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D M Waisman

Publications and source records attributed to D M Waisman.

At least 37 records · Page 2Linked to original sources
PubMed1995

Modulation of annexin II tetramer by tyrosine phosphorylation.

Annexin II tetramer (AIIt) is a Ca(2+)-dependent phospholipid-binding phosphoprotein. In cells either expressing transforming protein tyrosine kinases or treated with growth factors such as PDGF, AIIt has been shown to contain increased levels of phosphotyrosine. Therefore, we have examined the effects of the in vitro phosphorylation of AIIt by pp60c-src on several activities of the protein. AIIt was phosphorylated by pp60c-src to 0.91 +/- 0.07 mol of phosphate/mol of AIIt (mean +/- SD). The protein tyrosine phosphorylation of AIIt completely inhibited the ability of the protein to bind to and bundle F-actin. In contrast, the phosphoprotein and native protein bound to purified adrenal medulla chromaffin granules with similar affinity; however, the chromaffin granule bridging activity of the phosphoprotein was abolished. The inhibition of the chromaffin granule bridging activity of the phosphoprotein could be partially reversed by the addition of millimolar Ca2+. Furthermore, the phosphorylation of AIIt by pp60c-src inhibited the in vitro ability of this annexin to form a complex consisting of plasma membrane, chromaffin granules, and AIIt. In addition to binding to biological membranes, some annexin proteins have been shown to possess carbohydrate-binding activity. Although native AIIt bound to a heparin affinity column, tyrosine phosphorylation of AIIt blocked the ability of the protein to bind to the heparin affinity column. These results suggest that the tyrosine phosphorylation of AIIt is a negative modulator of AIIt and that the dephosphorylation of AIIt might be necessary for activation of the protein.

Animals↗
PubMed1994

Salt dependency of chromaffin granule aggregation by annexin II tetramer.

Annexin II tetramer (AIIt) is a Ca2+ and phospholipid binding protein that has been shown to reconstitute secretion in permeabilized adrenal medulla cells. In the present study, we have characterized the interactions of AIIt with biological membranes using isolated adrenal medulla secretory granules as a model system. Without added salt, maximal binding of AIIt to chromaffin granules occurred in the absence of AIIt-dependent chromaffin granule aggregation, whereas increasing the osmolality of the reaction mixture with sucrose did not activate AIIt-dependent chromaffin granule aggregation. As the KCl or potassium glutamate concentration of the reaction mixture was increased to between 30 and 50 mM salt, AIIt-dependent chromaffin granule aggregation increased to a maximum, while AIIt binding to chromaffin granules decreased. As the salt concentration was increased from 50 to 150 mM, both AIIt-dependent chromaffin granule aggregation and the binding of AIIt to chromaffin granules were decreased. Furthermore, at optimal salt concentration, KCl and potassium glutamate activated AIIt-dependent aggregation of chromaffin granules to maximum values of about 210% and 195% of control, respectively, whereas potassium phosphate supported AIIt-dependent aggregation of chromaffin granules to only 120% of control. The concentration of AIIt for half-maximal binding to chromaffin granules without added salt or at 50 mM KCl was 0.163 +/- 0.007 (mean +/- SD, n = 3) or 0.173 +/- 0.034 microM AIIt (mean +/- SD, n = 3), respectively, and binding of AIIt to chromaffin granules was not measurable at 150 mM KCl. In contrast, at 50 mM KCl, half-maximal AIIt-dependent chromaffin granule aggregation required 0.171 +/- 0.001 microM AIIt (mean +/- SD, n = 3) and was not measurable without added salt or in the presence of 150 mM KCl. Without added salt, at 50 mM KCl, or at 150 mM KCl, the Ca2+ concentrations for half-maximal aggregation of chromaffin granules and the maximal extent of chromaffin granule aggregation (Amax) were pCa2+ = 3.79 +/- 0.062 (mean +/- SD, n = 3) and Amax = 127% of control, pCa2+ = 6.07 +/- 0.021 (mean +/- SD, n = 3) and Amax = 185% of control, or pCa2+ 4.41 +/- 0.07 (mean +/- SD, n = 3) and Amax = 156% of control, respectively. The stimulation of chromaffin granule aggregation activity and the chromaffin granule binding activity of AIIt was reversible by removal of Ca2+. These results suggest that both ionic strength and salt composition modulate both AIIt-dependent chromaffin granule aggregation and binding to the membranes of these secretory granules.

Adrenal Medulla↗
PubMed1994

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis.

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.

Adrenal Glands↗
PubMed1993

Percoll purification of chromaffin granules inhibits their ability to take up and maintain calcium.

Secretory granules of the adrenal medulla have recently been shown to be able to sequester and release Ca2+, in addition to their previously established role as carriers of secretory products. In order to study the ability of these or any other secretory granules to participate in intracellular calcium homeostasis, it is imperative that they should be free of other contaminating Ca2+ sequestering organelles, and that the Ca2+ uptake and release mechanisms of those granules should remain intact throughout any chosen purification procedure. We report here that chromaffin granules which were purified by the isopycnic gradient medium Percoll, or even incubated with it, showed an attenuated ability to sequester Ca2+.

Adrenal Glands↗
PubMed1993

Regulation of annexin I-dependent aggregation of phospholipid vesicles by protein kinase C.

Annexin I is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins. The ability of this protein to aggregate and to mediate the fusion of various types of vesicles has supported the hypothesis that this protein might be involved in intracellular membrane fusion processes such as exocytosis. Although annexin I has been described as a major in vitro substrate of both protein kinase C and the epidermal-growth-factor-receptor protein tyrosine kinase, the functional consequences of these phosphorylation events have not been investigated. In this paper we examine the effect of the phosphorylation of annexin I by protein kinase C on the phospholipid aggregation activity of the protein. The stoichiometry of phosphorylation of the protein was affected by the method of preparation of the phospholipid. Under optimal assay conditions protein kinase C catalysed the incorporation of 2.83 +/- 0.23 mol of phosphate/mol of annexin I (mean +/- S.E.M., n = 21). Studies with the Ca(2+)- and phospholipid-independent form of protein kinase C suggested that the phosphorylation of annexin I was stimulated by phospholipid in the absence of Ca2+, although maximal phosphorylation was achieved in the presence of both phospholipid and Ca2+. Phosphorylation of annexin I resulted in a dramatic decrease in the rate and extent of phospholipid vesicle aggregation, without significantly disrupting the binding of the protein to the phospholipid vesicles. The phosphorylation of annexin I increased the EC50 (Ca2+) of phospholipid vesicle aggregation from 19 +/- 10 microM (mean +/- S.D., n = 7) for the native protein to 290 +/- 95 microM (mean +/- S.D., n = 5) for the phosphorylated protein. These results suggest that protein kinase C may act to inhibit the phospholipid vesicle aggregation activity of annexin I.

Animals↗
PubMed1992

Phosphorylation of annexin II tetramer by protein kinase C inhibits aggregation of lipid vesicles by the protein.

Annexin II tetramer (A-IIt) is a member of the annexin family of Ca2+ and phospholipid-binding proteins. The ability of this protein to aggregate both phospholipid vesicles and chromaffin granules has suggested a role for the protein in membrane trafficking events such as exocytosis. A-IIt is also a major intracellular substrate of both pp60src and protein kinase C; however, the effect of phosphorylation on the activity of this protein is unknown. In the current report we have examined the effect of phosphorylation on the lipid vesicle aggregation activity of the protein. Protein kinase C catalyzed the incorporation of 2.1 +/- 0.8 mol of phosphate/mol of A-IIt. Phosphorylation of A-IIt caused a dramatic decrease in the rate and extent of lipid vesicle aggregation without significantly effecting Ca(2+)-dependent lipid binding by the phosphorylated protein. Phosphorylation of A-IIt increased the A50%(Ca2+) of lipid vesicle aggregation from 0.18 microM to 0.65 mM. Activation of A-IIt phosphorylation, concomitant with activation of lipid vesicle aggregation, inhibited both the rate and extent of lipid vesicle aggregation but did not cause disassembly of the aggregated lipid vesicles. These results suggest that protein kinase C-dependent phosphorylation of A-IIt blocks the ability of the protein to aggregate phospholipid vesicles without affecting the lipid vesicle binding properties of the protein.

Amino Acids↗
PubMed1992

A nonapeptide to the putative F-actin binding site of annexin-II tetramer inhibits its calcium-dependent activation of actin filament bundling.

A synthetic nonapeptide, Val-Leu-Ile-Arg-Ile-Met-Val-Ser-Arg, corresponding to residues 286-294 of annexin-II tetramer (A-IIt), was shown to completely inhibit the Ca(2+)-dependent bundling of F-actin by this protein. The inhibitory effect of the nonapeptide required preincubation with F-actin and was reversed by the addition of excess A-IIt. Kinetic analysis suggested that the nonapeptide reduced the K(0.5) but not the Vmax of F-actin bundling. In contrast, addition of excess nonapeptide to A-IIt-bundled F-actin did not reverse F-actin bundle formation. Although the nonapeptide produced a dose-dependent inhibition of A-IIt-dependent F-actin bundling, the binding of A-IIt to F-actin was not affected. These results identify a domain of A-IIt that is involved in the bundling activity of the protein and suggest that this domain binds transiently with F-actin, resulting in activation of the bundling activity of A-IIt.

Actins↗
PubMed1992

Enolase is present at the centrosome of HeLa cells.

Antibodies raised against the C-terminus and N-terminus region of gamma gamma enolase, as well as a polyclonal antibody raised against bovine brain gamma gamma enolase, were used to study the distribution of this glycolytic enzyme during the cell cycle in HeLa cells. Enolase was found to be present throughout the cytoplasm of both interphase and dividing cells. In addition, a portion of cellular enolase was detected at the centrosome throughout the cell cycle. The capacity of glycolytic enzymes to play a structural as well as a glycolytic role suggests that the presence of enolase at the centrosome may be correlated with the organization of both the interphase cytoskeleton and the mitotic spindle.

Amino Acid Sequence↗
PubMed1991

Autoantibodies to the centrosome (centriole) react with determinants present in the glycolytic enzyme enolase.

Autoantibodies to cellular Ag are found in the sera of patients with systemic rheumatic diseases. Identification and characterization of the reactive autoantigens has helped clinicians to define subsets of rheumatic diseases and has assisted biologists in defining the function within the cell of these molecules. We have studied autoantibodies from patients that react with the centrosome (centriole) region of the cell. We found by immunoblotting techniques that these antibodies react with a 48-kDa protein. Additional immunoblotting and affinity purification studies indicate that the Ag may be the glycolytic enzyme enolase.

Autoantibodies↗
PubMed1990

Purification and characterization of annexin proteins from bovine lung.

Calcium-dependent association with a detergent-extracted particulate fraction was used as the first step in the purification of a group of phospholipid binding proteins. Elution of the detergent-insoluble fraction with excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) resulted in the release of several soluble proteins, termed calcium-activated proteins or CAPs. In the present paper, we describe the simultaneous purification of these CAPs and characterize their interaction with phospholipid, actin, and calmodulin. Partial sequence analysis has identified the majority of the CAPs as members of the annexin family of calcium and phospholipid binding proteins. Two additional CAPs may be novel proteins, one of which appears to be an annexin protein. All CAPs demonstrated Ca2(+)-dependent binding to phosphatidylserine vesicles but did not bind to phosphatidylcholine vesicles. The majority of CAPs exhibited Ca2(+)-dependent binding to F-actin; however, only CAP-III affected the rate of conversion of G-actin to F-actin. The interaction of CAP-III and lipocortin-85 with F-actin resulted in a Ca2(+)-dependent increase in both light scattering and sedimentation of F-actin under comparatively low centrifugal force. In contrast, only lipocortin-85 caused the formation of F-actin bundles. Although all of the CAPs bound to a calmodulin affinity column in a Ca2(+)-dependent manner, attempts to demonstrate binding of CAPs to native calmodulin were unsuccessful. These studies therefore document the similar behavior of the CAPs toward phospholipid and calmodulin but clearly show that F-actin binding or bundling is not a general property of these proteins. The reported purification procedure should allow further comparative studies of these proteins.

Actins↗
PubMed1990

Calcium-dependent regulation of actin filament bundling by lipocortin-85.

Lipocortin-85 (L-85, calpactin-I/lipocortin-II heterotetramer) binds to F-actin in the presence of calcium with high affinity and in a cooperative manner. Quantitative analysis of binding curves indicate an apparent Kd (L-85) of 0.226 microM +/- 0.153 (2 S.D., n = 3), a stoichiometry of L-85/actin of 1:1.9 and a Hill coefficient of 1.37 +/- 0.14 (2 S.D., n = 3). Large anisotropic bundles were visualized by electron microscopy under these conditions, and quantitation of bundling by both low speed sedimentation and light scattering yielded apparent Kd values between 0.12 and 0.27 microM L-85. Filament bundling was dependent upon calcium, and the calcium sensitivity was increased by raising the molar ratio of lipocortin-85/F-actin. At saturating levels of L-85, apparent K0.5 values of 0.1-2 microM Ca2+f were obtained. The monomeric heavy chain, lipocortin-II, bundled F-actin to a much lesser extent and at much higher concentrations than for lipocortin-85. Bundling of F-actin by lipocortin-I was not detected at molar ratios of lipocortin-I to actin as high as 2.5 mol/mol (lipocortin-I/actin). At 5-10 microM Ca2+f and saturating levels of L-85, F-actin bundling progressed very rapidly with a t0.5 of 6 s. The process was quickly reversed by the addition of excess EGTA, and bundles could be reformed by the addition of a second burst of 5-10 microM Ca2+f. Thus, our data suggest that lipocortin-85 can rapidly regulate F-actin bundling in a calcium-dependent manner at physiologically relevant calcium levels.

Actin Cytoskeleton↗
PubMed1990

Are many Z-DNA binding proteins actually phospholipid-binding proteins?

We used a Z-DNA affinity column to isolate a collection of Z-DNA binding proteins from a high salt extract of Escherichia coli. We identified one of the major Z-DNA binding proteins of this fraction, not as a protein involved in gene regulation or genetic recombination, but rather as an outer membrane porin protein. We then showed that several other known phospholipid-binding proteins (bovine lung annexins and human serum lipoproteins) also bind much more tightly to Z-DNA than to B-DNA. In all cases, this Z-DNA binding was strongly blocked by competition with acidic phospholipids, such as cardiolipin. Our results raise the question whether many of the Z-DNA binding proteins previously isolated are actually phospholipid-binding proteins.

Carrier Proteins↗
PubMed1989

The purification of a 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs.

The 100,000 g supernatant from the unfertilized egg of the sea urchin Strongylocentrotus purpuratus has been fractionated on DEAE-cellulose and analysed for Ca2+-binding activity by the Chelex-100 competitive Ca2+-binding activity assay. The major peak of Ca2+-binding activity was subjected to further purification and the Ca2+-binding protein responsible for this Ca2+-binding-activity peak has been isolated and characterized. Non-denaturing polyacrylamide-gel electrophoresis (PAGE) followed by 45Ca2+ autoradiography suggested a molecular mass of 80 kDa for the Ca2+-binding protein. SDS/PAGE revealed that the 80 kDa protein consisted of a 1:1 molar complex of proteins of 50 and 42 kDa. The 42 kDa protein was identified as actin. The complex was not dissociated by extensive dialysis against an EGTA-containing buffer. The EGTA-stable complex was named '50K-A'.

Actins↗
PubMed1989

The 50 kDa protein-actin complex from unfertilized sea-urchin (Strongylocentrotus purpuratus) eggs. Interaction with actin.

In the preceding paper [Golsteyn & Waisman (1989) Biochem. J. 257, 809-815] an EGTA-stable, Ca2+-binding heterodimer comprised of a 50 kDa protein and actin called '50K-A' was identified in the unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. In the present paper we have documented the binding of 50K-A to DNAase I and the effect of 50K-A on the kinetics of actin polymerization. When 50K-A was added to pyrene-labelled rabbit skeletal-muscle actin and the salt concentration increased, the initial rate of actin polymerization was inhibited by a very low molar ratio of 50K-A to actin. Furthermore, the steady-state level of G-actin was increased in the presence of 50K-A, suggesting that 50K-A caps the preferred end of actin polymer, shifting the steady-state concentration to that of the non-preferred end. Dilution of F-actin to below its critical concentration into 50K-A resulted in a much slower rate of depolymerization, consistent with capping of the preferred end. In contrast with the Ca2+-dependent binding to DNAase, the effect of 50K-A on the kinetics of actin assembly and disassembly was Ca2+-independent. These results suggest that 50K-A is a novel actin-binding protein with some similarities to the severin/fragmin/gelsolin family of F-actin-capping proteins.

Actins↗
PubMed1987

Identification of a new in vitro substrate of tyrosine protein kinase.

Recent studies in our laboratory [Tokuda, M., Khanna, N.C., Aurora, A., & Waisman, D. M. (1986) Biochem. Biophys. Res. Commun. 139, 910-917] have identified in membranes of rat spleen two tyrosine protein kinases named TPK-I and TPK-II. In this paper the identification of the Ca2+ binding protein CAB-48 as a major in vitro substrate of TPK-II is reported. TPK-II catalyzed the incorporation of 0.73 mol of phosphate/mol of CAB-48. Phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues. Phosphorylation of CAB-48 by TPK-I (rat spleen), protein kinase C, casein kinase I, casein kinase II, cAMP-dependent protein kinase, or calcium calmodulin dependent protein kinase was not observed.

Amino Acids↗
PubMed1987

The 48 kDa Ca2+-binding protein of bovine brain.

A Ca2+-binding protein of molecular mass 48 kDa and named 'CAB-48' has been purified from bovine brain 100,000 g supernatant. About 30 mg of CAB-48 was purified from 1 kg of bovine brain. The protein has been characterized with respect to its physical, chemical and Ca2+-binding properties. It has an apparent molecular mass of 48 kDa by SDS/polyacrylamide-gel-electrophoresis and 75.2 kDa from sedimentation-velocity and Stokes-radius data. The acidic nature of the molecule is suggested by its pI of 4.7. In the presence of 3.0 mM-MgCl2 and 150 mM-KCl, CAB-48 binds 1.0 mol of Ca2+/mol of protein with an apparent Kd of 15 microM. A tyrosine protein kinase partially purified from rat spleen catalysed the incorporation of 0.73 mol of phosphate/mol of CAB-48, and phosphoamino acid analysis revealed that phosphorylation of CAB-48 was specific for tyrosine residues.

Amino Acids↗
PubMed1987

Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain.

A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.

Amino Acids↗