Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram

Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7.     

Interaction between the Component Enzymes of the Glycine Decarboxylase Multienzyme Complex

The glycine decarboxylase multienzyme complex comprises about one-third of the soluble protein of the matrix of pea (Pisum sativum) leaf mitochondria where it exists at a concentration of approximately 130 milligrams protein/milliliter. Under these conditions the complex is stable with an approximate subunit ratio of 2 P-protein dimers:27 H-protein monomers:9 T-protein monomers:1 L-protein dimer. When the complex is diluted it tends to dissociate into its component enzymes. This prevents the purification of the intact complex by gel filtration or ultracentrifugation. In the dissociated state the H-protein acts as a mobile cosubstrate that commutes between the other three enzymes and shows typical substrate kinetics. When the complex is reformed, the H-protein no longer acts as a substrate but as an integrated part of the enzyme complex.     

Evidence for a role of high Km aldehyde reductase in the degradation of endogenous gamma-hydroxybutyrate from rat brain

gamma-Hydroxybutyrate (GHB) is a putative neurotransmitter in brain. We have already demonstrated that it is transformed into gamma-aminobutyrate (GABA) by rat brain slices incubated under physiological conditions. This conversion occurs via a GABA-transaminase reaction. Therefore, succinic semialdehyde, the oxidative derivative of GHB, appears to be the primary catabolite of GHB degradation. Apparently, the kinetic characteristics and pH optimum of GHB dehydrogenase (high Km aldehyde reductase) in vitro do not favor a role for this enzyme in endogenous brain GHB oxidation. However, in the presence of glucuronate, glutamate, NADP and pyridoxal phosphate, pure GHB dehydrogenase, coupled to purified GABA-transaminase does produce GABA from GHB at an optimum pH close to the physiological value and with a low Km for GHB.     

Extreme weather‐year sequences have nonadditive effects on environmental nitrogen losses

The frequency and intensity of extreme weather years, characterized by abnormal precipitation and temperature, are increasing. In isolation, these years have disproportionately large effects on environmental N losses. However, the sequence of extreme weather years (e.g., wet–dry vs. dry–wet) may affect cumulative N losses. We calibrated and validated the DAYCENT ecosystem process model with a comprehensive set of biogeophysical measurements from a corn–soybean rotation managed at three N fertilizer inputs with and without a winter cover crop in Iowa, USA. Our objectives were to determine: (i) how 2‐year sequences of extreme weather affect 2‐year cumulative N losses across the crop rotation, and (ii) if N fertilizer management and the inclusion of a winter cover crop between corn and soybean mitigate the effect of extreme weather on N losses. Using historical weather (1951–2013), we created nine 2‐year scenarios with all possible combinations of the driest (“dry”), wettest (“wet”), and average (“normal”) weather years. We analyzed the effects of these scenarios following several consecutive years of relatively normal weather. Compared with the normal–normal 2‐year weather scenario, 2‐year extreme weather scenarios affected 2‐year cumulative NO₃^? leaching (range: ?93 to +290%) more than N₂O emissions (range: ?49 to +18%). The 2‐year weather scenarios had nonadditive effects on N losses: compared with the normal–normal scenario, the dry–wet sequence decreased 2‐year cumulative N₂O emissions while the wet–dry sequence increased 2‐year cumulative N₂O emissions. Although dry weather decreased NO₃^? leaching and N₂O emissions in isolation, 2‐year cumulative N losses from the wet–dry scenario were greater than the dry–wet scenario. Cover crops reduced the effects of extreme weather on NO₃^? leaching but had a lesser effect on N₂O emissions. As the frequency of extreme weather is expected to increase, these data suggest that the sequence of interannual weather patterns can be used to develop short‐term mitigation strategies that manipulate N fertilizer and crop rotation to maximize crop N uptake while reducing environmental N losses.