Three micromethods are described for the assay of enzymes or products of the heme biosynthetic pathway in blood. A fluorometric method for the assay of protoporphyrin may be applied to the rapid screening of children for chronic lead poisoning. A colorimetric assay for delta-aminolevulinic-acid dehydratase may be applied to the detection of acute and chronic lead poisoning. A fluorometric assay for porphyrin formation from prophobilinogen is also described.

Acute Disease↗

PubMed1972

Development of chlorophyll and hill activity.

A sensitive luminometer is used to measure directly the low rates of oxygen evolution during greening of etiolated barley (Hordeum vulgare L. var. Wong) leaves. Oxygen evolution is measured in leaf segments infiltrated with p-benzoquinone. When illuminated, these leaves do not produce significant amounts of oxygen until the end of the lag phase of chlorophyll synthesis. Chlorophyll is increased by feeding delta-aminolevulinic acid to leaves in the lag phase, but this does not cause an earlier appearance of photosynthesis. Chloramphenicol, and to a lesser extent cycloheximide, when fed to leaves together with delta-aminolevulinic acid, strongly inhibit the development of oxygen evolution in the light while only slightly inhibiting chlorophyll synthesis. The ability to evolve oxygen develops to only a slight extent in darkness, even in the presence of high levels of chlorophyll.We conclude that the development of photosystem II is limited by the synthesis of proteins in both the cytoplasm and the plastid, not by chlorophyll synthesis. Prolonged illumination is necessary for the development of oxygen evolution.

Journal Article↗

PubMed1971

Nucleolar necklaces in chick embryo myoblasts formed by lack of arginine.

The round nucleoli of chick embryo myoblasts, when grown in a culture medium devoid of arginine, unravel in several days into 5-20 micro long, beaded strands termed nucleolar necklaces (NN). Addition of arginine reverses this change. The NN contain protein, RNA, and traces of DNA as determined cytochemically by enzyme digestion and by acridine-orange fluorescent staining. When a cell containing the beaded strand is treated with agents, such as actinomycin D, that prevent rRNA polymerase action, the strand collapses and condenses into a small dense nucleolus with segregated regions of ribonucleoprotein (RNP) and deoxyribonucleoprotein (DNP). The properties of the NN appear to resemble those of the nucleolar necklaces of amphibian oocytes. Cycloheximide or puromycin inhibition of general protein synthesis does not lead to NN formation. We suggest that NN formation during arginine starvation may be a result of a singular depletion of some rapidly turning over, arginine-rich proteins that normally attach to ribosomal RNA precursor molecules during their synthesis in the processing towards maturation of the ribosomes.

Acridines↗

PubMed1970

Induction of -aminolevulinic acid synthetase in chick embryo liver cells in cluture.

delta-Aminolevulinic acid synthetase is induced in chick embryo liver culture by the natural steroid, etiocholanolone, and by the foreign chemical, 3,5-dicarbethoxy-1,4-dihydrocollidine at the level of transcription. Alternatively, inducing chemicals such as allylisopropylacetamide and gamma-hexachloro-cyclohexane act primarily at the level of translation. Hemin (K(i) = 5 muM) inhibits the induction at the level of translation. In liver cell culture, the half-life of delta-aminolevulinic acid synthetase is 3 hr, that of mRNA about 5 hr.

5-Aminolevulinate Synthetase↗

PubMed1970

Rapid regeneration of protochlorophyllide(650).

The rate of regeneration of protochlorophyllide(650) was examined spectrophotometrically after a saturating light flash using 8- to 9-day-old dark-grown bean leaves. The regeneration occurred to the extent of 15% with a half rise time of about 20 seconds. Feeding delta-aminolevulinic acid to the excised leaves in the dark increased protochlorophyllides(635) but not the absorption at 650 nanometers, suggesting that the holochrome was normally saturated with protochlorophyllide and that the holochrome protein was not controlled by the level of protochlorophyllide. After a light flash, the excess protochlorophyllide, formed from exogenous delta-aminolevulinic acid, readily combined to regenerate the 650 nanometer absorbing species; the regeneration occurred to the extent of 60 to 80% with a half rise time of about 50 seconds. Regeneration was blocked at 0 degrees , suggesting that there was some enzymic process required for regeneration, possibly the formation of a reductant component of the protochlorophyllides(650) holochrome.

Journal Article↗

PubMed1970

Controls on chlorophyll synthesis in barley.

In 7- to 10-day-old leaves of etiolated barley (Hordeum vulgare), all of the enzymes that convert delta-aminolevulinic acid to chlorophyll are nonlimiting during the first 6 to 12 hours of illumination, even in the presence of inhibitors of protein synthesis. The limiting activity for chlorophyll synthesis appears to be a protein (or proteins) related to the synthesis of delta-aminolevulinic acid, presumably delta-aminolevulinic acid synthetase. Protein synthesis in both the cytosol and plastids may be required to produce nonlimiting amounts of delta-aminolevulinic acid. The half-life of a limiting protein controlling the synthesis of delta-aminolevulinic acid appears to be about 1(1/2) hours, when determined with inhibitors of protein synthesis. Acceleration of chlorophyll synthesis by light is not inhibited by inhibitors of nucleic acid synthesis, but is inhibited by inhibitors of protein synthesis. A model for control of chlorophyll synthesis is proposed, based on a light-induced activation at the translational level of the synthesis of proteins forming delta-aminolevulinic acid, as well as the short half-life of these proteins. Evidence is presented confirming the idea that the holochrome on which protochlorophyllide is photoreduced to chlorophyllide functions enzymatically.

Journal Article↗

PubMed1969

The occurrence of substances in human plasma capable of inducing the enzyme delta-aminolevulinate synthetase in liver cells.

We have demonstrated the presence, in the plasma of several patients with acute intermittent porphyria, of a substance which strongly induced the synthesis of porphyrins in chick embryo liver cells growing in primary culture. The induction response evoked by this humoral agent was in all respects similar to that elicited by drugs and hormones which are known to enhance porphyrin production by inducing the de novo formation of delta-aminolevulinate synthetase, the rate-limiting enzyme in this pathway. Inducing properties were not found in the plasma of normal individuals or in that from porphyric patients in remission. Significant inducing activity was, however, found in the plasma of some normal subjects ingesting drugs or contraceptive steroid mixtures. The occurrence of a potent inducer of delta-aminolevulinate synthetase in the plasma of certain porphyric patients may have clinical significance for these genetically susceptible individuals. It will also permit chemical characterization of those humoral agents which may be related to the episodic exacerbations of this hereditary liver disease.

Animals↗

PubMed1968