Use of a light-induced respiratory transient to measure the optical cross section of photosystem I in chlorella

A method has been developed whereby the magnitude of a transient in O₂ uptake attributable to photosystem (PS) I activity, following single-turnover laser flashes of varying energy, can be used to measure the optical cross section of PSI. As measurements are made under the identical physiological conditions for which the cross section of PSII has previously been determined (AC Ley, DC Mauzerall 1982 Biochim Biophys Acta 680: 96-105), it is now possible to simultaneously measure the cross section of both photosystems in intact, photosynthetically competent cells, without the use of inhibitors or artificial mediators of electron transport. Plots of light-saturation behavior of the respiratory oscillation following pulses at 596 nanometers indicate a mean optical cross section similar to that of PSII at this wavelength, but suggest significant heterogeneity in the cross section of PSI. If this method measures only PSI activity, this result implies that there exist units with different numbers of identical chromophores, or units having populations of chromophores with different absorption spectra.     

Mutational analysis of primosome assembly sites. Evidence for alternative DNA structures

Primosome assembly sites are complex DNA structures that share common functions (they elicit the DNA-dependent ATPase of replication factor Y from Escherichia coli and serve as origins of complementary strand DNA synthesis), but display little sequence homology. In order to ascertain a common basis for factor Y-DNA recognition, a primosome assembly site and its mutated derivatives have been functionally and structurally analyzed. Under conditions in which they lose the capacity to function as ATPase effectors these DNA templates have been (i) assayed for their ability to bind factor Y, and (ii) probed, with pancreatic DNase, for structural alterations. In this ATPase-inactivating environment (suboptimal concentrations of MgCl2 and NaCl, and high levels of the E. coli single-stranded DNA binding protein), factor Y does not bind to its cognate DNA and the DNase cleavage pattern characteristic of this site is perceptibly changed: compared to the DNase digest obtained under activating conditions, cleavage is notably decreased in the 5' half of the site and enhanced at the 3' end. The results of this study strongly indicate that the structure of the primosome assembly site under analysis consists of two hairpins which interact with each other. When the sites of pancreatic DNase attack are plotted on the proposed double hairpin structure, the 5' cleavage sites all map to one duplex while the 3' sites map to the other. The observation that, under factor Y ATPase-activating conditions, the 3' hairpin is largely refractory to the action of pancreatic DNase indicates that tertiary interactions between the two duplexes render a portion of the DNA structure inaccessible to the nuclease.     

Concentration of phosphoribosyl pyrophosphate in renal hypertrophy. Contrasting effects of early diabetes and unilateral nephrectomy.

Studies were made of the renal phosphoribosyl pyrophosphate (PPRibP) content and PPRibP synthetase (EC 2.7.6.1) activity in rats diabetic for 5, 14 or 20 days, or unilaterally nephrectomized (UN) for 5 days, and in doubly lesioned animals. Approximately equal degrees of renal enlargement were found after 5 days diabetes or 5 days UN. In the doubly lesioned animals the increment of growth was additive. Unilateral nephrectomy of 5 days duration, in contrast with diabetes, had no effect on the PPRibP content of the contralateral kidney, nor did it modify the renal PPRibP content when performed on animals diabetic for 5, 14 or 20 days. The activity of PPRibP synthetase was unaffected by diabetes, UN or diabetes +UN. The results pinpoint a stage of nucleotide synthesis which is differentially affected by the two stimuli, in line with evidence for differences in regulation of nucleic acid turnover in the two conditions.     

The pathway of biosynthesis of nicotinamide–adenine dinucleotide in rat mammary gland

1. The pathway of NAD synthesis in mammary gland was examined by measuring the activities of some of the key enzymes in each of the tryptophan, nicotinic acid and nicotinamide pathways. 2. In the tryptophan pathway, 3-hydroxyanthranilate oxidase and quinolinate transphosphoribosylase activities were investigated. Neither of these enzymes was found in mammary gland. 3. In the nicotinic acid pathway, nicotinate mononucleotide pyrophosphorylase, NAD synthetase, nicotinamide deamidase and NMN deamidase were investigated. Both NAD synthetase and nicotinate mononucleotide pyrophosphorylase were present but were very inactive. Nicotinamide deamidase, if present, had a very low activity and NMN deamidase was absent. 4. In the nicotinamide pathway both enzymes, NMN pyrophosphorylase and NMN adenylyltransferase, were present and showed very high activity. The activity of the pyrophosphorylase in mammary gland is by far the highest yet found in any tissue. 5. The apparent K(m) values for the substrates of these enzymes in mammary gland were determined. 6. On the basis of these investigations it is proposed that the main, and probably only, pathway of synthesis of NAD in mammary tissue is from nicotinamide via NMN.     

Influence of hormones on the nicotinamide nucleotide coenzymes of adipose tissue

The concentrations of the oxidized and reduced forms of the nicotinamide nucleotides were measured in the epididymal fat pads of normal, alloxan-diabetic and hypophysectomized rats. In both alloxan-diabetic rats and hypophysectomized rats the weight of the adipose tissue fell, as did the total content of NADH and NADPH; in addition, NAD⁺ was decreased in the alloxan-diabetic group. Of these changes the most marked was in NADPH and this was the only significant difference when the results were expressed as nicotinamide nucleotides/mg. of tissue protein. The concentration of NADPH in the hypophysectomized rats was not altered by treatment with growth hormone but was restored to normal by treatment with thyroxine. These results are discussed in relation to the known effect of these hormonal conditions on lipid synthesis in adipose tissue.     

The effect of treatment of rats with pituitary growth hormone on the activities of some enzymes involved in fatty acid degradation and synthesis

1. Measurements have been made of the activities of acyl-CoA dehydrogenase, enoyl-CoA hydratase, beta-hydroxyacyl-CoA dehydrogenase and ketothiolase in the livers of rats treated for either 12hr. or 3 days with pituitary growth hormone. 2. There was a significant increase in the activity of acyl-CoA dehydrogenase in rats treated with the hormone for 3 days. 3. Measurements were also made of the lipogenic enzymes acetyl-CoA carboxylase and palmitate synthase in the livers of similarly treated animals. 4. There was a depression of the activity of both enzymes after 12hr. treatment and a further decline after 3 days. 5. The results are discussed in relation to the known increase in the rate of fatty acid oxidation and inhibition of fatty acid synthesis in rats treated with growth hormone.