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D E Croall

Publications and source records attributed to D E Croall.

At least 37 records · Page 2Linked to original sources
PubMed1989

ATP-dependent mechanisms for protein degradation in mammalian cells.

The biochemical basis for ATP-dependent protein degradation observed in intact cells has been studied in cell-free extracts of baby hamster kidney fibroblasts. ATP plays at least two distinct roles in proteolysis. First, ATP is required for the covalent conjugation of ubiquitin to protein substrates. This modification markedly enhances the rates of degradation of some, but not all, proteins. Second, ATP appears to stimulate the activity of a protease capable of degrading both ubiquitinated and non-ubiquitinated proteins. This protease has several biochemical and catalytic features that resemble those of the previously described high molecular weight protease, macropain. Furthermore, antibodies against highly purified human erythrocyte macropain inhibit both ubiquitin-dependent and ubiquitin-independent pathways of ATP-dependent proteolysis. Such results provide evidence for an important role for macropain in ATP-dependent proteolysis in mammalian cells.

Adenosine Triphosphate↗
PubMed1988

An enzyme related to the high molecular weight multicatalytic proteinase, macropain, participates in a ubiquitin-mediated, ATP-stimulated proteolytic pathway in soluble extracts of BHK 21/C13 fibroblasts.

Soluble, cell-free extracts of BHK 21/C13 fibroblasts degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated the rates of proteolysis. Both the absolute rate of proteolysis and the magnitude of the ATP effect depended on the specific substrate. For example, casein was degraded approximately 10-fold faster than lysozyme, but lysozyme degradation was more highly stimulated by ATP than was casein degradation. Ubiquitin enhanced the ATP-stimulated proteolysis of each substrate in both postmicrosomal extracts and DEAE-cellulose fractionated extracts. In each extract, ubiquitin enhanced the ATP-stimulated degradation of lysozyme to a greater degree than that of casein. These results suggested that lysozyme was degraded by a pathway that was more dependent upon ubiquitin than was casein. Further evidence for this conclusion was obtained in studies using substrates whose amino groups were blocked by extensive methylation or carbamoylation. The high molecular weight proteinase, macropain, appears to be involved in the ATP-stimulated degradation of both substrates. Specific immunoprecipitation of macropain with polyclonal antibodies resulted in the inhibition of ATP-stimulated proteinase activity both in the absence and presence of ubiquitin. These results indicate that macropain plays a role in both ubiquitin-mediated and ubiquitin-independent ATP-stimulated proteolysis in BHK cell extracts.

Adenosine Triphosphate↗
PubMed1988

The calmodulin-binding site in alpha-fodrin is near the calcium-dependent protease-I cleavage site.

Fodrin (brain spectrin) binds calmodulin and is susceptible to proteolysis by calcium-dependent protease I (CDP-I, calcium-activated neutral protease I, or calpain I). Both events involve the central region of the alpha-fodrin subunit, and calmodulin binding enhances the sensitivity of fodrin to CDP-I mediated proteolysis. Fragments of fodrin, generated chemically or proteolytically, which retain calmodulin binding activity have been identified and analyzed by two-dimensional peptide mapping and by direct protein sequencing. Both CDP-I and calmodulin interact with the terminal portion of the eleventh repetitive unit in fodrin, which is at the center of the molecule. CDP-I cleavage occurs between Tyr104 and Gly105 and preserves the calmodulin binding activity of the carboxyl-terminal fragment. In contrast, chymotryptic cleavage at Trp120 reduces the ability of this fragment to bind calmodulin, and tryptic cleavage beyond Trp120 completely eliminates calmodulin binding activity. It is concluded that Ser-Lys-Thr-Ala-Ser-Pro-Trp-Lys-Ser-Ala-Arg-Leu-Met-Val-His-Thr-Val-Ala- Thr- Phe-Asn-Ser-Ile-Lys, a 24-residue peptide which bridges repeats 11 and 12 of brain alpha spectrin contains the high affinity calmodulin binding domain.

Binding Sites↗
PubMed1988

Proteolysis of the protein inhibitor of calcium-dependent proteases produces lower molecular weight fragments that retain inhibitory activity.

The specific inhibitor of calcium-dependent proteases was purified from soluble extracts of bovine heart. The protein had a molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gels and migrated on gel filtration chromatography with an apparent molecular weight of 250,000. The inhibitor specifically blocked the action of the two calcium-dependent proteases, CDP-I and CDP-II, but did not influence a variety of other proteases including trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. These latter enzymes extensively degraded the inhibitor to discrete lower molecular weight peptides as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by gel filtration chromatography. Under the conditions studied, proteolysis of the inhibitor had little or no effect on its inhibitory activity; isolated peptides with molecular weights as low as 17,000 retained inhibitory function. A number of various-sized inhibitor fragments were isolated by gel filtration chromatography and by SDS-PAGE. These fragments were compared with the intact inhibitor for their ability to inhibit CDPs. As suggested previously by us and others, one molecule of intact inhibitor appears to inhibit up to five molecules of calcium-dependent protease. The inhibitor fragments of decreasing size inhibited correspondingly fewer molecules of protease. These results suggest that the inhibitor protein contains multiple functional domains and may explain some of the discrepancies in reported molecular weights for this protein.

Animals↗
PubMed1988

ATP-stimulated proteolysis in soluble extracts of BHK 21/C13 cells. Evidence for multiple pathways and a role for an enzyme related to the high-molecular-weight protease, macropain.

Soluble extracts of cultured cells (BHK 21/C13) degraded a variety of exogenous proteins to acid-soluble peptides at pH 8.0. ATP stimulated this proteolytic activity up to 10-fold. The ATP effect was dependent on Mg2+ and was not elicited by nonhydrolyzable analogs of ATP. After the extract was fractionated on DEAE-cellulose, ATP-stimulated protease activity was in the fraction that bound to the resin and eluted in buffer containing 0.4 M NaCl. This activity had characteristics that were indistinguishable from those of the unfractionated extract but the degree of ATP stimulation was two- to three-fold lower. Although no protease activity was detected in the unbound fraction, reconstitution of this material with the bound fraction enhanced the ATP stimulation up to twofold. The component responsible for the enhancement of the ATP stimulation had properties similar to ubiquitin and purified ubiquitin enhanced the ATP-stimulated protease activity in the fractionated extract. Substrates whose amino groups were almost completely blocked by various chemical modifications were still degraded in an ATP-stimulated fashion, but the degradation of these substrates was not affected by ubiquitin. The protease activity isolated by ion-exchange chromatography was fractionated further by gel filtration chromatography on Sephacryl S-300. ATP-stimulated protease activity eluted with an apparent molecular weight of 750,000. Protease activity was enhanced up to eightfold by Mg2+-ATP but was not increased further by ubiquitin. An activity that hydrolyzed the synthetic peptide Z-Val-Leu-Arg-MNA coeluted with ATP-stimulated protease activity, but peptide hydrolysis was not affected by ATP. These and other catalytic and biochemical characteristics suggested that the protease might be related to the high-molecular-weight protease, macropain, recently purified by us from human erythrocytes (M. J. McGuire and G. N. DeMartino Biochim. Biophys. Acta (1986) 873, 279-289). Antibodies raised against macropain specifically reacted with proteins characteristic of macropain in the column fractions containing ATP-stimulated protease activity. These antibodies also specifically immunoprecipitated 70-100% of the ATP-stimulated protease activity as well as Z-Val-Leu-Arg-MNA hydrolyzing activity. Thus BHK cell extracts appear to contain both ubiquitin-mediated and ubiquitin-independent pathways for the ATP-stimulated degradation of proteins. Furthermore, at least one of these pathways appears to involve a high-molecular-weight, ATP-stimulated protease related to macropain.

Adenosine Triphosphate↗
PubMed1987

Colocalization of calcium-dependent protease II and one of its substrates at sites of cell adhesion.

Adhesion plaques, specialized regions of the plasma membrane where a cell contacts its substratum, are dynamic structures. However, little is known about how the protein-protein interactions that occur at adhesion plaques are controlled. One mechanism by which a cell might modulate its associations with the substratum is by selective, regulated proteolysis of an adhesion plaque component. Here we show that the catalytic subunit of the calcium-dependent protease type II (CDP-II) is localized in adhesion plaques of several cell types (BS-C-1, EBTr, and MDBK). We have compared the susceptibility of the adhesion plaque constituents vinculin, talin, and alpha-actinin to calcium-dependent proteolysis in vitro and have found talin to be the preferred substrate for CDP-II. The colocalization of a calcium-requiring proteolytic enzyme and talin in adhesion plaques raises the possibility that calcium-dependent proteolytic activity provides a mechanism for regulating some aspect of adhesion plaque physiology and function via cleavage of talin.

Actinin↗
PubMed1986

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.

Animals↗
PubMed1986

Limited proteolysis of the erythrocyte membrane skeleton by calcium-dependent proteinases.

The action of purified calcium-dependent proteinases on human erythrocyte membrane skeleton proteins has been examined. Preferential cleavage of proteins 4.1 a and b and band 3 and limited cleavage of alpha- and beta-spectrin occur when either calcium-dependent proteinase I or calcium-dependent proteinase II has access to the cytoplasmic side of the ghost membrane skeleton in the presence of calcium. Thus, when these proteinases are incubated with sealed ghosts they do not cleave these proteins. Leupeptin, mersalyl, the specific cellular protein inhibitor of these enzymes, and calcium chelators can inhibit proteolysis of the red cell ghost proteins by Ca2+-dependent proteinases. Each proteinase has also been loaded into erythrocyte ghosts in the absence of calcium at low ionic strength and subsequently trapped inside by resealing the ghosts. The proteinases were activated by incubating these ghosts in the presence of the calcium ionophore A23187 and calcium. Examination of the ghost proteins by electrophoresis demonstrated calcium-dependent proteolysis of Bands 4.1 and 3 and limited cleavage of alpha- and beta-spectrin similar to that observed on proteolysis of the open, leaky ghosts. In the presence of calcium each calcium-dependent proteinase appears to associate with the erythrocyte ghost membrane.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1986

Fractionation and quantification of calcium-dependent proteinase activity from small tissue samples.

Almost all mammalian tissues contain a cytoplasmic Ca2+-dependent proteolytic system consisting of two different proteinases (CDP I and CDP II) and an endogenous inhibitor specific for these proteinases. It was difficult to determine the relative activities of CDP I and CDP II directly and accurately without extensive purification, requiring relatively large amounts of tissue. We developed a simple technique based on Reactive Red-agarose affinity chromatography for quantitatively measuring CDP II activities in small (less than 100 mg) tissue samples. This technique is rapid, sensitive and highly reproducible. CDP II activities can be quantified in numerous tissue samples in a single day. Using this method, we analysed the components of the CDP system in various rat tissues and demonstrated quantitative differences in CDP II activities as well as relative differences in the amounts of CDP-inhibitor activity among the various tissues. This technique should prove useful in the efforts to define the currently unknown physiological function(s) of the Ca2+-dependent proteolytic system by allowing comparison of CDP activities in tissues under diverse conditions of protein metabolism.

Animals↗
PubMed1986

Calcium-dependent affinity purification of transglutaminase from rat liver cytosol.

Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases.

Animals↗
PubMed1984

Comparison of two calcium-dependent proteinases from bovine heart.

We have purified two calcium-dependent proteinases from bovine heart. Each enzyme was a heterodimer. One proteinase (designated CDP-I) contained subunits of 84 and 26 kDa. The other proteinase (designated CDP-II) contained subunits of 80 and 26 kDa. The large subunit of each proteinase accounted for the calcium-dependent proteolytic activity of the respective enzyme and could be isolated from the small subunit by casein-Sepharose affinity chromatography. The large subunits of CDP-I and CDP-II appeared to be distinctly different proteins, based on differences in peptide maps and on the lack of detectable immunologic cross-reactivity. On the other hand, the small subunits of the proteinases appeared to be identical peptides, based on peptide mapping and on two-dimensional gel electrophoresis. The function of the small subunit is unknown. The two calcium-dependent proteinases share many catalytic properties, including the nature of proteinase activity against several myofibrillar proteins. However, the proteinases were distinguished by different calcium-concentration requirements. CDP-II required 300 microM Ca2+ for half-maximal activity and 750 microM Ca2+ for full activity. CDP-I required 30 microM Ca2+ for half-maximal activity and 100 microM for full activity.

Animals↗
PubMed1984

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver.

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent Mr = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with Mr = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.

Animals↗
PubMed1983

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.

Animals↗
PubMed1983

Developmental regulation of mRNA in mouse heart.

The myocardium contains abundant translatable mRNAs that change during development. Maximal cell-free synthesis of [3H]leucine-, [35S]methionine-, and [35S]cysteine-labeled translation products directed by poly(A)-containing mRNAs from 12-, 14-, and 17-day fetal; 5-day-old neonatal; and 30-day-old adult mouse heart was determined by using one- and two-dimensional polyacrylamide gels. Three general developmental patterns of heart-specific mRNA translation products were observed: two translatable mRNAs were most abundant in 12-day fetal heart; five mRNAs were most abundant in 14- and 17-day fetal heart and occurred only at low concentrations in 12-day fetal and adult heart; four mRNAs, including mRNAs coding for actin, tropomyosin, and myosin light chains 1 and 2, were most abundant in the adult heart. Thus, differentiating cardiac muscle is characterized by a complex pattern of mRNA regulation.

Animals↗
PubMed1982

The steroid ligands of estrogen binding proteins in Xenopus laevis liver cytosol are primarily metabolites 17beta-estradiol.

The interactions in vitro between [3H]estradiol and liver proteins from Xenopus laevis have been examined to determine if the binding reaction meets criteria of steroid-receptors which may function in the induction of vitellogenesis. Estrogenic hormones associated with proteins is serum and liver cytosol from Xenopus laevis. However, the interactions between soluble liver proteins and estrogens apparently do not result from serum contamination of liver as specific binding was distinguishable by ligand affinity and by differential mobility on polyacrylamide gels. Steroid ligands bound by liver proteins during incubation in vitro were examined by solubility and by thin-layer chromatography. Only a small percentage (13%) of the bound radioactive ligand was recovered as the original tritium-labeled steroid, 17beta-estradiol. The major ligand was recovered as a water-soluble metabolite of estradiol which was identified tentatively as an estradiol-glucoside. To investigate whether the protein-bound estradiol metabolite(s) merely masks a small amount of authentic estradiol-receptor complexes or if the metabolite could be an intermediate in estrogen function, isolated liver nuclei were incubated with liver cytosol containing 3H-labeled steroid-protein complexes or with serum protein-steroid complexes relative to [3H]estradiol. Nuclei preferentially accumulated 3H-labeled steroids from liver cytosol protein-steroid complexes relative to [3H[estradiol from serum proteins. However, analysis of the steroids recovered in the nuclei after incubation with liver cytosol revealed that both 17beta-[3H]estradiol and the 3H-labeled water-soluble metabolite were retained in vitro by nuclei.U

Animals↗
PubMed1982

Calcium-dependent protease in neuroblastoma cells.

Extracts of neuroblastoma cells contained calcium-dependent proteolytic activity. This activity was accounted for by two distinct proteases. These proteases were separated by ion-exchange chromatography. Although each was totally dependent on calcium, the calcium requirements of the enzymes were different; one enzyme required approximately 40 microM Ca2+ for half-maximal activity and the other enzyme required approximately 150 microM Ca2+ for half-maximal activity.

Animals↗