The effects of pH and citrate on the activity of nicotinamide–adenine dinucleotide-specific isocitrate dehydrogenase from pea mitochondria

1. The effect of pH on the co-operative activation of the NAD-specific isocitrate dehydrogenase from pea mitochondria by isocitrate is shown. 2. The interlinked effects of pH on the affinity of the NAD-specific isocitrate dehydrogenase for isocitrate and the dependence of the pH optimum on the substrate concentration are presented. 3. A consideration of the conditions of pH and substrate concentration under which citrate activates the NAD-specific isocitrate dehydrogenase demonstrates similarities between the binding of isocitrate and citrate. 4. A comparison of the effects of citrate and pH on the gross structure of the enzyme is investigated by density-gradient centrifugation. 5. The kinetic interpretations of these results are briefly considered. 6. The metabolic significance of these studies is discussed.     

Epistatic interactions between four rad loci in yeast

Haploid yeast strains carrying mutations in two or more of four ad genes were contrusted by tetrad dissection, and the UV survival of these strains was measured. It was found that (with one exception) double mutant strains were not significantly more sensitive than the most sensitive single mutants, for strains involving mutant loci rad 1, rad 3 and rad 4. The exception was the double mutant rad 1–5 rad 4-4, but another double mutant involving different alleles of the the same loci did not show an enhanced UV sensitivity. Triple and quadruple mutants also failed to show a significantly increased UV sensitivity with respect to the single mutants. The results indicate that all these four mutant loci confer UV sensitivity by the same mechanism, and it is suggested that the wild-type alleles mediate excision-repair of UV-induced DNA lesions. Enhanced sensitivity of the genotype rad 1–5 rad 4-4 is attributed to leakiness of these alleles.     

An improved, leaf-disk method for determining the freeze-killing temperature of leaves

1.

1. An improved, half-leaf method is described for measuring the freeze-killing temperature (FKT) of a leaf at a specific stage of development, using six similar plants.     

Communication between normal and enzyme deficient cells in tissue culture

Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions.     

Optical activity of simple peptides. I. Glycine oligopeptides with internal L-alanyl or L-glutamyl residues

Oligopeptides containing glycine and one or two L -alanyl or L -glutamyl residues have been studied by circular dichroism (CD) and optical rotatory dispersion (ORD) in aqueous solution at pH 1.0, pH 6.0, and pH 10.0 and in aqueous ethanol. Two glycyl residues are required to remove effects of α-carboxyl or amino titration on the optical activity of the internal alanyl or glutamyl residues. The CD spectra of the alanyl and protonated glutamyl residues are similar, having two regions of negative ellipticity around 215 nm resulting in a spectrum reassembling that of poly-α-L -glutamic acid (PGA) at high pH. Another large positive band below 190 nm was observed for gly₂-glu₂-gly₂ in water at pH 6 and 10 and for several peptides in aqueous ethanol. Residue ellipticities were approximately additive in every case except for peptides containing intrenal glutamyl residu at pH 6.0.     

The effect of calcium ion concentration on myotube formation in vitro

The relationship of Ca^a+ concentration in in vitro tissue culture medium to the ability of the medium to support lizard and chick myotube formation was studied. Lizard myogenic cells (from lines established from the regenerating tail of the lizard, Anolis caroliensis) do not fuse in media with a Ca²⁺ concentration of below 650 μM; good fusion occurs at 1 750μM; and large anastomosing tubes result in media with concentrations of 2 750,μM. Chick myogenic cells from the limb of 11 day embryos do not fuse at Ca²⁺ concentrations below 260,μM, fuse well at 1 000 μM, and produce large anastomosing myotubes at concentrations above 1 700 μM. Colonies of myogenic cells from established lines plated at clonal densities in a medium with a 1 750 μM Ca²⁺ concentration grow more rapidly than those at 650 μM Ca²⁺; however, there is no increase in plating efficiency. Regardless of the Ca²⁺ concentration, lizard myogenic cells do not fuse until a large percentage of the cells in a colony have withdrawn from the mitotic cycle in Gl and entered GO. The similarities between in vivo and in vitro lizard myogenesis are discussed.     

Hexokinase from maize endosperm and scutellum

Hexokinase (EC 2.7.1.1) was isolated from endosperm and scutellum of developing and germinating maize (Zea mays) seeds. With fructose as the variable substate, Michaelis constant values for the scutellum enzyme were about onethird those of the endosperm enzyme (0.05 versus 0.15 mm), and no developmental differences were observed. With glucose as the variable substrate, Michaelis constant values were all in the range 0.1 to 0.2 mm. The enzyme preparation from germinating scutellum was studied further; when glucose was varied over a wide range, a Michaelis constant of 3.4 mm was observed in addition to the much lower Michaelis constant noted above. This low affinity binding of glucose may have regulatory significance and may indicate the presence of a glucokinase in addition to hexokinase.     

Thermal denaturation in acidic solutions of double-helical ribonucleic acid from virus-like particles found in Penicillium chrysogenum. A spectrophotometric study

1. Two species of double-helical RNA isolated from mycelium of Penicillium chrysogenum were titrated with acid at 25°C and 95°C (solvent 0.1m-sodium phosphate buffer). At 25°C denaturation occurred at about pH3. At 95°C in the denatured form cytosine residues titrated as a simple monobasic acid of pK3.9 compared with pK2.5 for the native form at 25°C. 2. On thermal denaturation in neutral and acidic solutions one species of RNA (38% rG·rC) `melted' in three distinct stages, equivalent to a mixture of three species, namely one of about 25% rG·rC, another of about 33% rG·rC and a third of about 46% rG·rC: the relative proportions were 0.25:0.35:0.40. 3. On thermal denaturation in acidic solutions the increase in the fraction of ionized cytosine residues concomitant with the `melting' of rG·rC base pair also affects the spectrum especially at 280nm and serves to enhance the contribution of rG·rC base pairs at this wavelength. The increment in ε_(P) at 280nm on `melting' an rG·rC base pair approaches 53501·mol<sup>−1</sup>·cm<sup>−1</sup> depending on pH, compared with 33501·mol<sup>−1</sup>·cm<sup>−1</sup> at pH7. In contrast ε<sub>(P)</sub> at 280nm is scarcely affected by `melting' rA·rU base pairs or by the protonization of adenine residues. 4. Changes in the spectrum of Escherichia coli rRNA on denaturation in acidic solutions were studied to yield the mole fractions of rA·rU and rG·rC base pairs `melting' at particular pH values.