation by thrombin was observed under conditions where no release of labeled glycoproteins was detected (i.e., brief incubations with low concentrations of CDP). Activation of the endogenous CDP with 2.5 mm calcium chloride plus the ionophore A23187 was accompanied by hydrolysis of actin-binding protein, a known substrate, and release to the supernatant solution of labeled glycocalicin and glycoprotein V plus a faster-migrating glycoprotein not released by exogenous protease. This effect was observed in the presence of leupeptin, which completely inhibited action of exogenous protease, suggesting that platelet calcium-dependent protease may modify the platelet surface in ways that can cause alterations of platelet function.     

Mechanisms of T cell activation by the calcium ionophore ionomycin

We have investigated signaling mechanisms that may underlie the T cell mitogenic properties of the Ca2+ ionophore ionomycin. Ionomycin induces highly purified resting human T cells to proliferate in the presence of monocytes with accompanying IL-2R expression and IL-2 synthesis. Treatment of T cells with ionomycin triggers the hydrolysis of phosphoinositides, as evidenced by the accumulation of the hydrolytic by-products phosphatidic acid and inositol phosphates. Ionomycin also induces the activation of protein kinase C (PKC), as demonstrated by the auto-phosphorylation of PKC and the phosphorylation of the PKC target proteins CD4 and CD8. Ionomycin synergizes with PMA in enhancing the activation of PKC. It is concluded that, in addition to its putative activation of Ca2+/calmodulin-dependent signaling pathways, ionomycin induces the hydrolysis of phosphoinositides and the activation of PKC in human T cells. The synergy of ionomycin with phorbol esters in triggering T cell activation may relate, at least in part, to enhanced activation of PKC.