Biomedical subjects

R L Mellgren

Publications and source records attributed to R L Mellgren.

At least 37 records · Page 2Linked to original sources

PubMed1991

Proteolysis of nuclear proteins by mu-calpain and m-calpain.

Purified calpains are capable of proteolyzing several high Mr nuclear proteins and solubilizing a histone H1 kinase activity from rat liver nuclei upon exposure to 10(-6) - 10(-5) M Ca2+. Major nuclear substrates displayed apparent molecular masses of 200, 130, 120, and 60 kDa on Coomassie Blue-stained SDS-PAGE gels. The nuclear proteins and the H1 kinase were released from Triton-treated nuclei following incubation with buffer containing 0.5 M NaCl. They therefore appeared to be internal nuclear matrix proteins. The nuclear H1 kinase activity solubilized by incubation with m-calpain was eluted in the void volume of a Bio-Gel A-1.5m column, indicating an apparent mass greater than 1,500 kDa. Treatment of the calpain-solubilized kinase with 0.5 M NaCl dissociated it to a form having an apparent mass of 300 kDa (Stokes radius = 5.6 nm), suggesting that the 300-kDa (Stokes radius = 5.6 nm), nuclei by calpain treatment as a large complex containing other internal matrix proteins. Purified human erythrocyte mu-calpain was capable of proteolyzing the nuclear matrix proteins at 10(-6) M Ca2+. In contrast, human erythrocyte multicatalytic protease complex produced little cleavage of the nuclear proteins. Proteolysis of nuclear proteins by either mu-calpain or m-calpain was inhibited by calpastatin. These experiments suggest a physiologic role for the calpains in the turnover of nuclear proteins.

Animals↗

PubMed1990

Interaction of human erythrocyte multicatalytic proteinase with polycations.

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.

Chromatography, Gel↗

PubMed1990

Calpain II-dependent solubilization of a nuclear protein kinase at micromolar calcium concentrations.

Incubation of isolated, Triton X-100 washed rat liver nuclei with purified bovine myocardial calpain II resulted in solubilization of a histone H1 kinase activity. The release of kinase from nuclei could be prevented by including the calpain inhibitors leupeptin or calpastatin in the incubation. Titration with Ca2+/EGTA buffers indicated that the calpain-dependent release of the kinase was half-maximal at approximately 3 microM Ca2+. In contrast, calpain II required at least 50 microM Ca2+ to produce detectable proteolysis of soluble or membrane-associated substrates. These results suggest that the cell nucleus is a site of calpain II activation and function.

Animals↗

PubMed1989

The binding of large calpastatin to biologic membranes is mediated in part by interaction of an amino terminal region with acidic phospholipids.

Animal cells contain a non-lysosomal proteolytic system which degrades various protein substrates in the presence of calcium ion. The calcium-dependent proteinases (calpains) co-exist in cells with a specific protein inhibitor called calpastatin. Distribution of this inhibitor to different subcellular sites could be important in overall regulation of the calpains. Previously, myocardial calpastatin was shown to be present in preparations of sarcoplasmic reticulum and sarcolemma. In the present work, we show that purified bovine myocardial calpastatin binds to the acidic phospholipids, phosphatidylinositol and phosphatidylserine, but not to the neutral phospholipids, phosphatidylcholine and phosphatidylethanolamine. Large forms of calpastatin from canine myocardium and rabbit liver also were bound to phosphatidylinositol. Smaller forms of calpastatin present in the preparations did not bind to acidic phospholipids. Bovine large calpastatin was subjected to CNBr digestion, and a phospholipid-binding fragment representing approximately one-sixth of the intact protein mass was purified. Amino acid sequence analysis indicated that the phospholipid-binding fragment was derived from the amino terminus of the large calpastatin.

Amino Acid Sequence↗

PubMed1989

The grid-blot: a procedure for screening large numbers of monoclonal antibodies for specificity to native and denatured proteins.

This report describes a procedure referred to as a grid-blot for simultaneously testing up to 30 monoclonal antibodies for specificity with an equivalent number of different proteins on a single sheet of nitrocellulose paper. Only 150 microliters of hybridoma culture supernatant is required for the screening and the entire procedure can be completed in less than five hours. This assay was developed to quickly identify those hybridoma cultures producing antibodies that preferentially recognize the native form of a protein and those that also recognize the SDS denatured form and were optimal for use in Western blots. Monoclonal antibodies raised against two distinct proteins, myofibril C-protein (120 antibodies) and the catalytic subunit of cyclic-AMP dependent protein kinase (240 antibodies) were tested. The grid-blot results indicated that 85 of the C-protein antibodies and 55 of the catalytic subunit antibodies were monospecific. Only 4 of the C-protein and 9 catalytic subunit antibodies showed a preferential staining for the appropriate native protein. The antibodies that stained the denatured protein most intensely in the grid-blot corresponded with those that produced the best immunostain in the Western blot. Finally, a version of the grid-blot was found to be an efficient means of determining antibody isotypes.

Animals↗

PubMed1989

The non-lysosomal, calcium-dependent proteolytic system of mammalian cells.

The intracellular calcium-dependent proteases (calpains) and their endogenous protein inhibitor (calpastatin) are present in many different mammalian cells. There is emerging evidence for their importance in the turnover of membrane-associated proteins. Accordingly, it is important to understand how these proteinases and their inhibitor interact within cells, in particular at membranes. Bovine myocardial calpastatin appears to be associated in part with intracellular membranes, where it may effectively block the activity of calpain II on membrane-associated proteins. Immuno-electron microscopic studies suggest that canine myocardial calpain and calpastatin are associated with a number of membranous organelles. During canine myocardial autolysis, the amount of calpain at various organelles decreased, but the amount of calpastatin decreased to an even greater extent. Thus there may be a high calpain to calpastatin balance during heart ischemia at these sites. Calpain II aggregation may contribute to localization of the proteinase at sites of high calcium concentration within cells. A model is presented for interaction of calpain II and calpastatin at cellular membranes in the presence of calcium.

Amino Acid Sequence↗

PubMed1988

Myocardial calpain 2 is inhibited by monoclonal antibodies specific for the small, noncatalytic subunit.

Calpains (EC 3.4.22.17) are nonlysosomal intracellular proteinases which require calcium ion for activity. The calpains are heterodimers composed of a large catalytic subunit and a small subunit which may have a regulatory function during the catalytic cycle. However, whether calpains remain in the dimeric form or dissociate upon exposure to calcium is controversial. To resolve this issue, two monoclonal antibodies which specifically recognize the small calpain subunit were prepared using bovine calpain 2 heterodimer as the antigen. Both antibodies, designated P-1 and P-2, were capable of inhibiting bovine or canine calpain 2, and partially purified human erythrocyte calpain 1. However, neither could produce full inhibition. Further studies with P-1 and bovine calpain 2 indicated that the antibody decreased the calcium requirement for the proteinase. The Km for casein was increased and the Vmax was decreased. The addition of P-1 to the assay mixture several minutes after initiation of proteolytic activity resulted in a rapid inhibition. The P-1 antibody was also capable of decreasing the ability of the protein inhibitor of calpains (calpastatin) to inhibit bovine calpain 2. These studies indicate that the small subunit remains bound to the large subunit during catalysis and may influence its activity.

Animals↗

PubMed1988

On the mechanism of binding of calpastatin, the protein inhibitor of calpains, to biologic membranes.

Bovine myocardial calpastatin, the endogenous inhibitor of the calcium-dependent proteinases, calpains, could bind to sarcoplasmic reticulum preparations at neutral pH and low ionic strength. Even in the presence of 100 to 200 mM KCl, 4 to 5 micrograms of calpastatin was bound per mg of membrane. Although calpastatin is found associated with bovine myocardial sarcolemma, neither canine nor human erythrocyte calpastatins were present in isolated erythrocyte membrane preparations. The bovine myocardial calpastatin, but not human erythrocyte calpastatin, could associate with purified phospholipid vesicles at low ionic strength. Thus, phospholipids appear to be involved in the binding of calpastatin to membranes.

Animals↗

PubMed1988

An improved purification procedure for calpastatin, the inhibitor protein specific for the intracellular calcium-dependent proteinases, calpains.

The specific inhibitor protein (calpastatin) for the calcium-dependent intracellular proteinases (calpains) is an important regulator of these enzymes. In this communication we describe a one day procedure for purifying 3 to 5 mg of calpastatin from a kilogram of bovine myocardium. This represents a substantial improvement over previously described methods, and should facilitate future studies of calpastatin structure and function. A key, novel step in the purification was dye-matrix chromatography on an Affi-Gel Blue column. Contrary to previous indications, calpastatin purified by the new method did not contain significant amounts of carbohydrate. However, the presence of covalently bound phosphate in purified bovine myocardial calpastatin was confirmed and co-migration of phosphate and calpastatin activity was demonstrated on Bio-Gel A-1.5m chromatography. Thus, it is possible that calpastatin function is regulated by phosphorylation.

Animals↗

PubMed1988

The influence of stimulated peritoneal feeder cells and mitogens upon antibody secreting hybridomas.

Peritoneal exudate cells from mice injected with immunostimulatory agents were evaluated for their ability to promote hybridoma growth. Peritoneal cells from mice receiving peritoneal injections of either Freund's incomplete adjuvant or pristane, seven days prior to harvesting, produced the greatest number of antibody-producing hybridomas. Freund's incomplete adjuvant produced 16 fold more peritoneal cells than unstimulated mice, thus reducing the number of mice needed to supply feeder cells for the hybridoma cultures. In separate experiments a number of B-lymphocyte stimulating lectins and factors were tested for their ability to promote hybridoma growth. 2-mercaptoethanol (25 microM) routinely increased the number of antibody producing hybridomas by 5 to 15 fold. 2-mercaptoethanol had a varying ability to increase the numbers of hybridoma colonies. The cloning efficiency, rate of cell growth and antibody production of hybridoma cell lines, previously produced in the absence of 2-mercaptoethanol could also be increased when this reducing agent was added to the culture medium.

Animals↗

PubMed1988

Discrimination learning in a foraging situation.

Rats were allowed to forage in a simulated natural environment made up of eight food sources (patches) each containing a fixed number of pellets. Two of the eight contained an extra supply of peanuts. The peanut patches were signaled by an olfactory/visual cue located at the bottom of the ladder leading to the patch. In successive phases the number of sessions per day, height of the patches, and availability of peanuts were manipulated. Subjects showed evidence of discrimination learning under these conditions, although the degree of discriminatory behavior varied as a function of environmental manipulations. Assessment of behavior within foraging sessions showed that subjects systematically changed their patterns of utilization of patches across time. Sampling or exploration, as well as food reinforcement, seem implicated in these results.

Journal Article↗

PubMed1987

A sarcolemma-associated inhibitor is capable of modulating calcium-dependent proteinase activity.

Purified bovine myocardial sarcolemma vesicles were shown to contain calcium-dependent proteinase inhibitor protein by direct assay and by immunoblot analysis following gel electrophoresis (Western blotting). Calcium-dependent proteinase (calpain, EC 3.4.22.17) was not detected in the sarcolemma vesicles. The inhibitor protein was not solubilized when the vesicles were ruptured by repetitive freezing and thawing. However, a large amount of latent inhibitor activity was exposed after freezing and thawing the sarcolemma, and the inhibitor was much more susceptible to removal by 1.0 M NaCl or proteolysis following this treatment. Since the vesicles were predominantly right-side-out, the latter observations suggested that the inhibitor was associated with the cytoplasmic face of the sarcolemma. The endogenous inhibitor was capable of protecting sarcolemmal protein kinase C from proteolytic conversion to soluble protein kinase M by type I or type II calcium-dependent proteinase. Thus, the inhibitor is probably important in controlling calcium-dependent proteolysis of sarcolemmal proteins.

Animals↗

PubMed1987

Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium-dependent protease inhibitor.

Bovine myocardial sarcolemma and sarcoplasmic reticulum vesicle preparations contained calcium-dependent protease inhibitor protein. No inhibitor was detected in mitochondrial membranes. The membrane-bound inhibitor co-purified with the marker enzymes for sarcolemma and sarcoplasmic reticulum, Na+,K+-ATPase and Ca2+,K+-ATPase respectively, on isopycnic ultracentrifugation through linear sucrose density gradients. Sarcolemma and sarcoplasmic reticulum vesicles contained about 1 mg of inhibitor per g of membrane protein. However, about one-half of the inhibitor in sarcoplasmic reticulum vesicles was not tightly associated with the membrane. The membrane-bound inhibitor may function to modulate calcium-dependent proteolytic cleavage of sarcolemmal or sarcoplasmic reticulum-associated proteins.

Animals↗

PubMed1987

Calcium-dependent proteases: an enzyme system active at cellular membranes?

Proteases having a neutral pH optimum and an absolute requirement for calcium ion are found in virtually all mammalian cells. Association of calcium-dependent proteases and a specific inhibitor protein with biological membranes seems to be an important regulatory feature of this proteolytic system, and it is likely that membranes are preferred sites for calcium-dependent protease action. Several recent hypotheses for the physiological function of calcium-dependent proteolysis are consistent with a membrane-associated protease action. Calcium-dependent proteases may participate in cell membrane fusion: the proteolysis of membrane proteins, which is required for the efficient fusion of erythrocytes, may be catalyzed by these enzymes. There is also evidence for the involvement of calcium-dependent proteolysis in postsynaptic membrane remodeling in the hippocampus after long-term potentiation. Although the relationship of the proteolysis to synaptic function is not known, it could have important physiological or pathophysiological consequences. Finally, it has recently been suggested that calcium-dependent proteolysis may be a physiologically significant mechanism for activating membrane-associated protein kinase C after exposure of some cell types to phorbol esters or other mitogens. Further pursuit of these hypotheses may reveal a novel role for intracellular calcium-regulated proteolysis in membrane-associated cell functions.

Calcium↗

PubMed1986

Catalytic subunit of the polycation-stimulated protein phosphatase. Effect of proteolysis on polycation stimulation.

The phosphorylase phosphatase activity of the holoenzyme form of phosphatase 2A isolated from extracts of porcine renal cortex or bovine heart was stimulated 600% and 500%, respectively, by the addition of histone H1. After conversion of the phosphatase to the catalytic subunit form by treatment with ethanol at room temperature, histone H1 stimulated activity by about 150% only. Purification of the catalytic subunit from porcine renal cortex yielded two forms of the enzyme which were separated by heparin-Sepharose chromatography. These forms were designated peak 1 and peak 2 according to their order of elution from the column. Peak 1 catalytic subunit was stimulated by more than 400% by histone H1, whereas peak 2 was stimulated by about 50% only. Based on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, peak 2 had a slightly higher Mr value than peak 1 (35,500 vs. 35,000). Incubation of the peak 2 phosphatase with trypsin converted it to a form that was similar to peak 1 with respect to Mr and stimulation by histone H1. When the catalytic subunit of phosphatase 2A was purified from bovine heart only one form was obtained. Bovine heart enzyme was similar to renal peak 2 in that it had an apparent Mr of 35,500 and was only slightly stimulated by histone H1. Treatment of the bovine heart catalytic subunit with trypsin, chymotrypsin or type 2 Ca2+-dependent proteinase decreased the apparent Mr by about 500 and increased histone H1 stimulation to about 500%. Thus, when a small peptide was removed by proteolysis, histone H1 stimulation of the 'nicked' catalytic subunit was similar to that obtained with the holoenzyme.

Animals↗

PubMed1986

Isolation and characterization of an inhibitor-sensitive and a polycation-stimulated protein phosphatase from rat liver nuclei.

Two protein phosphatases were isolated from rat liver nuclei. The enzymes, solubilized from crude chromatin by 1 M NaCl, were resolved by column chromatography on Sephadex G-150, DEAE-Sepharose and heparin-Sepharose. The phosphorylase phosphatase activity of one of the enzymes (inhibitor-sensitive phosphatase) was inhibited by heat-stable phosphatase inhibitor proteins and also by histone H1. This phosphatase had a molecular weight of approx. 35,000 both before and after 4 M urea treatment. Its activity was specific for the beta-subunit of phosphorylase kinase. Pretreatment with 0.1 mM ATP inhibited the enzyme only about 10%, and it did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as the catalytic subunit of phosphatase 1. The other phosphatase (polycation-stimulated phosphatase) was insensitive to inhibition by inhibitor 1, and it was stimulated 10-fold by low concentrations of histone H1 (A0.5 = 0.6 microM). This enzyme had a molecular weight of approx. 70,000 which was reduced to approx. 35,000 after treatment with 4 M urea. It dephosphorylated both the alpha- and beta-subunits of phosphorylase kinase. The enzyme was inhibited more than 90% by preincubation with 0.1 mM ATP and did not require divalent cations for activity. On the basis of these properties, this nuclear enzyme was identified as phosphatase 2A.

Animals↗

PubMed1986

Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex.

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.

Adenosine Triphosphate↗

PubMed1986

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.

Animals↗