Stanley M. Goldin

A superfusion system designed to measure release of radiolabeled neurotransmitters on a subsecond time scale.

A new method for subsecond measurement of release of neurotransmitters from nerve terminal preparations (e.g., synaptosomes) in vitro is described. Synaptosomes were prelabeled with [3H]GABA via a Na-dependent GABA uptake system. The prelabeled nerve terminals are retained on small glass fiber filters in a superfusion chamber accessed by three high speed, solenoid-driven valves. Microcomputer-programmed circuitry controls the timing of valve operation. Each valve controls the delivery of a separate solution to the chamber, permitting rapid and independent control of membrane potential, [Ca2+]e, and drug delivery. The minimal dead volume of the chamber and the relatively high solution flow rate afford time resolution for release of at least 60 ms. This time resolution was necessary to observe the most rapid of at least three components of GABA release.

Characterization and synthesis of a new calcium antagonist from the venom of a fishing spider

Abstract A new calcium antagonist, CNS 2103, is isolated from the venom of a fishing spider, Dolomedes okefinokensis . The structure of this compound is derived from spectroscopic data, including tandem mass spectrometry. A flexible, convergent synthesis of CNS 2103 is described.

[3] Transport-specific fractionation for purification of ATP-dependent Ca2+ pumps

Publisher Summary This chapter discusses transport-specific fractionation, which is a means for the identification of membrane transport proteins and as a purification procedure. The method depends on a specific biological property of a protein for isolation and employs a transport activity of the protein (rather than a ligand-binding property as would be used in affinity chromatography) as a physical tool for its purification. The most substantial success of this method in terms of a degree of purification has been its application by several laboratories to the purification of Ca 2+ -transport proteins, notably ATP-dependent Ca 2+ pumps and the Na + /Ca 2+ exchanger. The chapter discusses the methodology for the purification of reconstituted ATP-dependent Ca 2+ pumps from mammalian brain synaptosomes. Two critical variables to consider when solubilizing the Ca 2+ -ATPase for use in transport-specific fractionation are the phospholipid-to-protein ratio and the cholate-to-phospholipid ratio.

Biochemical and immunological evidence for a calcium pump in chromaffin granules.

ATP stimulated the accumulation of 45Ca2+ by chromaffin granule ghosts which contained 5 mM oxalate to trap transported calcium within the lumen. Inasmuch as the ATP-dependent 45Ca2+ transport was resistant to 25 mM ammonium acetate as well as the proton ionophore, carbonylcyanide-m-chlorophenylhydrazone, the chromaffin granule proton translocating ATPase does not provide the energy for this process. Instead, we suggest that chromaffin granules contain a calcium translocating ATPase which catalyzes the 45Ca2+ uptake directly. The observation that chromaffin granules bind to a monoclonal antibody raised against a calcium pump from bovine brain supports this hypothesis.

SYNAPTOSOME-DERIVED Ca TRANSPORT SYSTEMS: PROPERTIES AND PURIFICATION

Publisher Summary The regulation of intracellular calcium ion, [Ca], concentration in nerve terminals and its modulation helps to understand the neurotransmitter release process and the entire sequence of synaptic transmission. Small fluctuations in the free [Ca] of nerve terminals can cause large changes in the amount of neurotransmitter released. This chapter discusses some of the results obtained in study with calcium transporting molecules isolated from the rat brain synaptosome and their properties as determined in their native membrane and in the new phospholipid membrane into which they have been reconstituted. In the study, the transport specific fractionation technique had been used for reconstitution and purification of three calcium transport systems. One appears to be derived from internal membraneous structures—synaptosomal vesicles. The other two are from the synaptic plasma membrane: a Na dependent Ca transporter and an adenosine triphosphate (ATP) dependent Ca pump.