Substrate and metal ion binding to carbamate kinase: NMR and EPR studies

Metal ion and substrate binding to carbamate kinase from Streptococcus faecalis was studied by nuclear magnetic resonance (NMR) and electron paramagnetic resonance using Mn²⁺ as the paramagnetic probe. The enzyme binds Mn²⁺ weakly (K_D = 0.45 ± 0.05 mm) with a stoichiometry of one per two subunits. However, in the presence of nucleotides, tighter binding of Mn²⁺ was observed with K_D = 44 ± 4 μm in the presence of ADP and K_D = 23 ± 4 μm with ATP present. Proton relaxation rate enhancement studies were conducted on water molecules interacting with ternary enzyme-Mn²⁺-nucleotide and binary enzyme-Mn²⁺ complexes. Mn²⁺ bound to carbamate kinase enhances the proton relaxation rate of water giving a binary enhancement value of ?_b = 9.3 ± 0.4. When enzyme-Mn²⁺ was titrated with ADP or ATP, a bell-shaped titration curve was obtained typical of many other enzyme-Mn²⁺-nucleotide ternary complexes. Computer fits to the titration data gave ternary enhancement values of ?_t^ADP = 14 ± 1 and ?_t^ATP = 19 ± 1. The dissociation constants for Mn-ADP and Mn-ATP binding to carbamate kinase were also obtained from these data analyses and are K₁ = 2.5 ± 0.5 μm and K₁ = 50 ± 8 μm, respectively. Therefore, these data demonstrate the formation of a ternary enzyme-metal-nucleotide bridge complex at the nucleotide substrate site of carbamate kinase. Distance measurements were conducted by NMR techniques with ¹³C-enriched carbamate and demonstrate that carbamate is 4–8 Å from enzyme-bound Mn²⁺. Thus carbamate binds near the metal-nucleotide substrate site of carbamate kinase.     

Effect of tunicamycin on the glycosylation of rhodopsin

The incorporation of [³H]glucosamine, [³H]mannose, and [³⁵S]methionine into rhodopsin was investigated in retinas which had been incubated in the presence and absence of the antibiotic, tunicamycin. In its presence, the incorporation of glucosamine was inhibited 70% and mannose, 96% compared to controls. In the presence of tunicamycin the attachment of glucosamine to core-region sites was virtually eliminated. The formation of unglycosylated rhodopsin was also indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and concanavalin A-Sepharose chromatography. These findings are consistent with the participation of the lipid-linked pathway in the glycosylation of this well-characterized intrinsic glycoprotein of the membranes of the disk of the rod outer segment. As indicated by the incorporation of [³⁵S]methionine, the synthesis of rhodopsin apoprotein was inhibited by a much lesser amount. This suggests that the glycosylation of rhodopsin is not required for its insertion into the disk membrane.     

Crystallographic analysis of the phytoagglutinin from Abrus precatorius by X-ray diffraction and electron microscopy

The lectin from the seeds of Abrus precatorius has been crystallized and the crystals subjected to study by X-ray diffraction and electron microscopy. Three closely related crystal forms were obtained, of orthorhombic space group P2₁2₁2₁ with $\text{a = 138}\text{A}\text{?}$, $\text{b = 142}\text{A}\text{?}$, and $\text{c = 173}\text{A}\text{?}$, of tetragonal space group P4₁2₁2 with $\text{a = b = 136}\text{A}\text{?}$, $\text{c = 176}\text{A}\text{?}$, and a twinned intermediate of the first two. From electron microscopy and two-dimensional spatial filtering of electron micrographs of the crystals, the molecule appears to consist of four similar domains grouped in a roughly planar diamond-shaped arrangement having a local intramolecular dyad axis. The average diameter of the Abrus lectin molecule is 50 to 60 Å and the individual domains appear to have a diameter of about 25 Å.     

Studies of antibody affinity in plasmacytoma-bearing mice: Evidence for a maturational defect of B lymphocytes

The antibody response of plasmacytoma-bearing mice (PC-mice) is severely reduced. In order to understand the nature of the effect of the tumor on the cells making antibody, quantitative and qualitative studies of the humoral response of PC-mice were undertaken. In these studies, the affinity of the antibody produced by tumor-bearing and normal mice was compared to determine whether the small amount of antibody produced by PC-mice is the product of a normal or an altered population of B cells. Antibody to TNP-Ficoll made by PC-mice 3 days after immunization was less heterogeneous and of an affinity lower than that of antibody made by normal mice. However, at 7 days, the antibody made by PC- and normal mice did not differ significantly. These data suggest that, prior to antigenic stimulation, the B cells of PC-mice are relatively immature, reflecting a possible retardation in the generation and turnover of B lymphocytes. The process of antigen-driven selection of high-affinity antibody-producing cells, however, appears to function normally in PC-mice. These studies, then, reveal a qualitative as well as quantitative defect in the primary humoral response of PC-mice which may reflect an abnormality in the development and differentiation of B cells in these mice.     

Peter Brian Medawar

Patients with coccidioidomycosis manifest a wide variety of severity of infection. Patients with unifocal and particularly multifocal dissemination manifest a defect in host defense mechanisms. This study focuses on the in vitro response to PHA as an indicator of immunoregulatory mechanisms. Study subjects are divided into four groups: Group I—17, patients with active disseminated coccidioidomycosis and lesions in multiple organs; Group II—15, patients with active disseminated coccidioidomycosis and a single lesion; Group III—16, patients with active coccidioidomycosis confined to the lungs; and Group IV—18, patients in whom coccidioidomycosis is inactive. The response to PHA was heterogeneous, even within groups. When cells were cultured in the presence of indomethacin or R020-5720, Group I cells demonstrated highly significant augmentation of the response to PHA (P < 0.002), whereas cells from Groups II to IV were unaffected. When adherent cells were removed by passage through a nylon wool column, the effect of indomethacin could no longer be demonstrated. Direct measurement of prostaglandin production (PGE₂ and PGF_2α) and thromboxane suggest that there is no difference in the quantity of these compounds produced by unstimulated or PHA-stimulated cells by Group I versus Group IV cells (P = 0.2-0.4). Indomethacin or R020-5720 markedly inhibit the production of these compounds. Studies of the effect of exogenous prostaglandin on PHA-stimulated cells suggest that Group I cells may be more inhibited by PGF_1α and PGF_2α compared to Group IV cells. The data demonstrate that PHA cultures of cells from Group I patients are suppressed. The suppressor cell can be removed by adherence to nylon wool and is inhibited by indomethacin or R020-5720.     

Phorbol myristic acetate enhances the production of Interleukin 2

Interleukin 2 is an antigen-nonspecific factor produced by Concanavalin A (Con A)-activated mouse spleen cells that has a number of biologic activities including the capacity to stimulate thymocyte proliferation, support the growth of continuous T cell lines, augment the antibody response of nude mouse spleen cells, and support the generation of antigen-specific cytotoxic T cells. In order to obtain increased amounts of Interleukin 2 for further purification and biologic studies, we have examined the use of Phorbol Myristic Acetate (PMA) as a costimulant. In this report we demonstrate that the addition of PMA to Con A-induced mouse spleen cell cultures results in a 5- to 20-fold increase in the production of Interleukin 2 activity under serum-containing and serum-free culture conditions.     

Dexamethasone inhibition and phorbol myristate acetate stimulation of plasminogen activator in human embryonic lung cells

Treatment of human embryonic lung cells with dexamethasone resulted in a decrease in plasminogen activator activity measured in the fibrinolytic assay. The decrease in activity could at least partially be explained by the presence of an inhibitory substance(s) based on the following observations of lysates of dexamethasone-treated vs. control cells: a) an increase in specific activity following subcellular fractionation; b) an increase in fibrinolytic activity following separation by gel electrophoresis; c) an increase in fibrinolytic activity following mild acid-treatment; and d) a decrease in urokinase-directed fibrinolytic activity in mixing experiments. Phorbol myristate acetate increased plasminogen activator activity without affecting the level of inhibitory substance.     

Physicochemical analysis of reversible molybdate effects on different molecular forms of glucocorticoid receptor

Several distinct molecular forms of glucocorticoid receptor have been identified in a melanoma model system. We have used velocity sedimentation to monitor molybdate dependent alterations in receptor size and heterogeneity. In the absence of molybdate, native glucocorticoid receptor from dexamethasone-sensitive tumors sediments at 7–8 S and 12–13 S. Under identical conditions, receptor isolated from dexamethasone-resistant tumors sediments at 7–8 S only. However, when molybdate is introduced, either during homogenization or immediately prior to centrifugation, glucocorticoid receptors from both dexamethasone-sensitive and -resistant tumors sediment sharply at 9–10 S. These molybdate induced phenomena are reversible. The activated forms of glucocorticoid receptor isolated from both dexamethasone-sensitive and -resistant tumors by DEAE-cellulose chromatography have similar sedimentation coefficients (4–5 S) which are unaffected by molybdate.     

Model analysis of circadian rhythms in mouse epidermal basal cell proliferation

Computer simulations were made of circadian variations in six observed cell kinetic variables in the basal cell layer of hairless mouse epidermis. Different mathematical models were used and simultaneously fitted to all observed variables by an automatic method. The analysis shows that the origin of the circadian variations in cell kinetics in hairless mouse epidermis is not in the G₁ phase or at the $\text{G}{}_1\text{S}$ transition alone as concluded from other studies, but must result from circadian variations in the duration of S and G₂ phases. The results show that the data are compatible with the existence of circadian variations in the G₁- and M-phase durations, although such variations, unlike the S and G₂ variations, was not necessary to obtain a good fit of the model to the data. The results also indicate that the data are compatible with the existence of transient resting states of limited duration in S- and G₂-phases.     

Hemoglobin Rothschild (β37(C3)Trp → Arg): A high/low affinity hemoglobin mutant

In hemoglobin Rothschild arginine replaces the normal tryptophan at β37(C3), at α₁β₂ contact. Residue β37 is in close proximity to Argα92 (FG4). Substitution of Trp by Arg at β37 results in two positively charged Arg residues at FG4 and C3 facing each other, a situation that would destabilize the subunit constraints essential for the tetrameric integrity of the molecule and for the reduced ligand affinity of unliganded normal HB³ compared to isolated chains.Our studies show liganded HbR is extensively dissociated into dimers and has a high ligand affinity in phosphate buffer and a low ligand affinity in bis-Tris at alkaline pH. Kinetic studies indicate that in the T state HbR has a higher ligand affinity than HbA. This is explained by reduced subunit constraints in the T state and dissociation of the monoliganded species (Hb₄L) into dimers. Kinetic studies also show that R state Hb Rothschild has lower ligand affinity than R state HbA. These results are explained on the basis of extensive dissociation of R state Hb Rothschild into dimers and lower ligand affinity of dimers as compared to triliganded tetramers (α₂β₂(O₂)₃). Kinetic data indicate that the lower ligand affinity of dimers (Hb Rothschild) as compared to that of triliganded tetramers (HbA) is due to the increased ligand dissociation rates in the case of oxyhemoglobin and reduced ligand combination in the case of carboxyderivatives. Both the CO combination reaction time-course around 425 nm and the O₂ dissociation rates at 437.8 nm indicate the presence of large α,β-chain differences in Hb Rothschild.