Nerve growth factor stimulates tyrosine phosphorylation and activation of Src homology-containing protein-tyrosine phosphatase 1 in PC12 cells.

Rat PC12 cells respond to extracellular peptide growth factors in at least two distinct ways. When treated with nerve growth factor (NGF) PC12 cells exit the cell cycle and differentiate to a neuronal phenotype, whereas when treated with epidermal growth factor, they proliferate. We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatases (PTPs) in the differentiation process. PC12 cells express substantial amounts of both SH-PTP1 and 2. SH-PTP1, but not SH-PTP2, becomes tyrosine phosphorylated following NGF, but not epidermal growth factor treatment. The enzymatic activity of SH-PTP1 toward an exogenous substrate following NGF treatment is increased 2-fold. We found that SH-PTP1 binds to the NGF receptor TrkA in vitro and that anti-TrkA immunoprecipitates have PTP activity. These results show that SH-PTP1 is differentially phosphorylated and activated by NGF in PC12 cells and suggest that this activation may play a role in NGF-induced differentiation.

Isolation and partial characterization of calcium-activated chloride ion channels from thylakoids.

The goal of the present work was to characterize the thylakoid anion channel. Toward this purpose a new, more sensitive, assay was developed to measure chloride channel activity of solubilized and partially purified thylakoid membrane proteins. In this assay potassium channel function was reduced or eliminated by the use of cesium as the coion of chloride. Under these conditions calcium but not magnesium stimulated anion uptake, suggesting the presence of a calcium-activated anion channel in isolated thylakoid membrane proteins. A polyclonal antibody raised against an anion channel isolated from epithelial membranes immunoreacted with three thylakoid membrane proteins; a doublet in the 62 to 66-kDa range and a single band in the 57 to 59-kDa region suggesting that the thylakoid anion channel may be homologous to the mammalian chloride channel. Furthermore, the cDNA which encodes the epithelial chloride channel hybridized on Northern blots prepared from corn total RNA with three mRNA bands.

Identification and characterization of the developmentally regulated pattern of expression in the testis of a mouse gene exhibiting similarity to the family of phosphodiesterases.

A cDNA for a rat brain phosphodiesterase (PDE) was used to screen a mouse testis library to identify the murine PDEs which are expressed in this tissue. A clone of 981 bp, p4-6, was isolated and shown to exhibit limited identity at the amino acid level to the rat brain PDE (20%). The putative protein encoded by clone p4-6 also contains multiple potential modification sites, for phosphorylation, myristylation, and glycosylation, many of which are located at positions similar to those found for rat brain PDE. The gene identified by p4-6 yields 3 transcripts, an abundant 1.9 kb transcript, and less abundant transcripts of 3.8 and 6.7 kb. Of the nine tissues examined in this study, the expression of the corresponding gene was limited to the adult mouse testis. Furthermore, the expression in the testis was most abundant in the germ cell lineage, although low levels were detected in somatic cells of the testis as well. Analysis of RNA from testes at different stages of development suggested that the p4-6 gene is most abundantly expressed in germ cells that have completed the meiotic divisions.

Measurements of K+-driven Cl- uptake in thylakoids: inhibition of uptake by antibodies raised to the major polypeptides of the Cl(-)-efflux active particle(s).

The mechanism of a K+-driven Cl- accumulation against a concentration gradient was investigated by flow dialysis after addition of K+-Hepes. Non-specific chloride binding, measured in the presence of choline-Hepes, accounted for approximately 50% of the observed uptake in this system. The K+-Hepes-driven Cl- uptake was inhibited by poly-l-lysine and by two antibodies raised to the major polypeptides of the Cl(-)-efflux active particle. Poly-l-lysine had no effect on Cl- binding estimated with choline-Hepes.

Anion (and cation) requirements of the coupled electron flow in spinach thylakoids.

Proton uptake as well as coupled electron flow of chloroplasts swollen in dilute impermeable buffers became dependent upon the addition of exogenous permeable anions. This dependence was observed with both cyclic and noncyclic electron acceptors, suggesting that this anion requirement is associated with the electrogenic proton uptake step rather than with the oxygen-evolving reactions of photosystem II.

Isolation of protein(s) containing chloride ion transport activity from thylakoid membranes.

Extracts of detergent-treated thylakoids, when reconstituted into azolectin/cholesterol/dicetyl phosphate vesicles, stimulate chloride ion efflux as measured with a Cl--sensitive electrode. This stimulation is inhibited by piretanide, an active chloride ion transport inhibitor in fish intestinal epithelia. This stimulation is also abolished by pretreatment of extracted proteins with trypsin. Antiserum raised to efflux-active proteins inhibits a cation-driven Cl- influx in nonenergized thylakoids, as measured by a flow dialysis technique.

Chloride ion transport and its inhibition in thylakoid membranes.

Cl- translocation across energized and nonenergized thylakoid membranes was found to be inhibited by piretanide, an inhibitor of active Cl- transport in fish intestinal epithelia. Piretanide has no effect on photophosphorylation catalyzed by phenazine methosulfate or on Ca2+-dependent ATPase activity of isolated chloroplast coupling factor (CF1). Light-dependent Cl- uptake, contrary to H+ uptake, is severalfold greater at pH 8.0 than at pH 6.7.

AMP photophosphorylation and binding by chloroplasts.

Stoichiometric amounts of chloroplast thylakoids photophosphorylate free AMP to tightly bound ADP. Free ADP is a poor competitor for this AMP photoreaction, which saturates below 16 micronAMP. The inhibitor, diadenosine pentaphosphate, abolishes AMP photophosphorylation, and inhibits dark ADP binding. Taken together, these data imply that this photoreaction involves the high affinity nucleotide binding site(s) of chloroplast coupling factor CF1, and that little mixing with free nucleotides occurs.

Delayed light studies on photosynthetic energy conversion. VIII. Evidence from millisecond emission of chloroplasts for two adenylate binding sites on membrane-bound coupling factor, CF1.

Effects of adenylates on chloroplast delayed light emission, at millisecond dark times, are inverse to the previously characterized effects of adenylates on electron transport rates. Either ADP alone or ATP alone increase intensity of delayed light, while ADP plus Pi decrease it. ADP alone requires the presence of an electron acceptor to have this effect on delayed light, but ATP does not. All three adenylate effects are abolished by uncoupling with gramicidin, by partial removal of photophosphorylation coupling factor (CF1) with EDTA, and by antibody to CF1. Readdition of CF1 re-established the adenylate effects in EDTA-stripped membranes. The three adenylate effects are differentially sensitive to pH, and pH differentially affected their abolition by antibody to CF1. The two adenylate effects shown in the absence of Pi are exhibited at lower adenylate concentrations than the ADP plus Pi effect, and are also less sensitive to phloridzin. These results are discussed in terms of probable adenylate effects on membrane-bound chloroplast coupling factor, CF1. At least two ADP binding sites would differ with respect to adenylate concentration for half maximal binding; pH of optimal binding capacity; phloridzin sensitivity; and functional regulation of electron transport, proton uptake, and energy storage within the membrane as measured by delayed light emission. It remains unclear whether the high affinity ADP binding site is identical to a high affinity ATP binding site on CF1.