In vitro activation of bacteriophage P2 late gene expression by extracts from phage P4-infected cells.

We have used a cell-free, DNA-dependent protein-synthesizing system to study the stimulation of phage P2 late gene expression by satellite phage P4. An activity is present in extracts prepared from P4-infected cells, which, when added to the in vitro system with P2 DNA template, stimulates the synthesis of a number of P2 proteins. These stimulated proteins include the major P2 capsid protein (N gene product) and a major component of the P2 phage tail (FII gene product). Extracts prepared from P4-infected cells are also able to stimulate the synthesis from P4 DNA of two low-molecular-weight proteins (18,500 and 17,000 Mr). The stimulating activity has no effect on the synthesis of proteins from lambda plac5 template. Extracts prepared from cells infected with P4 alpha amber mutants lack this stimulating activity.     

Reduced osmotic potential inhibition of photosynthesis : site-specific effects of osmotically induced stromal acidification

The effects of reduced reaction medium osmotic potential (0.67 molar sorbitol as compared to a control treatment with 0.33 molar sorbitol) on the enzymic steps of the photosynthetic carbon reduction cycle were investigated using isolated spinach (Spinacia oleracea L. var Longstanding Bloomsdale) chloroplasts. Reversal of reduced osmotic potential inhibition of photosynthetic rates by a stromal alkalating agent (NH₄Cl) was associated with specific steps of the cycle. Low osmotic potential induced stromal acidification was found to be facilitated by osmotically induced chloroplast shrinkage. However, the action of the alkalating agent was found not to be associated with reversal of osmotically induced morphological changes of the stromal compartment.

Labeled metabolite analyses indicated that the osmotic stress treatment caused the substrate for fructose 1,6-bisphosphatase (FBPase) to build up in the absence of NH₄Cl, and the substrate for phosphoribulokinase to increase in the presence of NH₄Cl. These data were interpreted as indicating that the most severe effect of osmotic stress on photosynthesis is at the site of FBPase, and that this inhibition is mediated by osmotically induced stromal acidification. Phosphoribulokinase activity inhibition at the low osmotic potential treatment was apparently less severe and not mediated by stromal acidification. A third site of osmotic inhibition, which was reversed by NH₄Cl, and therefore was assumed to be mediated by stromal acidification, was at the step of ribulose 1,5-bisphosphate carboxylase.

Additions of NH₄Cl also enhanced the activity of the pH-insensitive phase of the photosynthetic carbon reduction cycle, 3-phosphoglyceric acid reduction, at the stress treatment. This effect was thought to be mediated by the removal of the block at FBPase. A model was proposed to outline the relative severity of osmotic stress effects at various sites of the photosynthetic carbon reduction cycle.

     

Interactions of porphyrins with nucleic acids

The interactions of nucleic acids with water-soluble porphyrins and metalloporphyrins have been investigated by stopped-flow and temperature-jump techniques. Both natural DNA (calf thymus) and synthetic homopolymers [poly(dG-dC) and poly(dA-dT)] have been employed. The porphyrins studied belong to the tetrakis(4-N-methylpyridyl)porphine (H2TMpyP-4) series and can be divided into two groups: (i) those which have no axial ligands when bound to nucleic acids [e.g., Ni(II), Cu(II), and the nonmetallic derivatives] and (ii) those which maintain axial ligands upon binding [e.g., Mn(III), Fe(III), Co(III), and Zn(II) derivatives]. The reaction of both axially and nonaxially liganded porphyrins at AT sites is too rapid to be measured by the kinetic methods utilized, whereas at GC sites the interaction of the nonaxially liganded porphyrins is in the millisecond time range and can be monitored by both stopped-flow and temperature-jump techniques. These results corroborate previous static studies, utilizing visible spectroscopy and circular dichroism, which indicate that the formation of an intercalated complex occurs only at GC base pair sites with porphyrins which do not possess axial ligands. With all the porphyrins investigated, the complexes formed at AT sites are envisioned as being of an "external" type involving some degree of overlap between the porphyrin and the bases of the duplex. In relaxation experiments of poly-(dG-dC) with H2TMpyP-4, a large, reproducible effect is observed which can be analyzed as a single exponential. Rate constants for association and dissociation of the H2TMpyP-4/poly(dG-dC) complex are 3.7 X 10(5) M-1 s-1 and 1.8 s-1, respectively. Relaxation studies of mixtures of poly(dA-dT) and poly(dG-dC) with H2TMpyP-4 indicate that the transfer of the porphyrin from one homopolymer to another occurs via a mechanism involving dissociation rather than direct transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of glycolate by a reconstituted spinach chloroplast preparation

A reconstituted preparation requiring fructose 6-phosphate, transketolase, triphosphopyridine nucleotide, ferredoxin, fragmented spinach chloroplasts, and light capable of forming glycolate at rates of about 10 micromoles per milligram of chlorophyll per hour has been characterized. The glycolaldehyde-transketolase addition product could be substituted for fructose 6-phosphate and transketolase. The stoichiometry of the reaction was: 1 mole of fructose 6-phosphate consumed for each mole of glycolate and of reduced triphosphopyridine nucleotide produced. Evidence was presented indicating that glycolate formation was coupled to the photosystems of the photosynthetic electron transport chain. Synthesis of glycolate is envisaged as the result of either (a) a reaction between the upper two carbon atoms derived from fructose 6-phosphate and an uncharacterized oxidant generated by photosystem 2 or (b) hydrogen peroxide produced by the reoxidation of reduced triphos-phopyridine nucleotide or reduced ferredoxin by molecular oxygen.     

Involvement of Photosynthetic Carbon Reduction Cycle Intermediates in CO(2) Fixation and O(2) Evolution by Isolated Chloroplasts

The photosynthetic carbon reduction cycle intermediates can be divided into three classes according to their effects on the rate of photosynthetic CO₂ evolution by whole spinach (Spinacia oleracea) chloroplasts and on their ability to affect reversal of certain inhibitors (nigericin, arsenate, arsenite, iodoacetate, antimycin A) of photosynthesis: class I (maximal): fructose 1, 6-diphosphate, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, ribose-5-phosphate; class 2 (slight): glucose 6-phosphate, fructose 6-phosphate, ribulose-1, 5-diphosphate; class 3 (variable): glycerate 3-phosphate. While class 1 compounds influence the photosynthetic rate, they do not lower the Michaelis constant of the chloroplast for bicarbonate or affect strongly other photosynthetic properties such as the isotopic distribution pattern. It was concluded that the class 1 compounds influence the chloroplast by not only supplying components to the carbon cycle but also by activating or stabilizing a structural component of the chloroplast.     

CHLOROPLAST DEVELOPMENT IN OCHROMONAS DANICA

When dark-grown cells of Ochromonas danica are placed in the light, the amount of chlorophyll a per cell increases 82-fold; the content of carotenoid pigment, 24-fold. Concomitantly with this increase in chlorophyll and carotenoid pigment, the small proplastid of dark-grown cells develops into a large lamellate chloroplast. During the first 12 hours in the light, vesicles appear within the loose clusters of dense chloroplast granules, enlarge, align themselves into rows (plates in three dimensions), and fuse into discs. Double discs may form from the more or less simultaneous fusion of two adjacent plates of vesicles or by the addition of vesicles to an already formed single disc. Three-disc bands arise by the addition of a disc to an already formed two-disc band through the approach and fusion of more vesicles. After 24 hours in the light, most of the chloroplast bands contain three discs, but the chloroplasts are still small. After 48 hours in the light, almost all the cells contain full-sized chloroplasts with a full complement of three-disc bands. However, at this time the amount of chlorophyll a and carotenoid pigment is only one-half of maximum. During the next 3 days in the light, as the number of chlorophyll and carotenoid molecules per chloroplast approximately doubles, there is a compression of the discs in each band (from 180 to 130 A) and a precise alignment of their membranes. Changes also occur in the nucleus when dark-grown cells are placed in the light. There is an increase in the number of small nucleolar bodies, many of which lie directly against the nuclear envelope, and in a few cells a dense mass of granules is seen between the two membranes of the nuclear envelope.     

An Example of Information Retention in Rabbit Ventricular Muscle Fibres

The preparation was stimulated externally and transmembrane action potentials were recorded with intracellular microelectrodes. The relationship between the area of the first action potential after a pause in stimulation and the duration of the pause was examined. It was found that the area retained its dependence on the pattern of stimulation prior to the pause. These experiments confirm one of the predictions of a mathematical model (Gibbs et al., 1963) which describes the relationship between the area of action potentials and the pattern of stimulation.     

Purification and properties of erythro-β-hydroxyasparate dehydratase from Micrococcus denitrificans

1. The novel enzyme, erythro-beta-hydroxyaspartate dehydratase, a key enzyme of the beta-hydroxyaspartate pathway (Kornberg & Morris, 1963, 1965), has been purified 30-fold from extracts of glycollate-grown Micrococcus denitrificans. The purified preparation was devoid of erythro-beta-hydroxyaspartate-aldolase activity, and free from enzymes that act on oxaloacetate. 2. Properties of the purified dehydratase were studied by direct assay of the enzymic formation of oxaloacetate and ammonia from added erythro-beta-hydroxyaspartate. 3. The enzyme was highly substrate-specific, utilizing only the l-isomer of erythro-beta-hydroxyaspartate (K(m), 0.43mm, and V(max.), 99mumoles of oxaloacetate formed/min./mg. of protein at pH9.15 and 30 degrees ). Of many compounds tested, only maleate was a competitive inhibitor (K(i), 32mm at pH7.6). 4. The optimum pH for activity was about 9.5. The K(m) varied with pH, showing a marked optimum at pH7.8. The V(max.) also varied with pH in a manner suggesting the presence in the enzyme-substrate complex of a dissociable group of pK'(a) about 8.5. 5. Carbonyl reagents were inhibitory, but of three thiol reagents tested only p-chloromercuribenzoate was inhibitory. 6. A partially resolved preparation of the enzyme was activated four-fold by the addition of pyridoxal phosphate and thereby restored to half activity. 7. EDTA (0.1mm) was almost completely inhibitory, activity being restored by bivalent cations (Mg(2+), Ca(2+) and Mn(2+)); no activation by univalent cations was observed. 8. The findings are discussed in the light of reported properties of related hydroxyamino acid dehydratases.