Brain CCK receptors are structurally distinct from pancreas CCK receptors

Brain and pancreas cholecystokinin (CCK) receptors differ markedly in their selectivity for CCK analogs. To determine the size and subunit structure of the brain CCK receptor and compare it to that of the pancreas, 125I-CCK33 was covalently cross-linked with ultraviolet light to its receptor on mouse brain particles and purified pancreatic plasma membranes. When CCK was crosslinked to brain membranes, a single consistent major labeled protein band of Mr = 55,000 was observed in both the presence and the absence of DTT. These data with brain receptors contrast to results with pancreatic receptors where two bands of Mr = 120,000 and 80,000 are labeled in the absence and presence of DTT, respectively. These studies indicate, therefore, that the brain and pancreas CCK receptors are structurally and functionally distinct.     

ATP and the processivity of Xenopus laevis DNA polymerase α

We use specific restriction fragments as defined primers for DNA synthesis on single-stranded circular phage fd DNA. These structures are relatively poor templates for a highly purified DNA polymerase α from Xenopus laevis eggs. However, DNA synthesis is stimulated about 5-fold by addition of ATP to the reaction mixture. We show that the deoxynucleotide polymers, synthesized in the presence of ATP, are significantly longer than those produced in the absence of ATP. We also show that this effect is due to a more tenacious binding of DNA polymerase α to DNA and conclude that ATP increases the processivity of the enzyme.     

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Differentiation of cells producing polypeptide hormones (ACTH,MSH, LPH, α- and β-endorphin,GH and PRL) in the fetal porcine anterior pituitary

Summary The aim of the present study on the fetal porcine pituitary was (1) to detect by means of the immunoperoxidase technique the earliest stages of cells producing polypeptide hormones: -MSH, ACTH, -LPH, - and -endorphin, growth hormone (GH) and prolactin (PRL), (2) to study the development of the synthesis and the storage of these hormones during fetal life, and (3) to detect whether several hormones can be located in one and the same cell.The corticotropic cells were revealed as the earliest functional elements of the fetal anterior pituitary. Our results indicate clearly that ACTH, -MSH, -LPH, - and -endorphin appear at 34 days in the same regular, round or ovoid cells; no differences in the time of their appearance could be observed. The ACTH-cells, irregular or angular in shape and endowed with cytoplasmic processes such as described in the adult pituitary, were not seen until day 50. The first GH-cells were detected between 40 to 45 days of fetal life. From day 45 to 90, the GH-cells greatly increased in number and in staining intensity of their progressively extending cytoplasmic area, but they displayed the same regular and round shape. The PRL-cells were the last cell type to appear in the fetal pituitary. The first PRL-cells, small in size and round or ovoid in shape with a high nucleus/cytoplasm ratio, were detected at day 70. At day 80, the PRL-cells increased in size and staining intensity. They displayed an irregular elongated or stellated shape and cytoplasmic processes resembling those characteristic of the adult pituitary. These data suggest that in the fetal porcine pituitary: (1) ACTH, -LPH and related peptides are synthesized and stored in the same cells, and (2) PRL and GH appear in individual cellular elements.     

Purification and some properties of a soluble cytochrome (cytochrome c-551) from Erythrobacter species strain OCh 114

A soluble cytochrome, cytochrome c-551 was purified from an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942) by ammonium sulfate fractionation, ion-exchange chromatography and gel-filtration. The cytochrome had absorption maxima at 277, 410, and 524–525 nm in the oxidized form, and at 415, 522, and 550.5 nm in the reduced form. At 77 K, the -band of the absorption spectrum of the reduced form split in two at 547 and 549 nm. The millimolar absorption coefficient at 550.5 nm was 26.8 mM^-1 cm^-1 in the reduced form. This cytochrome was an acidic protein with an isoelectric point of 4.9. Its molecular weight was determined to be 15,000 by gel-filtration on Sephadex G-100 and 14,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The midpoint potential of this cytochrome was +250 mV at pH 7.0. This cytochrome did not bind CO.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Photooxidation reactions of diphenylcarbazide and their DCMU-sensitivity in thylakoids of the blue-green alga Oscillatoria chalybea

Thylakoids of Oscillatoria chalybea are able to split water. The Hill reaction of these thylakoids is sensitive to DCMU. Diphenylcarbazide can substitute for water as the electron donor to photosystem II with these fully functioning thylakoids. However, the diphenylcarbazide photooxidation is completely insensitive to 3-(3,4-dichlorophenyl)-N-N-dimethyl urea (DCMU) at high diphenylcarbazide concentrations. In with Tris-treated Oscillatoria thylakoids the water splitting capacity is lost and diphenylcarbazide restores electron transport through photosystem II as occurs with higher plant chloroplasts. However, also these photoreactions are insensitive to DCMU. If diphenylcarbazide acts in Oscillatoria as an electron donor to photosystem II the result suggests that diphenylcarbazide feeds in its electrons behind the DCMU inhibition site. This in turn indicates that in Oscillatoria the site of inhibition of DCMU is on the donor side of photosystem II.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-N-N-dimethyl urea - DPC diphenylcarbazide - DCPiP 2,6-dichlorophenol indophenol - TMB tetramethyl benzidine - A-2-sulf anthraquinone-2-sulfonate