Throughout this paper, protochlorophyll (Pchl) will be used to designate the epiphasic pigment on petroleum ether-alkaline acetone partition, assumed to be an esterified form of Mg vinylpheoporphyrin a5 which moves on chromatography in a similar manner to chlorophyll a (Chl). Protochlorophyllide (Pchlide) designates the hypophasic pigment assumed to be unesterified at the propionic acid residue of ring IV, which moves in a similar manner to Pchlide extracted from etiolated barley leaves.Pchl(ide) will be used when the exact nature of the protopigment has not been determined or when a mixture of the two species is present. Chlide designates chlorophyllide a.It has recently been demonstrated that both Pchl and Pchlide are present in dark-grown cells of Euglena and that both protopigments are transformed to Chl-like pigments upon illumination (9, 10). Klein and Schiff (19) investigated proplastid development in 2-to 9-day-old etiolated bean leaves and observed that (a) the 3-day-old bean proplastids are about the same size as 'This research was supported by Grant GM14595 from the National Institutes of Health. Because of the marked similarities between plastid precursors in young dark-grown bean leaves and those of Euglena, we undertook a study of the forms of protopigments present and their phototransformation at various stages in the devef7opment of etiolated bean seedlings, particularly at times preceeding 7 to 9 days, the material commonly employed in such studies. In the usual material, Pchlide is the predominant protopigment which is phototransformed to Chlide a and then finally esterified to yield Chl a. Pchl photoconversion is extremely low and is not regarded as a significant route of Chl formation (8,12,16,31,32,35,37,39). In this work we show that Pchl conversion in young etiolated bean seedlings represents a highly significant fraction of the convertible protopigments, and results in the direct formation of Chl. A brief abstract of this work has appeared (22). MATERIALS AND METHODSSeeds of Phaseolus vulgaris L. var. Red Kidney were germinated in wet vermiculite at 26 C in total darkness. The vermiculite was kept wet at all times by uniformly watering the trays, each containing 200 beans, with 1 liter of water each day. After time periods ranging from 2 to 17 days, a specified number of seeds, depending on the age, were removed, washed with water, and the cotyledons separated to expose the primary leaf pair. All manipulations were performed under dim green safelights (27) or in complete darkness. Roughly 1000 leaf pairs were required for each analysis of 3-day-old material. The number of leaf pairs required could be reduced to 35 at 5 days, 20 at 9 days, 15 at 13 days, and 10 by the 17th day.The methods for pigment extraction were modified from Shlyk (31). Before pigment extraction, the leaves were either (a) frozen in approximately 25 ml of liquid N2, (b) steamed for 10 min, or (c) extracted directly. All solvents used were reagent grade (Fisher).For experiments in which the leaves were heated, the spec...

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A set of monoclonal antibodies (MAb) to human GH (hGH) was used to study the hormone binding orientation to its receptors (R) from female rat liver. The hGH antigenic region left exposed after its binding to liver microsomes was detected by measuring the ability of various [125I]MAb to bind to the preformed hGH-R complexes. Results indicated that a cluster of epitopes defined by the MAb, termed AE5, AC8, and AE12, remains accessible in the hGH-R complex whereas overlapping epitopes 3C11 and HG3 would define a hGH region involved in the binding site. Supporting these findings, solubilization and HPLC gel filtration of [125I]MAb-hGH-R complexes showed a radioactive peak of about 450,000 mol wt for MAb AE5 or AC8, but not for MAb 3C11 or HG3. [125I]MAb AE12 behaved differently, suggesting that epitope AE12 may be masked or altered in hGH-R-solubilized complexes. MAb directed to the putative hGH-binding site (MAb 3C11, HG3, and the closely related MAb 10C1 and NA71) failed to inhibit binding of the preformed [125I]MAb AE5-hGH complex to the receptors, suggesting a hormone modification after MAb AE5 binding. Accordingly competition experiments indicated an increase in the affinity of hGH for its receptors induced by this MAb. A higher hGH concentration was required to obtain 50% [125I]hGH binding to liver microsomes in the presence of MAb AE5 than in its absence. As the MAb used define epitopes that were previously correlated with the hGH structure, we concluded that a high flexible region (sequences 134-150) is exposed in the hGH-R complex. Furthermore, some MAb directed to this region enhance the hormone affinity for its rat liver receptors, probably through an induced conformational change.

Abstract— The small amounts of chlorophyll precursors in dark‐grown cells of Euglena gracilis Klebs var. bacillaris Pringsheim and the presence of contaminants which interfere with their purification have made their isolation and characterization difficult. We now extract cells with acetone: 0.1 M NH4OH (9:1 v/v). Protochlorophyll is obtained by extracting this solution with petroleum ether (30–50° b.p.) and extracting this petroleum ether fraction with 80% acetone to remove substances which interfere with subsequent chromatography. Protochlorophyllide remaining in the original acetone: NH4OH fraction is extractable into diethyl ether after adjusting the pH to approximately 5.5. Both pigments are verified and further purified by chromatography on Whatmann 3 MM paper using benzene:petroleum ether:acetone (30:10:1) or cellulose MN 300 thin layers with methanol:methylene chloride:water (100:18:20). These pigments resemble their well‐described barley counterparts in solubility properties, spectral absorption maxima in ether (432, 530, 571 and 623 nm) and chromatographic behavior. Substantial amounts of protopheophytin and protopheophoribide are also found along with the Mg2+— containing pigments in cell extracts even when precautions are taken to avoid loss of Mg2+ during extractions and purification.Using heated cells before and chilled solvents after illumination to preclude enzymatic modification of pigments, chlorophyll and very small amounts of chlorophyllide are found as products of the photo‐conversion of the protopigments suggesting that both protochlorophyll and, to a much smaller extent, protochlorophyllide are photoconvertible in these organisms. These properties join several others which suggest that the Euglena chorophyll‐forming system more closely resembles that found in young bean leaves rather than the chlorophyll‐forming system of the older leaf material usually studied.