Eric S. Holdsworth

The effect of vitamin D on enzyme activities in the mucosal cells of the chick small intestine.

SummaryA search was made for enzyme activities that are increased after vitamin D treatment of rachitic chicks. Three enzyme activities located in the brush borders of the mucosal cells of the intestine — ATPase, p-nitrophenyl phosphatase, and pyrophosphatase — were found to approximately double in activity 48 hr after vitamin D was given. The ATPase and the p-nitrophenyl phosphatase required Mg++ for activity but could be further stimulated by addition of Ca++. The three activities are probably caused by the same enzyme since 20 mM phenylalanine inhibited all three activities. It is unlikely that the Ca++-stimulated ATPase is concerned with Ca++ translocation since phenylalanine, which inhibits this enzyme, had no effect on45Ca transport from mucosal to serosal fluids of everted sacs of intestine.

Enzymes concerned with β-carboxylation in marine phytoplankter: Purification and properties of phosphoenolpyruvate carboxykinase

Abstract The enzyme resonsible for β-carboxylation, with eventual incorporation of CO2 into amino acids, has been studied in three diatoms and a dinoflagellate. The enzyme from Phaeodactylum tricornutum has been purified to homogeneity and has an absolute requirement for ADP and Mn2+. This enzyme is best described as phosphoenolpyruvate carboxykinase (carboxylating), but it differs considerably from enzymes with the same name, isolated from mammalian and bacterial sources, in that the reaction of phosphoenolpyruvate with bicarbonate lies strongly in the direction of formation of oxaloacetate and ATP. This conservation of the high-energy phosphate groups of phosphoenolpyruvate in ATP would be advantageous to diatoms living at low levels of light intensity in seawater.

A manganese-copper-pigment-protein complex isolated from the photosystem II of Phaeodactylum tricornutum

Abstract The diatom Phaeodactylum tricornutum has been grown in defined medium with known amounts of 54 Mn and Cu. The diatoms were then fractionated by mild procedures to isolate a metallo-pigment-protein complex. This complex had a molecular weight of 850,000 and probably was made up of 40 subunits of protein, 40 mol of chlorophyll a , 20 mol of chlorophyll c , 20 mol of fucoxanthin, 8 g-atoms of Cu, and between 0.6 and 2.0 g-atoms of Mn per molecule. Detergents break the complex down to small units with molecular weights of approx 25,000. The metallo-pigment-protein is probably part of the PSII system of chloroplasts since (i) it photoreduces dichlorophenolindophenol in the presence of diphenylcarbazide, (ii) its behavior on electrophoresis after treatment with sodium dodecylbenzene sulfonate is similar to photosystem II, (iii) its fluorescence in the presence of reducing and oxidizing agents resembles chlorophyll-protein complexes from photosystem II, and (iv) its electron paramagnetic resonance spectrum is similar to that of photosystem II in whole chloroplasts. A theory is put forward to show how this Cu-Mn-pigment-protein complex may react in the early steps of the O 2 -evolving system of photosystem II.

ß CARBOXYLATION ENZYMES IN MARINE PHYTOPLANKTON AND ISOLATION AND PURIFICATION OF PYRUVATE CARBOXYLASE FROM AMPHIDINIUM CARTERAE (DINOPHYCEAE)1

The diatoms, Phaeodactylum tricornutum Bohlin, Cylindrotheca closterium var. californica (Meres.) Reim. & Lewin, Thalassiosira pseudonana Hasle & Heimdal and the prymnesiophyte Pavlova lutheri (Droop), Green, have been shown to contain phosphoenolpyruvate carboxykinase E.C.4.1.1.49. Another diatom Chaetoceros calcitrans (Paulsen) Takano, the chlorophyte Dunaliella tertiolecta Butcher, a rhodophyte Porphyridium cruentum Naegeli and the cyanophyte Anabaena cylindrica Lemmermann, all possessed phosphoenolpyruvate carboxylases E.G.4.1.1.31, which were stimulated by Mn ions and inhibited by malate and aspartate. Two dinoflagellates, Amphidinium carterae Hulbert and Gymnodinium sp., were shown to contain pyruvate carboxylase E.G.6.4.1.1., not previously reported in plants or marine algae. Pyruvate carboxylase was isolated and purified and found to contain biotin and it was inhibited by avidin and could be distinguished from the other two enzymes by complete inhibition by 5 mM Mn ions. The β carboxylating enzymes account for the anaplerotic formation of amino acids and intermediates of the tricarboxylic acid cycle formed during short‐term fixation of CO2.

The Use of High Pressure Liquid Chromatography for the Identification and Preparation of Pigments Concerned in Photosynthesis

Abstract The chlorophylls and carotenoids present in preparations from chloroplasts of marine algae can be extracted and separated by high pressure liquid chromatography (H.P.L.C.). The reverse-phase columns; Partisil 10 ODS (Whatman), μ Bondapak C18 and μ Bondapak CN (Waters Associates) gave good separation of the different pigments. Addition of the ion-pairing agent tetrabutylamnonium phosphate to the methanol/water solvents gave improved separations with complex mixtures and identification was facilitated by examination of the column eluant at 440nm where a ll the pigments absorbed and at 650nm where only chlorophylls absorbed. The method allowed the isolation of individual pigments for further study.

Stimulation of calcium efflux from rat liver mitochondria by adenosine 3'5 cyclic monophosphate.

SummaryThe uptake or release of Ca2+ from rat liver mitochondria was studied by means of a sensitive Ca-electrode. It was found that using palmitoyl coenzyme A together with carnitine and ATP as substrates that Ca2+ was released gradually from mitochondria by adenosine 3′5′ cyclic monophosphate. The effect was obtained with either mitochondria preloaded with Ca2+ or with their physiological content of Ca2+. No such release was obtained with the usual substrates used to provide energy for Ca2+ uptake by mitochondria.

Assays of glucose tolerance factor and its mode of action, studied with brewer's yeast.

Glucose tolerance factor (GTF) has usually been assayed by manometric measurement of CO2 evolved when glucose was metabolizing glucose. By using 14C labeled substrates it has been shown that GTF increases the decarboxylation of pyruvate to ethanol and CO2. Thus in addition to measuring CO2 evolution, the enzymatic estimation of the increased ethanol production can be used to assay GTF. A further effect of GTF was to cause increased carboxylation of pyruvate to substrates that are used in the biosynthesis of cell substance. The metabolic sites of action of GTF are discussed.

A method for the estimation of proteins in colored or turbid solutions.

Abstract The many photometric methods of protein estimation give unsatisfactory results with highly colored solutions. Some plant pigment-protein complexes are not precipitable with trichloracetic acid; therefore the dye binding methods are not applicable. A rapid procedure has been devised where the protein is complexed with excess copper, the noncomplexed copper removed by an ion-exchange resin, and the copper complexed to polypeptide then estimated by atomic absorption spectrophotometry. The copper complex is proportional to the amount of protein and different proteins give the same result in the range of 0.05 to 1 mg.

Calcium uptake and release by rat liver mitochondria in the presence of rat liver cytosol or the components of cytosol.

SummaryA study has been made of factors present in rat liver cytosol that might regulate the calcium content of mitochondria. A cytosol preparation containing all the components of molecular weight greater than 10,000 prevented uptake and caused early release of accumulated calcium. These effects were due to free long-chain fatty acids and their coenzyme A derivatives present in the cytosol, and these inhibitory effects were controlled by inclusion of Mg2+, carnitine, and adenosine triphosphate at physiological levels in the incubation medium. Palmitoyl carnitine was a good substrate for calcium uptake and did not cause release of calcium from mitochondria. A specific fatty acid-binding protein was found in cytosol which may be the intracellular transport protein for fatty acids.