Dependent Proteolysis Research Articles

Squid neurofilaments. Phosphorylation and Ca2+-dependent proteolysis in situ.

Three major polypeptides co-purify with neurofilaments from squid (Loligo forbesi) axoplasm: P60 (apparent Mr 60,000), P200 (apparent Mr 200,000) and Band 1 (apparent Mr 400,000). Anti-IFA, a monoclonal antibody that recognizes an epitope common to all classes of intermediate filaments, binds to P200 and P60. When axoplasm is incubated with [32P]Pi, the major phosphorylated polypeptides are P200 and Band 1. We have investigated Ca2+-dependent proteolysis of [32P]phosphorylated axoplasm in order to localize the major sites of phosphorylation and to probe the arrangement of the polypeptides in the filament. The proteinase preferentially cleaves P200 and Band 1, liberating the phosphorylated domains. Analysis of proteolysed filaments by electron microscopy and gel electrophoresis shows that most of P200 and Band 1 can be cleaved while still maintaining intact filaments. We suggest that P200 is initially cleaved within a single highly sensitive region, generating two major fragments called P100p (apparent Mr 100,000) and P110s (apparent Mr 110,000). P100p contains the Anti-IFA epitope and co-sediments with filaments, whereas P110s is highly phosphorylated and does not sediment with filaments. Band 1 is cleaved to produce a soluble high-Mr fragment that is phosphorylated and that represents a major portion of the undigested component, whereas P60 is relatively resistant to limited proteolysis. Thus proteolysis appears to define two major filament domains: a conserved core that forms the backbone of the filament, and a highly phosphorylated peripheral region that is not essential for filament integrity.

Selective localization of calpain I (the low-Ca 2+-requiring form of Ca 2+-dependent cysteine proteinase) in B-cells of human pancreatic islets

An immunohistochemical study was performed to localize two distinct Ca 2+-proteases (low-Ca 2+-requiring calpain I and high-Ca 2+-requiring calpain II) and their specific inhibitor (calpastatin) in human pancreas using the respective monospecific antibodies. Strongly positive staining by anti-calpain I antibody was found in pancreatic islets, specifically in B-cells, whereas the exocrine pancreatic tissue showed essentially no positive immunostaining. No such specific staining was found with anti-calpain II antibodies or anti-calpastatin antibodies. The results suggest that the Ca 2+-dependent proteolysis in B-cells can be triggered by a small rise of the intracellular Ca 2+ concentration without serious interference by the endogenous inhibitor.

Reconstitution of calpain I and calpain II from their subunits: Interchangeability of the light subunits

Calpain (Ca 2+-dependent cysteine proteinase) is known to be a heterodimer, composed of one heavy (called 80K) and one light (called 30K) subunit. Calpain I, a low-Ca 2+-requiring form from porcine and human erythrocytes, and calpain II, a high-Ca 2+-requiring form from porcine kidney, were separated into their respective 80K and 30K subunits by high-performance liquid chromatography on a TSK-Gel G3000SW column in 1 m NaSCN. The isolated 80K subunits from porcine calpains I and II showed Ca 2+-dependent proteolysis, though much depressed and requiring higher Ca 2+ concentrations compared with the respective parent calpains. The optimal conditions were set for the reconstitution of a heterodimeric molecule from the once denatured and separated 80K and 30K subunits. In such reconstitution, the 30K subunits of calpains I and II were found to be functionally identical and interchangeable; either a homologous or heterologous 30K subunit lowered the Ca 2+ requirement of an 80K subunit significantly and enhanced the proteolytic activity. Subunit interchange could also be demonstrated between porcine and human calpains.

Multiple forms of phosphoinositide-specific phospholipase C of different relative molecular masses in animal tissues. Evidence for modification of the platelet enzyme by Ca2+-dependent proteinase.

The Mr distribution of phosphoinositide-specific phospholipase C in the supernatants isolated from a variety of animal tissues was analysed by high-performance gel-filtration chromatography. In most tissues, at least four peaks of activity were resolved. However, different tissues showed quite marked differences in the distribution of activity between these peaks. In rat heart, lung and kidney, the predominant form had Mr approx. 90000, whereas the predominant form in brain had Mr approx. 290000. In liver, the Mr-90000 form predominated, but this tissue also contained relatively large amounts of a form of Mr approx. 150000. Phospholipase C in these tissues from other animal species gave similar distributions of activity between the peaks. In supernatants prepared from platelets sonicated in the presence of leupeptin (0.5 mM) or EGTA (20 mM), the Mr-290000 form predominated. However, when leupeptin or EGTA (inhibitors of Ca2+-dependent proteinase) was omitted from the sonication buffer, the Mr-290000 form appeared to be replaced by a form of Mr 100000. Similar changes in Mr were not demonstrated with the other tissues. These results may be relevant to the intracellular regulation of phospholipase C, since Ca2+-dependent proteolysis has been reported to occur during platelet activation.

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by [formula omitted] (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O- octadecyl-2-O- acetyl-sn- glyceryl-3- phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β- aminoethyl ether)-N,N,N′,N′- tetraacetate to prevent Ca 2+-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl > LiCl > KCl > choline chloride . NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.

Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin.

A total of 53 different cell lines originating from a variety of mammalian species were cultured in vitro and analysed for the presence of vimentin, employing polyacrylamide gradient slab gel electrophoresis in urea/acetic acid as buffer system. Irrespective of the cell culture conditions, and the growth potential and morphology of the cells, vimentin was expressed in all cell lines examined, with two exceptions: MPC-11 mouse myeloma and MOPC-31C mouse plasmacytoma cells. Immunoblotting with the monoclonal antibody alpha-IFA, which is directed against an antigenic determinant shared by all classes of intermediate filaments, did not detect any other of the known intermediate filament proteins in MPC-11 and MOPC-31C cells. Vimentin synthesized by various cell lines was characterized by four different criteria: (1) its extractability with Triton X-100 under various ionic conditions; (2) its behaviour in (NH4)2SO4 fractionation of cellular extracts; (3) its electrophoretic mobility in polyacrylamide gel electrophoresis in urea/acetic acid; and (4) the co-isolation of polypeptides of higher electrophoretic mobility, which, by comparison with degradation products of vimentin obtained with the Ca2+-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca2+-dependent proteolysis of vimentin. Although the degradation products occurred in different ratios in extracts of different cell lines, they constituted the same characteristic set of proteins whenever degradation of vimentin was observed. The formation of proteolytic breakdown products could be partially to totally suppressed when the cells were harvested, washed and processed in the presence of EGTA and proteinase inhibitors. The experimental data show that: (1) vimentin, as well as the Ca2+-activated proteinase specific for intermediate filament proteins, is highly conserved during the evolution of mammalian species; (2) the proteolytic breakdown products of vimentin, which give rise to a characteristic 'staircase' in two-dimensional gel electrophoresis, are probably artefacts of isolation; (3) the expression of vimentin is neither a prerequisite for nor necessarily indicative of rapid cell proliferation in vitro; and (4) the techniques described can be used for the routine identification of vimentin in cells and tissues in case vimentin-specific antibodies are not available.

Evidence for the presence of high- Mr microtubule-associated proteins and their Ca 2+-dependent proteolysis in synaptosomal cytosol

Calcium-dependent proteolysis of several polypeptides from rat brain and synaptosomal cytosol was observed including proteolysis of polypeptides of M r 340 000 and 300 000. These latter polypeptides comigrated with high- M r microtubule-associated proteins of microtubule preparations from brain or synaptosomal cytosol. Calcium influx into intact synaptosomes due to depolarisation with high potassium or veratridine or treatment with the ionophore A23187 did not result in Ca 2+-dependent proteolysis of any polypeptides. This may be due to the low calcium sensitivity of the protease since no proteolysis of the M r 340 000 and 300 000 polypeptides was seen in synaptosomal cytosal at < 10 μM free Ca 2+.

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells.

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.

Subcellular distribution of cyclic AMP-dependent protein kinase activity and of cyclic AMP-binding proteins in human platelets: Modification by Ca 2+-dependent proteolysis

Subcellular distribution of cyclic AMP-dependent protein kinase activity and of cyclic AMP-binding proteins in human platelets: Modification by Ca 2+-dependent proteolysis

Detection of factor IX antibodies by radioimmunoassay: effect of calcium on antibody-factor IX interaction

A radioimmunoassay for alloantibodies (inhibitors) and heteroantibodies to human factor IX has been developed using radioiodinated human factor IX and formalin-fixed, heat-killed Staphylococcus aureus cells (Staph A). Staph A was used as a solid-phase adsorbent for immune complexes. The assay is specific, shows excellent correlation with factor IX coagulant neutralization assays in detecting alloantibodies (r = 0.98), and is 60 times more sensitive. The Staph A method allows rapid separation of immune complexes (within 10 min of addition) and binds immunoglobulin with equivalent efficiency in the presence and absence of calcium. These characteristics have made the Staph A binding method useful for observing the Ca++ effect on the antigenicity of factor IX as detected by hetero- and alloantibodies. All antisera investigated showed some increase in antibody titer when measured in the presence of Ca++. One particular alloantibody showed the greatest increase in titer (&gt; twofold). The reversibility of the calcium effect by excess EDTA indicates that it was not caused by Ca++-dependent proteolysis of the factor IX molecule. The Staph A procedure can also be used as a sensitive competitive radioimmunoassay for human factor IX using alloantibody. The assay could detect factor IX antigen to a dilution of 1:1280 of normal plasma and correctly classified hemophilia B plasmas as cross-reacting material positive or negative.

Detection of Factor IX Antibodies by Radioimmunoassay: Effect of Calcium on Antibody–Factor IX Interaction

AbstractA radioimmunoassay for alloantibodies (inhibitors) and heteroantibodies to human factor IX has been developed using radioiodinated human factor IX and formalin-fixed, heat-killed Staphylococcus aureus cells (Staph A). Staph A was used as a solid-phase adsorbent for immune complexes. The assay is specific, shows excellent correlation with factor IX coagulant neutralization assays in detecting alloantibodies (r = 0.98), and is 60 times more sensitive. The Staph A method allows rapid separation of immune complexes (within 10 min of addition) and binds immunoglobulin with equivalent efficiency in the presence and absence of calcium. These characteristics have made the Staph A binding method useful for observing the Ca+ + effect on the antigenicity of factor IX as detected by hetero- and alloantibodies. all antisera investigated showed some increase in antibody titer when measured in the presence of Ca++. One particular alloantibody showed the greatest increase in titer (>twofold). The reversibility of the calcium effect by excess EDTA indicates that it was not caused by Ca+ + -dependent proteolysis of the factor IX molecule. The Staph A procedure can also be used as a sensitive competitive radioimmunoassay for human factor IX using alloantibody. The assay could detect factor IX antigen to a dilution of 1:1280 of normal plasma and correctly classified hemophilia B plasmas as cross-reacting material positive or negative.

Calcium-dependent proteins in platelets response of calcium-activated protease in normal and thrombasthenic platelets to aggregating agents

Recent studies have suggested a role for Ca 2+-dependent proteolysis in the regulation of microfilament disassembly by high molecular weight actin-binding protein. A Ca 2+-activated protease similar to myofibrillar Ca 2+-activated protease has been described in platelets. To explore the role of Ca 2+-activated proteolysis of actin-binding protein in platelet function, we have examined the effects of platelet aggregating agents on platelet Ca 2+-activated protease-like activity. The hydrolysis of actin-binding protein by Ca 2+-activated protease was determined electrophoretically. The calcium ionophore, A23187, produced a dose-dependent stimulation of Ca 2+-activated protease-like activity in the presence of exogenous calcium but had no effect in the absence of external calcium. Both normal and thrombasthenic platelets generated Ca 2+-activated protease-like activity in response to A23187. Ionophore-induced stimulation of Ca 2+-activated protease-like activity was not affected by prior incubation of platelets with 8-bromo cyclic GMP, 8-bromo cyclic AMP, prostaglandin E 1, prostaglandin I 2, indomethacin or tetracaine, but was inhibited by the sulfhydryl inhibitor N-ethylmaleimide. These results confirm the presence of Ca 2+-activated protease in platelets and indicate that the source of calcium important in Ca 2+-activated protease stimulation is in part extracellular. Other aggregating agents, thrombin, epinephrine, and ADP, were not accompanied by hydrolysis of actin-binding protein, indicating that the alteration in ionic calcium that occurs during aggregation by these other agents is insufficient to generate Ca 2+-activated protease-like activity as measured by the present analytical technique.

Nuclear translocation and interactions of the estrogen receptor in uterus and mammary tumors

The mechanism of the nuclear translocation of the estrogen receptor (ER) and the nature of the acceptor sites interacting with the ER-estrogen complex are unknown. We report here results of our laboratory aiming to solve the following problems in the uterus and mammary tumors systems. 1. Mechanism of the activation of the cytosol ER leading to its nuclear translocation: The nuclear translocation of ER is triggered by all ligands tested thus far either agonist or antagonist. From the physico chemical characteristics of the nuclear ER extracted by micrococcal nuclease digestion, we propose that the mechanism of the activation of ER is not the Ca 2+-dependent proteolysis of the receptor and might be a simple conformational change. 2. Is DN A a biological acceptor of ER? our results are in agreement with, but do not prove such an hypothesis, (a) The native nuclear ER is extracted after micrococcal nuclease digestion in a form which is still able to bind DNA. (b) Intercalating drugs inhibit 50% of translocation of the ER to the nucleus, (c) Preliminary experiments with 5-bromodeoxyuridine aiming to cross-link in vino the nuclear ER to DNA in MCF 7 cells are also consistent with this hypothesis. 3. Acceptor and Effector sites: The relationship between the nuclear components interacting with the receptor and possibly responsible for its nuclear retention (acceptor sites) and the nuclear components interacting with the receptors to give a response (effector sites) is discussed on the basis of results obtained in estrogen responsive and unresponsive systems. Results obtained in independent mammary tumors of C3H mice and dimethylbenzanthracene treated rats, and with the antiestrogens in MCF, cells strongly suggest that the nuclear translocation step is required but not sufficient to induce the estrogenic response.