Human phosphoserine 31 corticotropin1-39. Isolation and characterization

Two distinct forms of corticotropin1-39 (ACTH) were isolated and purified from an extract of three adult human pituitaries by reversed-phase chromatographic techniques. Structural studies indicated that the more polar form of ACTH was phosphorylated at serine residue 31. Approximately 30% of the ACTH was found in the phosphorylated form. A similar proportion of phosphorylated ACTH was observed in extracts of three pituitaries from human fetuses of 15, 17, and 20 weeks gestation. Phosphorylated and nonphosphorylated human ACTH1-39 were found to be steroidogenically equipotent using both an isolated rat adrenal cell bioassay and a cultured human fetal adrenal cell bioassay.     

Carbohydrate changes in pre- and peri-implantation mouse embryos as detected by a monoclonal antibody

We have examined the tissue and embryonic distribution of an antigen on a large polysaccharide that is recognized by a monoclonal antibody, IIC3, prepared against F9 teratocarcinoma cells. By immunofluorescence the antigen is first detected on compacted morulae and early blastocysts. It is strongly expressed on the primary endoderm and trophoblast of expanded blastocysts, but then disappears from the trophoblast of attached blastocysts in vitro. The binding of the antibody is completely inhibited by D-galactose and N-acetylgalactosamine. Fluoresceinated lectins were used to study further the changes in cell surface carbohydrates on trophoblast during implantation. Ricinus I, specific for terminal galactose, binds to preimplantation stages but does not bind to the trophoblast of the attached blastocyst. On the other hand, wheat germ agglutinin, specific for N-acetylglucosamine and sialic acid, binds to all preimplantation embryos and also to attached blastocysts (embryo proper and trophoblast). Neuraminidase treatment of blastocyst outgrowths enhances binding of both IIC3 and Ricinus I to the trophoblast; conversely, the binding of wheat germ agglutinin is decreased by this treatment. The results obtained in this study show changes of cell surface carbohydrates during early mouse development and suggest that sialic acid may be masking molecules on the surface of the trophoblast at the time of implantation.     

Cis-trans test shows a functional relationship between non-allelic lethal mutations in the T/t-complex

Recessive lethal mutations in the T/t-complex of the mouse characteristically show defective genetic complementation, even when they affect very different stages of embryogenesis and are known to be nonallelic. To address the question of their genetic or functional relationship, we have applied the cis-trans test, using several well defined recombinant t-chromosomes that carry two or more lethal mutations, and others that are devoid of specific lethals. We show here that the defective complementation that occurs between different t-lethals is a specific result of the trans configuration; thus these genes, which may map as much as 15 cM apart, constitute a functional unit. Some speculations are presented to interpret this enigma in terms of DNA plasticity.     

Differentiation in mouse melanoma cells: initial reversibility and an on-off stochastic model

Various proposals that a stochastic event, "commitment," is the first and rate-limiting step in mammalian cell differentiation were tested in one cell type, B16C3 mouse melanoma cells. Differentiation (pigment production) was observed in time-lapse films and in cloned single cells. As predicted by all the theories, onset of differentiation was at widely variable times in different cells after stimulation; and selection experiments showed that little of the variability was genetic. Contrary to some theories, differentiation appeared unrelated to cell division. Two properties of the melanoma cells did not fit any of the theories: times of differentiation were highly correlated in sister cells; and differentiation could be reversed in a proportion of cells, which was highest at the lowest levels of pigmentation. Dedifferentiation was associated with cell proliferation, so that most pigmented clones were small and most unpigmented clones large. These findings are accommodated by a model in which functions associated with differentiation can switch on and off, but an inhibition of the off transition builds up in the on state.     

Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.     

Fate of corticotrophins in an isolated adrenal-cell bioassay and decrease of peptide breakdown by cell purification

1. The fate of corticotrophins in a trypsin-dispersed rat adrenal-cell assay system was investigated with a view to establishing whether differences in the rate of inactivation might contribute to potency differences observed between analogues. 2. Corticotrophin-(1-24)-tetracosapeptide and to a lesser extent synthetic 1-39 corticotrophins were found to be inactivated during incubation with cell suspension. 3. Peptide fragments were isolated by using [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide as a marker. The fragments indicate a peptidase with a predominantly tryptic specificity. 4. The peptidase is present in the extracellular fluid and is released from cells when they are damaged. 5. Cells were fractionated on an albumin gradient. Cells from the zona fasciculata and the zona intermedia or reticularis were present in fractions which produced fluorogenic steroids in response to corticotrophin. 6. Purification of the cells by centrifugation through albumin decreased degradation by peptidases, so that if the assay is carried out with a dilute suspension of purified cells peptide breakdown should not affect the observed potencies of adrenocorticotrophin analogues. 7. No binding of [[(3)H(2)]Tyr(23)]corticotrophin-(1-24)- tetracosapeptide to cells could be detected at low concentrations of the peptide. This indicated that less than 120 receptors/cell are occupied during stimulation by a dose that would elicit approx. 80% of the maximal response.     

`Single addition'' and `transnucleotidation'' reactions catalyzed by polynucleotide phosphorylase. Effect of enzymatic removal of inorganic phosphate during reaction

The reaction of the tetranucleotide, pA-A(2)-A, with 2'(3')-0-(alpha-methoxyethyl)uridine 5'-diphosphate, Mg(2+) ions, and M. luteus polynucleotide phosphorylase followed by mild acid treatment to remove the blocking groups results in a 49% yield of the desired single addition product, pA-A(3)-U, together with smaller amounts of pA-A-U, pA-A-A, pA-A(2)-U, pA-A(2)-A, pA-A(3)-A, pA-A(4)-U, and pA-A(4)-A. The side products are thought to arise from the phosphorolysis of the acceptor molecule by the inorganic phosphate formed in the reaction mixture and from subsequent additions to the various oligonucleotide species by the resulting adenosine 5'-diphosphate. A system developed for the removal of inorganic phosphate as it is formed in the synthesis involves the addition to the reaction mixture of calf spleen nucleoside phosphorylase and nicotinamide riboside and, under these conditions, pA-A(3)-U can be prepared in 90% yield with essentially no side products. Under similar conditions, pA-A(3)-A, pA-A(3)-G, and pA-A(3)-C may be prepared from pA-A(2)-A and the appropriate blocked nucleoside diphosphate in yields of 85-94%. The incubation of pA-A(2)-A alone with polynucleotide phosphorylase exhibits the phenomenon of "transnucleotidation" in that the molecule is partially converted to oligonucleotides of smaller and larger chain lengths. In the presence of the phosphate removal system, however, the tetranucleotide is not attacked by the enzyme, and thus, "transnucleotidation" appears to be simply a combination of phosphorolytic and addition reactions catalyzed by trace amounts of inorganic phosphate contaminating the enzyme and/or the substrate.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.