Martha Kirk

Detection of steroids in paper chromatography

Summary o 1. Methods for the detection of steroids in paper chromatography have been described and evaluated for 31 compounds. 2. A new method of detection of steroids involving phosphomolybdic acid has been reported. 3. A method using silicotungstic acid has been extended to cover many new compounds.

Photosynthesis by isolated chloroplasts I. Diffusion of labeled photosynthetic intermediates between isolated chloroplasts and suspending medium

Abstract The diffusion of 14 C- and 32 P-labeled photosynthetic intermediate compounds from isolated chloroplasts has been investigated. Those intermediate compounds of the photosynthetic carbon reduction cycle lying between the carboxylation reaction and the diphosphatase reaction were found to diffuse rapidly from the chloroplast to the suspending medium. In contrast, those intermediates lying between the diphosphatase reaction and the carboxylation reaction, with the exception of the pentose monophosphates, tend to be retained by the chloroplasts during the 15 min or more when the photosynthesis of CO 2 by the isolated chloroplasts is most active. An experiment in which changes in the level of ATP were observed on addition of CO 2 shows that ATP and ADP, which had diffused from the choroplasts, can apparently re-enter the chloroplasts and be used metabolically. It is proposed that the diphosphatase reaction plays a general role in metabolic regulation in biosynthesis as well as in the light-dark transition.

REGULATORY EFFECTS OF AMMONIA ON CARBON METABOLISM IN PHOTOSYNTHESIZING CHLORELLA PYRENOIDOSA

Abstract Addition of ammonia to Chlorella pyrenoidosa , photosynthesizing under steady-state conditions, causes changes in the metabolism which are due not only to the increased availability of NH 4 + for reductive amination but also to regulation of controlled enzymes. One such effect is an increased rate of the reaction which converts phosphoenolpyruvate to pyruvate in vivo . This regulatory effect was revealed by kinetic tracer studies with 14 CO 2 , paper chromatography and radioautographic analysis, which showed that upon addition of NH 4 + (1) the levels of both 3-phosphoglycerate and phosphoenolpyruvate drop, with the ratio of 3-phosphoglycerate/phosphoenolpyruvate increasing, (2) the level of labeled pyruvic acid increases and the rate of formation of alanine increases rapidly, while the rate of formation of serine is unaffected, (3) the rate of flow of carbon into the tricarboxylic acid cycle acids, malate and citrate, increases along with the increased rates of formation of glutamate, glutamine and aspartate and (4) the rate of labeling of lipids increases. The increased flow of carbon into amino acids is mostly at the expense of sucrose synthesis; starch synthesis decreases only slightly. The interruption of sucrose synthesis apparently is due to stopping the reaction between UDP-glucose and fructose 6-phosphate. The rate of conversion of fructose 1,6-diphosphate to fructose 6-phosphate is also decreased upon NH 4 + addition.

DYNAMICS OF THE PHOTOSYNTHESIS OF CARBON COMPOUNDS II. AMINO ACID SYNTHESIS

Further kinetic studies have been made of the rates of appearance of 14C in individual compounds formed by Chlorella pyrenoidosa during steady state photosynthesis with 14CO2. Total and “active” pools of several amino acids have been determined. The effects of adding unlabeled acetate and of turning off the light have been studied in this system. From these experiments it is concluded that synthesis and utilization of alanine, serine, aspartic acid, glutamic acid and several other amino acids are most active within the chloroplasts during photosynthesis and that these amino acids are formed rather directly from intermediates of the carbon reduction cycle. The major portion of the carbon utilized for amino acid synthesis is accounted for in the synthesis of these compounds. Evidence for the presence of at least two separated pools of these amino acids is given, and the effect of light and dark and of the addition of unlabeled acetate upon the synthesis of these amino acids is discussed.

Regulatory effects of ammonia on carbon metabolism in Chlorella pyrenoidosa during photosynthesis and respiration.

Abstract Addition of ammonia to Chlorella pyrenoidosa , respiring in the dark following a period of photosynthesis, causes a stimulation of the flow of carbon into the synthesis of amino acids similar to that observed upon addition of ammonia during photosynthesis. In both cases, this stimulation is due not only to the increased availability of NH 4 + for reductive amination of α-ketoglutarate to glutamate but is also due to stimulation of the rate of conversion of phosphoenolpyruvate to pyruvate. Addition of NH 4 + in the dark causes a large increase in the formation of 6-phosphogluconate, beyond the increase in 6-phosphogluconate already seen when the light is turned off. When the light is turned off, the level of starch begins to decrease, and the rate of this decrease is not changed by the subsequent addition of ammonia. In contrast, the level of sucrose becomes nearly constant when the light is turned off, but begins immediately to decline when ammonia is added. As observed before, the level of ATP drops temporarily when the light is turned off and then rises to a steady state similar to that seen in the light. Upon the addition of ammonia, a similar transient drop and re-establishment in the level of ATP is seen. These and other reported results are discussed with respect to sites and mechanisms of light-dark metabolic regulation leading to increased flow of carbon from carbohydrate reserves into mitochondrial metabolism in the dark, and the sites and mechanisms by which ammonia affects the rate of this flow.

Dynamics of the photosynthesis of carbon compounds I. Carboxylation reactions

Abstract Kinetic studies have been made of the rates of appearance of 14 C in individual compounds formed by Chlorella pyrenoidosa during steady state photosynthesis with 14 CO 2 . These rates have been compared with rates of CO 2 and 14 C disappearance from the gas phase during the same experiments. 1. The following results were obtained: 2. 1. After the first few seconds, the rate of appearance of 14 C in compounds stable to drying on planchets at room temperature is 95 to 100% of the rate of uptake of carbon from the gas phase. 3. 2. After the first few seconds, the rate of appearance of carbon in compounds isolable by usual methods of paper chromatography constitutes at least 73 to 88% of the rate of uptake of carbon from the gas phase. Compounds formed from the carbon reduction cycle via the carboxylation of ribulose diphosphate account for a least 70 to 85% of the uptake, while carboxylation of phosphoenolpyruvic acid appears to account for at least another 3%. 4. 3. The induction period in the appearance of 14 C in stable compounds may be due to a reservoir of intracellular CO 2 and HCO 3 − or to some other volatile or unstable compound. If so, this reservoir contains no more than 1.5 μmoles of carbon, corresponding to about 7 sec carbon fixation in the experiment in which it was measured. 5. 4. No other carboxylation reactions, such as the carboxylation of γ-aminobutyric acid, could be observed. The rate of labeling of glutamic acid after 5 min of exposure of the algae to 14 CO 2 reached a maximum rate of about 5% of the total uptake rate, but this labeling appears to be due to conversion of labeled intermediates formed from the carbon reduction cycle or phosphoenolpyruvic acid carboxylation. 6. 5. The in vivo carboxylation of ribulose diphosphate in the light appears to be followed by conversion of the product to one molecule of phosphoglyceric acid, containing the newly incorporated 14 CO 2 and one molecule of some other (kinetically distinguishable) three carbon compound. This reaction would be different from the one reported for the isolated enzyme system and the in vivo reaction in the dark, which produces two molecules of 3-phosphoglyceric acid.

Inhibition of photophosphorylation and photosynthetic carbon cycle reactions by fatty acids and esters.

Abstract Photosynthesis in Chlorella pyrenoidosa is quickly inhibited by the addition of 3 × 10 −4 to 6 × 10 −4 M lipoic acid, octanoic acid, or methyl octanoate. These inhibitions are wholly or partially reversible. Studies with 32 P-labeled phosphate and 14 C-labeled carbon dioxide of transient changes in the levels of photosynthetic intermediate compounds during the time following addition of inhibitor indicate blocking of (1) the carboxylation reaction of the photosynthetic carbon reduction cycle, (2) photophosphorylation (formation of ATP in the light), and (3) conversion of fructose-1,6-diphosphate to fructose-6-phosphate and of sedoheptulose-1,7-diphosphate to sedoheptulose-7-phosphate. Blocking of photophosphorylation results in decreased rates of conversion of ribulose-5-phosphate to ribulose-1,5-diphosphate, of glucose-6-phosphate to polysaccharides and sucrose, and other reactions. These metabolic effects, together with other published results, lead us to conclude that at least several key steps in the photosynthetic carbon reduction cycle may be mediated by enzymes in some organized system associated with the lamellae and photophosphorylation.

PHOTOSYNTHESIS OF AMINO ACIDS

Abstract Methods have been developed for performing dual tracer experiments with 15 N and 14 C on the photosynthesis and biosynthesis of amino acids in Chlorella pyrenoidosa . These methods involved the development of apparatus for maintaining algae in a condition of steady-state photosynthesis, including amino acid synthesis, and of adding tracers in a stepwise fashion so that specific activity of the tracer is maintained at a constant level without alteration of other environmental factors. This method has been applied, and the kinetics of the labeling of several primary amino acids with 14 C and 15 N have been studied. The results indicate that the reductive amination to form glutamic acid is the primary route for incorporation of 15 N in this organism during photosynthesis of amino acids. Certain relationships between the site of the photosynthesis of carbon skeletons of primary amino acids and the site of 15 N incorporation have been proposed and discussed.

Protein synthesis during photosynthesis

Abstract Kinetic studies have been performed on the appearance of 14 C in free amino acid pools and in the amino acid moieties of protein in Chlorella pyrenoidosa during steady-state photosynthesis with 14 CO 2 . At the same time, we have determined the total concentrations of free and bound amino acids, permitting us to calculate the specific radioactivity of the free and bound pools of certain primary amino acids. Comparison of the specific activities of the free amino acids pools with the rate of 14 C-labeling of the corresponding pools of bound amino acids shows that there is a direct kinetic precursor-product relationship between the actively-turning-over pools of free amino acids and the bound amino acids in the protein. In the case of certain amino acids, notably glycine, the actively-turning-over pool of free amino acid is very small, leading to “saturation” when the average specific activity of the entire free amino acid pool of glycine is only 0.015. The total rate of labeling with 14 C of the bound amino acids in protein appears to account for only a fraction of the steady-state rate of labeling of the free amino acid pools. Explanations are offered for the apparent discrepany.

Carbon‐14 Respiration Pattern Analyzer for Clinical Studies

A description of an instrument for measuring C14, percent CO2, and C14 specific activity in the breath of human subjects is presented. Ionization chamber assay for the C14 is used. The design parameters of radioactivity sensitivity and response time are discussed. For simple carbon‐14 metabolites some 1 to 10 μC are required for an eight‐hour respiration analysis with this instrument.