Role of calmodulin in the activation of tryptophan hydroxylase

Tryptophan hydroxylase can be activated 2.0- to 2.5-fold in vitro by ATPa dn Mg2+. This apparent phosphorylation effect is not dependent on cyclic nucleotides but is dependent on the presence of calcium. The activation of tryptophan hydroxylase by ATP-Mg2+ reduces the apparent Km of the enzyme for its cofactor, 6-methyltetrahydropterin, from 0.21 to 0.09 mM. The addition of certain antipsychotic drugs known to bind to calmodulin in a phosphorylation reaction mixture prevents the activation to tryptophan hydroxylase by ATP-Mg2+ in the concentration-dependent fashion. External addition of purified calmodulin protects the enzyme from the drug-induced effects. Preparation of calmodulin-free tryptophan hydroxylase by affinity chromatography on fluphenazine-Sepharose 4B yields an enzyme that is no longer activated by ATP-Mg2+, whereas the readdition of calmodulin to a calmodulin-free enzyme restores the responsiveness of tryptophan hydroxylase to ATP-Mg2+. This restoration is dependent on Ca2+. Taken together, these results indicate that the activation of tryptophan hydroxylase by phosphorylating conditions is dependent on both calcium and calmodulin.     

Gene 67, a new,essential bacteriophage T4 head gene codes for a prehead core component,PIP: II. The construction in vitro of unconditionally lethal mutants and their maintenance

We have obtained frameshift mutations of the bacteriophage T4 gene 67 by manipulating restriction cleavage sites within the gene cloned onto small plasmids. When these mutated genes were recombined back into the T4 genome the resulting phages were inviable. They could only be propagated by complementation in strains carrying a cloned, non-mutated copy of the gene on a plasmid. These experiments demonstrate that gene 67 is essential for T4 growth. Electron microscopy of bacteria infected with 67^? phages revealed that phage head morphogenesis was blocked at an early stage and particles resembling abnormal preheads were found in large numbers. The gene 67 product, PIP, is therefore essential for correct prehead assembly.     

The role of tyrosine in the binding of steroid by progesterone binding globulin from the guinea pig

Treatment of progesterone binding globulin (PBG) with tetranitromethane (TNM) resulted in a loss of steroid binding activity (inactivation) which was dependent on both time and concentration of reagent. Scatchard analysis of binding revealed that inactivation was due to a decrease in binding site number with no effect upon the affinity of PBG for steroid. Incorporation studies demonstrated that the loss of binding activity correlated with the incorporation of 1.3 nitro groups per molecule of PBG. The involvement of the steroid binding site in the reaction was shown by the ability of progesterone, but not cortisol, to protect against inactivation. Treatment with N-acetylimidazole did not inactivate PBG nor did the conversion of nitrotyrosyl residues to amino-tyrosines regenerate binding activity, suggesting that the pheolic hydroxyl is not involved in steroid binding. These studies suggest that inactivation was due to the incorporation of a bulky group into the aromatic ring of a tyrosine present at the steroid binding site thus blocking its ability to participate in hydrophobic interactions with the ligand.     

Partial characterization of a Trypanosoma cruzi-released decomplementing factor

Trypanosoma cruzi releases a factor (SCAF) when grown in vitro which decomplements normal mouse, human, and guinea pig sera. The production and potency of SCAF was dependent on the density of cultured parasites, parasite viability and proliferative capacity, and duration of culture. The in vitro interaction between SCAF and serum complement (C') occurred rapidly and was complete within 30 min of mixing. The administration of SCAF to normal mice resulted in up to 50% reduction in hemolytic C' activity, whereas SCAF had no effect on the C' levels in mice infected wit T. cruzi for more than 10 days. The active moiety of SCAF was shown to be a nonproteinaceous substance(s) with a molecular weight of approximately 23,000 daltons.     

Trypanosoma cruzi-induced suppressor substance III. Activation of suppressor cells

Serum from mice infected with Trypanosoma cruzi (SSS) is known to interact with normal spleen cells to induce an immunosuppressed condition and activate splenic suppressor cells. The induction of immunosuppression by SSS was shown to be independent of, and precede the activation of, suppressor cells. Suppressor-cell activation, however, was demonstrable only after the induction of immunosuppression. Furthermore, mice that were given two aliquots of SSS at different intervals of time, exhibited suppression of humoral responses of similar duration and magnitude, regardless of the SSS transfer regimen, whereas both the length and degree of suppressor-cell activity was critically dependent on the interval of time between SSS transfers. SSS interacted with spleen cells via a trypsin-sensitive membrane site which was regenerable within a 4- to 5-hr period, yet the suppressive effects of SSS on spleen cells following interaction was resistant to treatment with trypsin. The interaction between SSS and spleen cells during brief adsorption protocols leads to immunosuppression only because extensive washing of SSS-treated spleen cells did not reverse the immunosuppression process, but did prevent the development of detectable suppressor cells. The phenomenon of suppressor-cell activation was further distinguished from immunosuppression in that supernates from culture of spleen cells derived from SSS-treated mice or T. cruzi-infected contained a factor that activated suppressor cells, but did not directly induce a state of suppression in the responding cell population.     

Differential effects of placental lactogen,growth hormone and prolactin on rat liver ornithine decarboxylase activity in the perinatal period

Ovine placental lactogen, (oPL), ovine growth hormone, (oGH), and ovine prolactin, (oPRL) are present in high concentrations in the fetal circulation late in gestation. To determine if these hormones stimulate the activity of ornithine decarboxylase (ODC), an enzyme widely implicated in the control of cellular growth, rat fetuses were injected $\text{in utero}$ with 100 μg of oPL, oGH, oPRL, rat growth hormone (rGH) or rat prolactin (rPRL) and ODC activity in the livers, hearts, and brains of the fetuses was measured 2, 4, and 6 hours after injection. OPL stimulated fetal liver ODC activity by 282 ± 45% (mean ± SEM) as compared to litter mates injected with buffer alone but oGH, oPRL, rGH and rPRL had no effect on fetal liver ODC activity. However, in neonatal rats 24–48 hours old all five hormones significantly increased liver ODC activity. ODC activities in the hearts and brains of the fetuses and neonates were unaffected by any of the five hormones. In other experiments 50 μg of oPL significantly stimulated fetal liver ODC activity while 250 μg of oGH were without effect. However 25 μg of oGH significantly stimulated liver ODC activity in rat pups 1–2 days after birth. These results suggest that oPL, by its stimulation of ODC activity, has somatotropic effects in the fetus and that rat liver ODC activity becomes responsive to growth hormone and prolactin in the perinatal period.     

Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

Substrate specificity of citrate lyase deacetylase of Rhodopseudomonas gelatinosa and Rhodopseudomonas palustris.

Citrate lyase (EC 4.1.3.6) isolated from Rhodopseudomonas palustris was investigated with regard to its kinetic properties and its subunit composition. This enzyme was inactivated by citrate lyase deacetylase (EC 3.1.2.-) of Rhodopseudomonas gelatinosa. A corresponding cross-reaction was measured with partially purified deacetylase of R. palustris and citrate lyase of R. gelatinosa. The three different subunit types (alpha, beta, and gamma) of citrate lyase from R. gelatinosa wee purified to homogeneity, and antibodies were prepared against each of the three subunits and against the native enzyme complex. In corresondence with the enzymatic interactions, immunological cross-reactions were found between anti-enzyme and anti-large subunit antibodies and citrate lyase from R. palustris. On the other hand, no immunological cross-reactions were detectable among each of the antibodies and citrate lyases from Enterobacter aerogenes, Streptococcus diacetilactis, and Clostridium sphenoides. Antibodies against the large subunit of citrate lyase inhibited the deacetylase, but antibodies against the middle and small subunits did not, indicating that the large subunits of citrate lyase are involved in binding the deacetylase.