In vivo cGMP levels in frog photoreceptor cells as a function of light exposure.

By employing a combination of highly sensitive radioimmunoassays and histochemical techniques, an in vivo time course of cGMP levels has been determined in the outer segment, photoreceptor cell and outer plexiform layers of frog retina. Frogs (Rana pipiens) were dark-adapted overnight and either frozen rapidly (approximately 3 sec) in liquid nitrogen or exposed to periods of light varying between 0.1 sec and 2 hr before freezing. Frozen retinal sections were cut, freeze-dried, and samples of individual layers dissected out and analysed for cGMP. In the outer plexiform layer, there was a 42% drop in cGMP concentration after 2 sec of light (250 ft candles) followed by a 34% rise after 2 min; a steep concentration gradient formed around the layer after the 2 min exposure. In both the outer-segment layer and photoreceptor-cell layer (which includes outer segments, inner segments and outer nuclear layers), cGMP levels declined from a dark value of 56 mumol kg-1 (dry) to 9 mumol kg-1 (dry) as a result of increasing exposure to several types of light source: levels appear to be primarily a function of total ft candle min. Cyclic GMP concentrations at the longest exposures (2 min with a fiber optic light source or 2 hr with fluorescent room light) reached identical minimum levels. In the outer segments, a 15% decrease in cGMP was observed after 0.1 sec of light exposure. Although the freezing time is too long to be able to say whether the 15% decrease in cGMP at the 0.1 sec exposure is involved in transduction, the low identical levels reached gradually after longer exposures appear to indicate that a light-induced biochemical adjustment in cGMP metabolism occurs over a relatively long time period separate from the msec time course of the transduction process.

Adenine nucleotide and P-creatine levels in layers of frog retina as a function of dark and light adaptation.

Eight layers of frog retina were analyzed for ATP, P-creatine, ATP + ADP, and AMP under conditions of dark, 2 sec, 2 min, and in the case of AMP, 2 hr of light adaptation. Samples of each layer, usually ranging between 5 and 50 ng, were dissected from lyophilized frozen sections. After brief light exposure, ATP dropped while ADP rose sharply in the pigment epithelium, outer segments, and inner segments; ADP was too low to be measured accurately in the inner retina. The profile of ATP, P-creatine, and ATP + ADP concentrations showed peaks in the inner nuclear and ganglion layers. AMP, by contrast, was highest in the two plexiform layers. Levels in the inner retina dropped after only 2 sec of light but rose after 2 hr to levels that were higher than dark values in all retinal layers. AMP was often characterized by a non-uniform distribution: adjacent areas of a layer agreed very closely in value to each other but could vary several-fold from a different section of the same frog or from another frog exposed to the same conditions. This distribution produced clusters of values, particularly prominent in dark-adapted animals, something not observed with the other metabolites measured. The peaks of AMP in the plexiform layers suggest that AMP may be a by-product of dopamine-stimulated adenylate cyclase which also has peaks in these same layers.

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas.

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.

An examination of the efficiency of glucose and glutamine as energy sources for cultured chick pigment epithelial cells.

Embryonic chick pigment epithelial cells in culture require glucose as their major energy source for long-term growth, pigment formation, and colony organization. Cell number increases with glucose concentration at least up to 5.0 mM. Cells can be grown with glutamine as the major energy source but produce comparable cell numbers for only the first 3 days in culture, after which they cease growing. However, they are able to metabolize glutamine at a two to sixfold higher rate than cells grown in the presence of glucose as measured by CO2 release and by incorporation into protein. In cells grown in the presence of both glucose and glutamine, basal ATP levels were 31.1 nmoles/mg protein; P-creatine averaged 15.2 nmoles/mg protein and showed marked variability between experimental groups. During starvation, P-creatine levels fell while ATP levels remained relatively constant. Glucose was required for the recovery of P-creatine to prestarvation levels when measured 5 min after refeeding. Because of these marked changes in P-creatine concentration as a function of nutritional status, the ATP/P-creatine ratio becomes a useful measure of the energy state of the cell.

Characterization of polynucleotide phosphorylase from Micrococcus luteus and isolation of the 13,000 base poly(A) product of the polymerization reaction.

A new purification procedure for polynucleotide phosphorylase from freeze-dried Micrococcus luteus cells gives approximately 20% yield of nearly homogeneous, primer-independent enzyme which is free of nucleic acid. The physicochemical properties of M. luteus polynucleotide phosphorylase are similar to those previously described for the enzyme from Escherichia coli in terms of Mr, subunit structure, and amino acid composition. The purified enzyme appears to be a trimer composed of three identical subunits (Mr 92,000), but it probably does not exist as such in the cell. Ferguson plot analyses of enzyme in cell extracts indicate that prior to purification the enzyme exists in oligomeric forms characterized by both higher charge and greater Mr. Changes in size and charge of oligomers which occur during purification are probably due to the dissociation of proteins and/or nucleic acids. Dissociation of the oligomers is achieved by dilution and electrophoresis, but reassociation does not occur after concentration. The poly(A) product of the initial polymerization stages migrates as a single band on both nondenaturing and urea-agarose gels. It is 13,000 +/- 2,000 nucleotides long, as measured by electron microscopy, and 8,000 nucleotides long by gel electrophoretic analysis. This poly(A) product remains bound to the enzyme after synthesis, yet can be easily obtained free of protein by proteinase K digestion.

Measurement of metabolites in single preimplantation embryos; a new means to study metabolic control in early embryos.

Methods are described for preparing and analyzing single preimplantation mouse embryos for a variety of metabolites and cofactors (glucose-6-P, fructose-6-P, fructose-1,6-bisphosphate, ATP, AMP, Pi, citrate, isocitrate, alpha-ketoglutarate, and malate). Oil-well and enzymatic cycling techniques are combined to provide the sensitivity needed to measure the amounts present (10(-12) to 1o(-15) moles). After experimental treatment, embryos are collected on glass slides and freeze-dried. They can then be stored indefinitely under vacuum at -25 degrees C without deterioration. With these procedures, the embryos were collected at successive stages of development and subjected to starvation and refeeding with glucose, pyruvate or both. The results confirm the existence of a block at early stages at the P-fructokinase step. This may be due to inhibition by the very high citrate levels present. The data suggest that glycolysis is turned on late in preimplantation development by the rise in fructose-6-P, a deinhibitor of P-fructokinase. In the citrate cycle, no step between citrate and alpha-ketoglutarate is rate-limiting, but a step between alpha-ketaglutarate and malate appears to impede the flux at early embryonic stages.

The explanation for the blockade of glycolysis in early mouse embryos.

The reason for the failure of early-stage mouse embryos to grow on glucose alone was investigated by measurement of glucose-6-phosphate, fructose-1,6-bisphosphate plus triose phosphates, citrate, and malate in individual embryos during starvation and refeeding with glucose or glucose plus pyruvate. The results indicate a block at the 6-phosphofructokinase (EC 2.7.1.11) step at early stages, which is later removed. Although there seems to be no early difficulty in phosphorylation of glucose, maximum glucose-6-phosphate levels (and probably fructose-6-phosphate levels) are much lower at early stages than at later stages. The increase in fructose-6-phosphate with age may be the major cause of the increase in 6-phosphofructokinase activity. Unusually high citrate levels at all ages may help to keep this enzyme strongly inhibited until the increase in fructose-6-phosphate occurs. The changes in metabolite levels also indicate an early defect in mobilization of glycogen and a probably less important defect in the citrate cycle.