Saul Puszkin

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.

Characterization of Brain Synaptic Vesicle Phospholipase A2 Activity and Its Modulation by Calmodulin, Prostaglandin E2, Prostaglandin F2α, Cyclic AMP, and ATP

Abstract: Brain synaptic vesicle phospholipase A2 (PLA2) activity was characterized. It is Ca2+‐dependent and has a pH optimum of 9.0. The enzyme has a Km of 60 μM and a Vmax of 2.0 nmol/mg/h. Calmodulin, prostaglandin F2α, and cAMP, and ATP all increased the Vmax of the enzyme. Prostaglandin E2 inhibited the Vmax in the presence or absence of calmodulin. Light‐scattering techniques in conjunction with phase‐contrast and electron microscopy demonstrated that an increase in Vmax of PLA2 was correlated with synaptic vesicle aggregation, lysis, and possible fusion. In vitro synaptic vesicle‐vesicle association that was stimulated by conditions that in creased PLA2 activity could be diminished when synaptic vesicles were preincubated with PLA2 inhibitors. It is suggested that endogenous synaptic vesicle PLA2 activity may be an important mechanism underlying Ca2+ ‐mediated neurotransmitter release.

Regulation of endogenous calcium-dependent synaptic membrane phospholipase A2.

Synaptic plasma membrane preparations from brain tissue have endogenous Ca2+-dependent phospholipase A2 activity. Characterization of this activity revealed that it was maximally active at 10(-7)-10(-5) M Ca2+ and pH 7.0. The enzyme had a Km of 62.0 microM and a Vmax of 98.0 nmol/mg/h. Calmodulin and prostaglandin F2 alpha stimulated phospholipase A2 activity, whereas prostaglandin E2, cyclic AMP and ATP were inhibitory. Addition of exogenous phospholipase A2 to synaptic plasma membrane and synaptic vesicle preparations led to their disruption and/or lysis. We suggest that Ca2+-dependent regulation of phospholipase A2 activity may be required for synaptic vesicle and synaptic plasma membrane interaction.

Erythrocyte actin and spectrin. Interactions with muscle contractile and regulatory proteins.

Actin and spectrin were isolated from washed red blood cell membranes. Spectrin bound and polymerized erythrocyte actin in the absence of potassium. Spectrin coated into polystyrene latex particles bound 8--9 mol of erythrocyte actin per mol of spectrin when actin was in its depolymerized state. Spectrin enhanced the interaction of erythrocyte actin with muscle myosin as manifested by changes in Mg2+-ATPase activity. A similar enhancement also was observed with muscle alpha-actinin while muscle tropomyosin abolished these effects. The data suggest that spectrin may play the role of polymerizing factor as well as the anchoring site for erythrocyte actin just as alpha-actinin is the anchoring site for actin filaments in muscle and other non-muscle cells.

Compartmentalization of adriamycin and daunomycin in cultured chick cardiac myocytes. Effects on synthesis of contractile and cytoplasmic proteins.

The affinity of two anthracycline antineoplastics ([MC]adriamycin and [14C]daunomycin) for cultured embryonic chick heart cells was determined by measuring their uptake, compartmentalization into subcellular fractions, and effects on the synthesis of cytoplasmic and cytoskeletal proteins. Both drugs, at micromolar concentrations, were readily uptaken by myocytes and found to be concentrated in a light-buoyant-density fraction containing no lysosomes. Nuclear UC drug content accounted for 20–25% of the drug incorporated. Binding of adriamycin was saturable within 90 minutes of drug exposure, and the uptake of [14C]adriamycin was inhibited 50% by verapamil and adenosine triphosphate. Uptake of [uC]daunomycin was not influenced by these compounds. Cytosolic and contractile protein synthesis measured by [35S]methionine incorporation into proteins was blocked 70% overall in each fraction after 6 hours of incubation with 2 jtM adriamycin. Sodium dodecyl sulfate-polyacrylarrude gel electrophoresis followed by autoradiography and quantitative densitometry, revealed that actin synthesis was the least affected of the major proteins. Cardiac myocytes incubated for short periods of time with 2 JZM adriamycin revealed subtle cytoplasmic changes in their organelles with the appearance of clear zones of cytoplasm containing short unorganized microfilaments. The deleterious effects of anthracyclines in heart cells are manifested early by rapid drug incorporation into myocytes and inhibition of cytoplasmic protein synthesis.

Phosphorylation of brain synaptic and coated vesicle proteins by endogenous Ca2+/calmodulin- and cAMP-dependent protein kinases.

Abstract: Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5‐15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity‐purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin‐dependent protein kinase. Affinity‐purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin‐dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP‐dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.

Phosphorylation of Tubulin by Casein Kinase II Regulates Its Binding to a Neuronal Protein (NP185) Associated with Brain Coated Vesicles

Abstract: We recently described a new protein associated exclusively with neuronal clathrin‐coated vesicles (CCVs), and characterized two monoclonal antibodies that react with it (S‐8G8 and S‐6G7). In this report, the association of neuronal protein of 185 kilodaltons (NP185) with CCV kinases and its interaction with tubulin are described. The affinity of NP185 for tubulin is significantly enhanced when tubulin is phosphorylated by CCV‐associated casein kinase II. In contrast, phosphorylation of tubulin by a kinase activity associated with purified brain tubulin decreases its affinity for NP185. Together, these data suggest that the interaction of NP185 with tubulin is modulated by protein phosphorylation. Recent evidence has suggested that tubulin is phosphorylated by casein kinase II during neurite development. The enhanced affinity of NP185 for tubulin phosphorylated by casein kinase II could be important for proper intracellular sorting of this protein in the developing neuron.

Calmodulin Affinity for Brain Coated Vesicle Proteins

Abstract: A systematic characterization of the affinity of calmodulin for brain coated vesicles was undertaken. Binding of [125I‐labeled calmodulin to coated vesicles was saturable and competed with unlabeled calmodulin, but not with troponin‐C. Scatchard analysis revealed one high‐affinity, low‐capacity binding site, KD= 3.9 ± 0.6 nM, Bmax= 16.3 ± 2.4 pmol/mg, and one low‐affinity, high‐capacity binding site, KD= 102 ± 15.0 nM, Bmax= 151 ± 23.0 pmol/mg. Radioimmunoassay revealed that coated vesicles contain 1.05 μg calmodulin/mg protein. Because this value remained constant even after removal of clathrin, the major coat protein, from the coated vesicle, it is apparent that calmodulin is associated with the vesicle per se rather than with its clathrin lattice. When a Triton X‐100‐treated extract of coated vesicles was passed through a Sepharose 4B‐calmodulin affinity column, polypeptides with Mrs (molecular weights) of 100,000, 55,000, and 30,000 bound in a Ca2+‐dependent manner. A 30,000 Mr, protein doublet purified from coated vesicles was completely eluted by EGTA from the calmodulin affinity column, confirming that this protein doublet represents one of the coated vesicle calmodulin binding sites. Because calmodulin stimulated [Ca2+‐Mg2+]‐ATPase activity as well as Ca2+ uptake in coated vesicles, it is postulated that the 100,000 and 55,000 Mr calmodulin binding proteins represent the [Ca+2‐Mg2+]‐ATPase complex, the other coated vesicle calmodulin binding site.

Brain clathrin. Viscometric and turbidimetric properties of its ultrastructural assemblies.

Clathrin was isolated in highly purified form from bovine brain preparations rich in coated vesicles and by some improvements of our previous procedures. At pH 7.5, clathrins solution was viscous, but clear. At pH 6.5, clathrins solution was less viscous, but turbid. By electron microscopy, clathrins turbidity at pH 6.5 correlated with the presence of numerous basket-like lattices or cages; the higher viscosity observed at pH 7.5 correlated with a mixture of various polymeric forms of clathrin having linearly assembled filaments or filamentous bundles of cross-linked clathrin molecules. In vivo, clathrins capacity for assembling or disassembling itself into baskets or cage-like structures is compatible with a mechanism that retrieves areas of the plasma membrane containing protein molecules, smaller stimulatory or inhibitory compounds bound on the external cell membrane surface.

Effects of ADP, ethanol and acetaldehyde on the relaxing complex of human muscle and its adsorption by polystyrene particles☆

Abstract The ATPase activity and interaction of actin with myosin were altered by ADP, ethanol, and acetaldehyde in partially purified human muscle actomyosin. By contrast, reconstituted actomyosin from purified actin and myosin did not exhibit a similar response to these compounds. The addition of regulatory proteins, tropomyosin and the troponin complex, under controlled conditions, restored the response to ADP, ethanol, and acetaldehyde. When tropomyosin and/or troponin were adsorbed on the surface of polystyrene Lytron particles, ADP, ethanol, and acetaldehyde were found to influence calcium binding of the regulatory complex by altering the number of calcium binding sites. These results may explain, at least in part, the physiological effects of these agents on the contractile protein system of muscle.