Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Secretion of proteoglycans by chondrocytes. Influence of colchicine, cytochalasin B, and beta-D-xyloside.

Chondrocytes obtained from epiphyseal cartilage of fetal guinea pigs or ear cartilage of young rabbits were cultured in monolayer. The influence of colchicine, cytochalasin B, and p-nitrophenyl-β-d-xylopyranoside on secretion of proteoglycans was investigated. Radioactive sulfate was used as a precursor. As observed previously in other systems, β-d-xylosides initiated the synthesis of free chondroitin sulfate chains, competing with the endogenous proteoglycan core protein acceptor. The molecular weights of the chondroitin sulfate chains synthesized both on the xyloside and on the core-protein acceptor in maximally stimulated cells were similar and significantly lower than in proteoglycans synthesized in the absence of xyloside. The size of the chondroitin sulfate chains synthesized on the xyloside was inversely related to the concentration of this compound. This finding suggests that the chain length is dependent on the ratio between available acceptor and chain-lengthening enzymes or precursors. Cytochalasin B, a microfilament-modifying agent, inhibited proteoglycan synthesis, without any effect on secretion. Cells treated with cytochalasin B could be stimulated with β-d-xyloside to synthesize free chondroitin sulfate chains to the same relative degree as cells with intact microfilaments. Colchicine, an antimicrotubular agent, partially inhibited synthesis and secretion of proteoglycan. However, cells treated with colchicine could be stimulated with β-d-xyloside to synthesize and secrete free chondroitin sulfate chains to about the same relative degree as cells with intact microtubules. The data suggest that microtubules may have a facilitatory rather than an obligatory role in the secretion of proteoglycans and that at least part of the effect of colchicine is located at or after the site of glycosaminoglycan synthesis.     

Relaxation studies on complex formation of macrocyclic and open chain antibiotics with monovalent cations

The stability constants for the 1 : 1 complexes of macrocyclic antibiotics (nonactin, monactin, dinactin and trinactin) with Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺, NH⁺₄ and for the Na⁺-complexes with the open chain compounds nigericin and monensin in methanol solution have been determined. The relaxation amplitude method was employed to obtain both the equilibrium constants and the enthalpies of reaction. The kinetics were studied with the help of temperature-jump, electric-field pulse and ultrasonic absorption techniques. Although complex formation of the metal ions with the antibiotics involves multidentate ligand chelation, the formation rates are in general very high, i.e. close to the limits imposed for diffusion controlled processes. The data for the macrotetrolides indicate the existence of conformational transition prior to complexation. A sequential substitution or “redressing” mechanism is proposed which is in accord with the high rates of complex formation. The selectivity patterns, as expressed by the equilibrium constants, are similar to those observed for the transport of metal ions across membranes in presence of the antibiotics. Selectivity results from an optimal balance between the strength of metal ion solvation and the stability of the individual metal complex, which in turn is governed by the conformational flexibility of the antibiotics.     

Conformation studies of 13 trinucleoside diphosphates by 360 MHz PMR spectroscopy. A bulged base conformation. I. Base protons and H1' protons

The 360 MHz NMR spectra of the base protons and the H1 protons of thirteen trinucleoside diphosphates have been analyzed. The sequences chosen represent all purine-pyrimidine sequences. The chemical shifts of the base protons give evidence for strong next nearest-neighbor effects in some oligonucleotides. Although increasing chain length usually increases nearest-neighbor base-base stacking, it is not always so. Comparing ApCpG, ApUpG and GpUpG to their component dimers, one finds a decrease in stacking of the center pyrimidine with the purine on either side. The coupling constants J 1'2' also show that these three trimers show less stacking for their terminal residues than expected from their component dimers. We conclude that the sequence Pu-Py-Pu favors a conformation in which the pyrimidine is bulged out and the two purines stack on each other.     

Induction of anti-hapten antibody response by hapten-isologous carrier conjugate. II. Specificity of hapten-reactive helper T cells.

Anti-hapten antibody production was elicited by the immunization of hapten-isologous carrier conjugate (PAB-MGG) in mice. Spleen and lymph node cells taken from these primed mice could demonstrate their helper activity for anti-DNP antibody production when transferred intravenously into 600R X-irradiated recipient mice along with DNP-primed B cells and the double hapten conjugated carrier, DNP-MGG-PAB. Isologous carrier (MGG)-primed cells could not demonstrate this helper activity. Accordingly, helper cells reactive for a haptenic group are considered to develop by the immunization of hapten-isologous carrier conjugate. Hapten-reactive helper activity was also induced by the immunization of other hapten-isologous carrier conjugates, e.g., MAB-MGG, PABS-MGG or PAB-MSA. These hapten-reactive helper cells were T lymphocytes, as the helper activity of PAB-MGG-primed cells was completely abolished by in vivo ATS-treatment. Helper activity of PAB-MGG-primed cells for DNP-primed B cells was also demonstrated through the double hapten conjugated heterologous carrier DNP-HGG-PAB to be the same as with DNP-MGG-PAB, but weakly through DNP-KLH-PAB. As HGG but not KLH resembles MGG in composition, almost all hapten-reactive helper T cells can be considered to recognize not only haptenic groups but also physicochemical properties of the hapten-conjugated carrier site. However, these helper T cells could discriminate structural differences among related haptenic groups, because PAB-MGG-primed cells clearly responded to DNP-MGG-PAB to demonstrate their helper activity for DNP-primed B cells, but responded only weakly to DNP-MGG-PABS or DNP-MGG-MAB. When the specificity restrictions of T and B cells to the same haptenic group were compared by responsiveness measured after the antigenic stimulation (B cell function by anti-hapten antibody production and T cell function by helper activity), differences were noted, as PAB-MGG-primed T cells could respond not only to DNP-MGG-PAB but also fairly well to DNP-MGG-MAB to demonstrate their helper activity, but PAB-MGG-primed B cells responded to only PAB-MGG. Thus, hapten specificity appears to be much more restricted for B cells than T cells. The difference of this responsivity between B cells and helper T cells was thought to derive from the specificity difference of B cell and helper T cell receptors rather than from any sensitivity differences of the experimental procedure. The differences in the specificity restrictions of receptors of B and helper T cells were discussed in the light of hapten-specificity.     

The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts.

Collagen secretion by chick embryo fibroblasts was measured by incorporating [¹⁴C]proline into proteins and then analyzing the amount of collagen in the cell and medium separately by using purified bacterial collagenase. In order to produce varying levels of hydroxylation, cells were incubated with varying concentrations of ascorbate or with varying concentrations of α,α′-dipyridyl in the presence of saturating ascorbate. Ascorbate stimulated both the hydroxylation of proline in collagen and the secretion of collagen; the concentration of ascorbate required for half-maximal stimulation of both proesses was approximately 4.5 × 10^?7, m. Since the cells could concentrate ascorbate 10-fold, this K_M for proline hydroxylation is 100-fold lower than values reported for purified prolyl hydroxylase (Abbot, M. T., and Udenfriend, S. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.), p. 173, Academic Press New York; Kivirikko K. I., et al. (1968) Biochim. Biophys. Acta, 151, 558–567). Conversely, α,ga′-dipyridyl inhibited both proline hydroxylation and collagen secretion; half-maximal inhibition of both processes was observed at 7 × 10^?5, m. The results of the two types of experiments show that the secretion of collagen becomes directly proportional to proline hydroxylation when approximately 30% of the proline residues in collagen have been hydroxylated compared to maximal hydroxylation of 50%. Since the stability of triple-helical collagen at 37 °C has been shown to be dependent on the hydroxyproline content of the molecule (Rosenbloom, J., et al. (1973) Arch. Biochem. Biophys., 158, 478–484), we suggest that the observed proportionality between secretion and hydroxylation is a reflection of the increased amount of stable triple helical collagen at 37 °C. When the cells were incubated with a concentration of ascorbate that was saturating for secretion and hydroxylation, there was no significant activation of prolyl hydroxylase as measured in a cell-free extract. These experiments suggest that ascorbate effects collagen secretion by acting at the site of proline hydroxylation but not by increasing the activity of prolyl hydroxylase.     

Mechanisms involved in the expression of Jones-Mote hypersensitivity: II. Lymph node morphology and in vitro correlates

Lymph node morphology, in vitro lymphocyte transformation, and inhibition of macrophage migration were studied at varying intervals after sensitization for Jones-Mote hypersensitivity (JMH) with ovalbumin (OA) in Freund's incomplete adjuvant (FIA). The effect of cyclophosphamide (CY, 300 mg/kg), given 3 days before sensitization with OA in FIA, was also studied in an attempt to clarify further its role in increasing the intensity of skin reactions and its effect on the passive transfer of skin reactivity described in the preceding paper. There were increased numbers of large pyroninophilic cells in paracortical areas of draining lymph nodes and increased in vitro DNA synthesis, by lymph node cells, in animals treated with CY 3 days before sensitization with OA in FIA. There was no inhibition of macrophage migration of PEC from animals sensitized with OA in FIA, whether or not these guinea pigs had been treated with CY before sensitization.     

Altered interaction between binding and catalytic subunits of a cyclic AMP-stimulated protein kinase from hepatoma cells.

Binding activity obtained from an established line of hepatoma tissue culture (HTC) cells has a lower apparent affinity for cyclic AMP at physiological pH than has the analogous binding activity from rat liver. However, the apparent binding affinity of HTC preparations can be reversibly increased by adding NaCl or guanidine · HCl. In the presence of such activating substances, a macromolecular inhibitory activity has been chromatographically separated from the cyclic AMP-binding activity. Removal of this inhibitory component causes the apparent affinity of the cyclic AMP-binding activity from HTC cells to increase and resemble that observed with liver preparations. Before treatment with salt, the inhibitory activity seems to be physically associated with the binding activity. Adding the isolated inhibitory component back to a suitably activated binding preparation from HTC cells results in a decrease in the apparent affinity for cyclic AMP. The isolated inhibitory component is devoid of cyclic AMP-binding and cyclic AMP phosphodiesterase activities and has an apparent minimal molecular weight of about 30,000 by gel filtration. It possesses protein kinase activity and seems to be identical to the catalytic subunit of a cyclic AMP-stimulated protein kinase on the basis of chromatographic properties and sensitivities to heat and low pH. This catalytic subunit represents only a minor portion of total cellular protein kinase activity and is also present in liver extracts. However, the binding activity from liver is not inhibited significantly under conditions where the binding from HTC cells is affected by the catalytic subunit. The difference in this inhibitory response between liver and HTC preparations appears to reflect differences in the cyclic AMP-binding proteins themselves.     

Sodium-dependent amino acid transport in preimplantation mouse embryos. III. Na+-k+-atpase-linked mechanism in blastocysts.

Mouse blastocysts collapse in cytochalasin B (CB), reexpand (accumulate fluid) in control medium, but cannot reexpand in ouabain, an inhibitor of Na⁺K⁺-ATPases. These ATPases, then, seem to be necessary for fluid accumulation in blastocysts. Since intact blastocysts are relatively insensitive to ouabain, CB seems to make it possible for ouabain to reach the Na⁺K⁺-ATPases localized on the blastocoelic surface. CB-Collapsed blastocysts were found to transport alanine and lysine at the same rate as intact blastocysts, indicating that, in 1 hr, amino acids are transported into the cells of the intact blastocyst, and not into the fluid-filled blastocoel. Transport rates in CB-collapsed blastocysts do not exceed those in intact blastocysts, suggesting that hypothetical amino acid carriers are located only on the external blastocyst surface. Most important, ouabain strongly inhibits sodium-dependent alanine transport in CB-collapsed blastocysts, but not in intact blastocysts, providing strong evidence that Na⁺K⁺-ATPases, localized on the blastocoelic surface, are necessary for this transport. Ouabain does not inhibit sodium-independent lysine transport in CB-collapsed blastocysts. Thus, the dependency of both sodium-dependent amino acid transport and fluid accumulation upon Na⁺K⁺-ATPases, and the separate localization of amino acid carriers and these ATPases, provides functional evidence for an epithelial tissue type of mechanism for sodium-dependent amino acid transport in mouse blastocysts.     

Creatine kinase, myokinase, and acetylcholinesterase activities in muscle-forming primary cultures of mouse teratocarcinoma cells.

Multipotential mouse teratocarcinoma cells in embryoid bodies were explanted on plastic or collagen substrates. Various modes of cell determination, including myogenesis, occurred. The predominant avenue of differentiation soon became myogenesis: many multinucleated myotubes formed and yielded an extensive network of skeletal muscle fibers. The process does not proceed to normal completion, as the fibers have a paucity of striations and are not contractile. Activities of several enzymes ordinarily associated with muscle differentiation were examined. Acetylcholinesterase activity increases, especially during myotube formation, as in normal myogenesis. However, creatine kinase activity rises during myotube formation and then drops abnormally, and myokinase activity fails to increase appreciably. The fetal isozymic form of creatine kinase is expressed in the cultures, although well differentiated solid tumors taken from mice show attainment of the adult muscle isozyme type if skeletal muscle is demonstrably present. The results are consistent with the interpretation that coordinately regulated changes in gene expressions controlling these functions may be required for later stages of myogenesis.     

Structural studies of human chorionic gonadotropin and its subunits using tyrosine fluorescence.

The fluorescence properties of the tyrosyl residues of human chorionic gonadotropin (hCG) and its α and β subunits have been examined. The effects of pH, guanidine, and disulfide cleavage on the intensity and polarization of the fluorescence suggest that the isolated subunits possess little, if any, tertiary structure beyond that which is stabilized by the disulfide bonds. Essentially all of the fluorescence of hCG and its subunits was accessible to quenching by iodide ions. Similar results were observed for several other proteins whose fluorescence originates from tyrosyl residues. Thus, we have confirmed and extended the conclusion of R. W. Cowgill ((1966) Biochim. Biophys. Acta120, 196) that the buried tyrosyl residues in ribonuclease fluoresce with a much lower quantum yield than those which are exposed. The dissociation of hCG into its subunits was accompanied by an increase in fluorescence, suggesting the exposure of tyrosyl residues. This was confirmed by difference absorption measurements which indicate a net exposure of two to three tyrosyl residues upon dissociation of the subunits. An additional 0.6 tyrosine was exposed when the disulfide bonds of the β-subunit were cleaved. The polarization of the fluorescence of hCG-β was high (P = 0.19) and, unlike several other proteins with high polarization, could not be lowered by denaturing conditions. Only by cleavage of the disulfide bonds could the fluorescence polarization of either subunit be lowered to a value (P = 0.08) characteristic of a random polypeptide. It appears that the disulfide bonds play an important role in maintaining the rigidity of the fluorescent tyrosyl residues, located at or near the surface of the protein.     

Electron spin resonance spin label studies of intercalation of nitrobenzene in DNA.

Nitrobenzene-DNA intercalation mechanisms have been studied by means of electron spin resonance spin label techniques. Of the seven derivatives prepared and examined, 2,4-dinitrobenzene analogs with amine linkage to the nitroxide reporter demonstrate the strongest binding with DNA by intercalation, and the reporter nitroxide is oriented 45 ° to the plane of the benzene ring and is due primarily to the steric hindrance of the 2-nitro substituent. This binding is found to be largely dependent upon the number of nitrosubstituents, their relative position on the benzene ring, and the type of linkage between the ligand and the nitroxide reporter, suggesting that polarization bonding is a major driving force in their complex formation with DNA.     

Failure of sensory adaptation in bacterial mutants that are defective in a protein methylation reaction.

Chemotactic bacteria, such as E. coli, detect changes in the chemical composition of the environment. Addition of an attractant or repellent leads to an immediate response, characterized by a change in the swimming behavior of the cells--a process known as sensory excitation. However, the response gradually disappears with time, despite the continued presence of the chemical--a process known as sensory adaptation. We report here the behavior of a class of nonchemotactic mutants (cheX) that can carry out sensory excitation but are defective in the process of sensory adaptation. These mutants are also defective in the ability to carry out a protein methylation reaction which has previously been implicated in the adaptation process (Goy, Springer and Adler, 1977). The results presented here establish a firm relationship between the methylation reaction and sensory adaptation.     

Cytostatic and cytolytic activities of macrophages regulation by prostaglandins

Murine peritoneal macrophages (M phi), activated in vivo or in vitro, remarkably inhibited the uptake of thymidine by a lens epithelial cell line, while resident M phi, or M phi induced by thioglycollate, exhibited much lower or no cytostatic capacity. The target cells were partially protected from the cytostatic activity by the anti-inflammatory agents indomethacin, aspirin, and dexamethasone, but not by lipoxygenase inhibitors. The protective activity of indomethacin and aspirin, but not of dexamethasone, was completely counteracted by prostaglandin E2 (PGE2). Yet, PGE2 alone has no effect on the uptake of [3H]thymidine by lens epithelial cells. PGE1 resembled PGE2 in its effect on this system, whereas PGA2, PGB2, or PGF2 alpha had no detectable activity. The counteracting effect of PGE2 was mimicked by dibutyryl cAMP or by cholera toxin, an agent which increases cAMP levels. These findings suggest that PGEs are not direct cytostatic agents, but rather, are essential mediators for the development of the cytostasis. Activated M phi did not lyse cells of the original lens epithelial cell line, but caused substantial cytolysis of cells of a subline derived from it. In contrast to its aforementioned effect on the cytostasis, PGE2 inhibited the cytolytic activity of M phi. Thus, this study provides a first demonstration in a single system of the opposite effects of PGEs on M phi activity on target cells, i.e., mediating the cytostasis and inhibiting the cytolysis.     

Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues

The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH₃ (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ were found to be 0.05 × 10^?7 (A), 0.02 × 10^?7 (B), and 1.6 × 10^?7 (C) [(NIH units/ liter)s]^?1. The value of$\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ for peptide C is less than 1% of that for fibrinogen [although the value of k_cat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P₉ and P₈, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin.     

A facile tritium release assay for mammalian L-dihydroorotate dehydrogenase

Proteinase B activity in crude yeast extract is difficult to detect, unless the firmly bound peptide inhibitor is previously inactivated. Methods currently used to accomplish this inactivation suffer from serious limitations, particularly when applied to crude extracts obtained from yeast grown in complete media. These limitations are discussed on the basis of the kinetics of activation. A novel procedure to unmask proteinase B activity, based on pepsin treatment of crude extracts, is described. Specific activity of proteinase B, determined after pepsin A treatment, is always two to three times higher than that obtained by other methods. The method proposed is simple, reliable, and can be applied to crude extracts stored at ?20°C for up to 1 week, repeatedly giving the same specific activity for proteinase B.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Isolation of a circular derivative of yeast chromosome III: implications for the mechanism of mating type interconversion.

We describe genetic and physical characterization of rearrangements of chromosome III which result in changes of cell type in S. cerevisiae. Two types of rearrangements were obtained as rare events which caused a change at the locus controlling cell type, MAT, associated with a recessive lethal mutation, in one case from MATalpha to MATa-lethal, and in the other case from MATa to MATalpha-lethal. The MATa-lethal mutation is a deletion on the right arm of chromosome III, which we demonstrate extends to (or near) HMalpha. We suggest this deletion removes MATalpha and activates cryptic MATa information stored in HMalpha as proposed in the cassette model of mating type interconversion. The MATalpha-lethal mutation is the result of the formation of a circular chromosome III, which we interpret to remove MATa and activate the cryptic MATalpha information stored at HMa. Strains carrying the MATalpha-lethal chromosome contain a circular chromosome of length 62.6 plus or minus 5.7 mum, which is absent in related strains. This chromosome was confirmed to be chromosome III by hybridization of specific yeast DNA fragments to supercoiled DNA obtained from MATalpha-lethal strains. The isolation of a large circular derivative of chromosome III allows correlation of genetic and physical distance based on large distances-1 centimorgan corresponds to approximately 2700 base pairs.     

Gas chromatography--mass spectrometry studies of tetramethyl uric acids

Of the 12 possible isomers of tetramethyl uric acid, 6 are obtained by methylation with diazomethane and 4 by on column methylation with trimethylphenyl ammonium hydroxide. Elution patterns on three different columns, quantitative data, and mass spectra are presented. In addition, elution data and the mass spectrum of 8-methoxy-1,3,8-trimethylxanthine are given. The results may be useful for studies of incorporation of [¹³C]glycine and other stable isotope precursors into uric acid.