Stoichiometric binding of diacylglycerol to the phorbol ester receptor

The major phorbol ester receptor is the Ca++-activated, phospholipid-dependent protein kinase C. Diacylglycerol stimulates protein kinase C in a fashion similar to the phorbol esters. Likewise, it inhibits phorbol ester binding competitively. Both results suggest that diacylglycerol is the/an endogenous phorbol ester analogue. Alternatively, the diacylglycerol might simply be acting to modify the phospholipid environment of the protein. If diacylglycerol were indeed functioning as an analogue, it should interact with the receptor stoichiometrically. This interaction can be quantitated by measuring the perturbation in apparent diacylglycerol binding affinity as a function of the ratio of diacylglycerol to receptor. We report here that 1,2-dioleoylglycerol interacts with the receptor with the predicted stoichiometry.     

Triterpenes from Periandra dulcis roots

Three oleanane triterpenes were isolated from the roots of Periandra dulcis,and identified as 3β-hydroxy-25-al-olean-18-en-30-oic acid (periandric acid I), 3β-hydroxy-25-al-olean-12-en-30-oic acid (periandric acid II) and 3-oxo-25-hydroxy-olean-12-en-30-oic acid. The former two compounds (periandric acids I and II) were identical with the aglycones obtained by hydrolysis of periandrin I and II, respectively and the latter one was a new triterpene.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

Characterization of chlorophyll fluorescence quenching in chloroplasts by fluorescence spectroscopy at 77 K II. ATP-dependent quenching

In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 3}\text{min}$) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F₇₃₅) and a decrease in PS II fluorescence (F₆₉₅) were observed in the low-temperature spectra. The change in the $\text{F}{}_{735}\text{F}{}_{695}$ ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (F_v) and initial (F₀) fluorescence at 690 nm and an increase in F₀ at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.     

Rabbit kidney function in vitro: the effect of colloids, energy substrate, a vasodilator, perfusion pressure, and bovine serum albumin

An in vitro perfusion system at 37 degrees C for the assessment of rabbit kidney function is described. The purpose of this assay system is to evaluate the effects of cryobiological manipulation on kidney function. The effect of the colloids dextran (MW = 70,000, 80,000, and 180,000) in the perfusate at 110 mm Hg were compared to a reduced perfusion pressure, colloid-free perfusate. Better function was obtained at lower perfusion pressure with the colloid-free perfusate. Less damage was noted histologically on light and electron microscopy. Investigation of energy substrates on rabbit kidney function demonstrated that butyrate, or lactate, in addition to glucose resulted in increased sodium and glucose reabsorption over glucose alone. Substrate-free perfused kidneys exhibited depressed Na transport. Lactate, and to some extent butyrate, decreased net glucose utilization. An alpha-adrenergic blocking agent, isoxsuprine, in the initial flush solution did not appear to be beneficial. An increase of perfusion pressure from 50 to 75 mm Hg resulted in an increase in GFR. Tubular function was enhanced by inclusion of small amounts of BSA in the perfusate.     

Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

The use of amphipathic maleimides to study membrane-associated proteins

A series of amphiphilic polymethylenecarboxymaleimides has been synthesized for use as sulfhydryl reagents applicable to membrane proteins. Physical properties of the compounds which are relevant to their proposed mode of action have been determined. By comparing rates of reaction in aqueous and aprotic solvents, the compounds have been shown to react exclusively with the thiolate ion. The effects of the reagents on three membrane-associated proteins are reported, and in two cases a comparative study has been made of the effects on the proteins in the absence of membranes. A mechanism is proposed whereby the reagents are anchored at the lipid/water interface by the negatively charged carboxyl group, thus siting the reactive maleimide in a plane whose depth is defined by the length of the reagent. Supporting evidence for this model is provided by the inability of the reagents to traverse membranes, and variation of their inhibitory potency with chain length when the proteins are embedded in the membrane, but not when extracted into solution. As examples of general use of the reagents to probe sulfhydryl groups in membrane proteins, the reagents have been used to (a) determine the depths in the membrane at which two populations of sulfhydryl groups occur in the mitochondrial phosphate transporter; (b) locate a single sulfhydryl associated with the active site ofD--hydroxybutyrate dehydrogenase in the inner mitochondrial membrane; (c) examine sulfhydryl groups in theD-3-glyceraldehyde phosphate dehydrogenase associated with the human red blood cell membrane.