How does lysozyme penetrate through the bacterial outer membrane?

Lysozyme fails to penetrate through the outer membrane of stationary phase cells of Escherichia coli when it is simply added to suspensions of plasmolyzed cells. Lysozyme penetrates the outer membrane only when these cells are exposed to a mild osmotic shock in the presence of EDTA and lysozyme.In the presence of Mg²⁺, the outer membrane is stabilized sufficiently so that there is no lysozyme penetration during osmotic shock. If Mg²⁺ is added after an osmotic shock has been used to cause lysozyme to penetrate a destabilized outer membrane, the outer membrane is stabilized once again. In this case however, cells are converted to spheroplasts by the lysozyme which has gained access to the murein layer prior to the addition of Mg²⁺. Mg²⁺ stabilizes the outer membranes of these spheroplasts sufficiently so that they remain immune to lysis even in the absence of osmotic stabilizers such as sucrose.These results are discussed in terms of current information on the structure of the murein layer and the outer membrane.     

Effects of phosphatase inhibitors and a protein phosphatase on norepinephrine secretion by permeabilized bovine chromaffin cells

A protein phosphatase and phosphatase inhibitors were used to examine the role of protein phosphorylation in the regulation of norepinephrine secretion in digitonin-permeabilized bovine chromaffin cells. Addition of okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, or 1-naphthylphosphate, a more general phosphatase inhibitor, to digitonin-permeabilized chromaffin cells caused about a 100% increase in the amount of norepinephrine secreted in the absence of Ca²⁺ (in 5 mM EGTA) without affecting the amount of norepinephrine secreted in the presence of 10 μM free Ca²⁺. This stimulation of norepinephrine secretion by protein phosphatase inhibitors suggests that in the absence of Ca²⁺ there is a slow rate phosphorylation and that this phosphorylation triggers secretion. Addition of an exogenous type 2A protein phosphatase caused almost a 50% decrease in Ca²⁺-dependent norepinephrine secretion. Thus, the amounts of norepinephrine released both in the absence of Ca²⁺ and in the presence of Ca²⁺ appear to depend upon the level of protein phosphorylation.     

The crystallographic study of left-handed Z-DNA d(CGCGCG)2 and thermine complexes crystallized at various temperatures and at various concentration of cations

In crystals of complexes of thermine and d(CGCGCG)₂ molecules grown at 4, 10, and 20 °C, the numbers of thermine molecules connected to the DNA molecule were dependent on the temperature of the crystallization. Two molecules of thermine and one Mg²⁺ ion were connected to DNA molecule when thermine and d(CGCGCG)₂ were co-crystallized at 4 and at 20 °C. When an increased concentration of magnesium and thermine molecules were co-crystallized with d(CGCGCG)₂ molecules at 10 °C, three Mg²⁺ ions and only one thermine molecule were bound with a d(CGCGCG)₂ molecule. The number of polyamines and of Mg²⁺ ions connected to DNA was dependent on the atomic values of the polyamine and of the metal ion. The binding of more Mg²⁺ ions occurred when the atomic value of Mg²⁺ exceeded that of the corresponding mono- or polyamine, and when the Mg²⁺ ion concentration was elevated. Furthermore, this study is the first documentation of a naturally occurring polyamine bound to the minor groove of DNA in a crystal structure.     

Effect of inotropic agents on the calcium binding to isolated cardiac sarcolemma

Ca²⁺ binding to fragmented sarcolemma isolated from canine heart was measured by an ultracentrifugation technique. Two classes of binding site with dissociation constants of 2.0 · 10^?5 and 1.2 · 10^?3 M were identified. The capacities of the high- and low-affinity sites were 15 and 452 nmol/mg, respectively. These sites were not affected by treatment with neuraminidase. The effects of various cations and drugs on Ca²⁺ binding were studied. All cations tested inhibited Ca²⁺ binding with the following order of potency: trivalent > divalent > monovalent cations. The order of potency for the monovalent ions was: Na⁺ > K⁺ > Li⁺ ? Cs⁺ and for the divalent and trivalent ions: La³⁺ ? Mn²⁺ > Sr²⁺ ? Ba²⁺ > Mg²⁺. 1 · 10^?3 M caffeine and 1 · 10^?8 M ouabain increased the capacity of the low-affinity sites to 1531 and 837 nmol/mg, respectively. 1 · 10^?7 M verapamil, acidosis (pH 6.4), 1^?10^?5 M Mn²⁺ and 1 · 10^?4 M ouabain depressed the capacity of the low-affinity sites to a range of 154–291 nmol/mg. The dissociation constants of the high- and low-affinity sites and the capacity of the high-affinity sites were not affected by these agents.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Androgen receptor complex binding in murine skeletal muscle nuclei

Examination of binding of androgen-receptor complexes from murine skeletal muscle cytosol was performed by modified nuclear retention assay and modified nuclear acceptor assay. The experiments showed the binding of androgen-receptor complexes to the nuclear acceptor sites to be a cooperative process. Hill analysis of the data obtained resulted in a Hill coefficient of 3,6. The apparent dissociation constant for binding of cytosolic [³H]-testosterone-receptor complexes to nuclei was found to be in the range of K_D = 6 ? 8 × 10^?11 M. The nuclear matrix was able to bind androgen-receptor complexes in a saturable way, too.     

Inhibitory effect of ethylenediaminetetraacetate (EDTA) on the porcine lymphocyte response to mitogens and reversal of the effect with metal ions

The effect of ethylenediaminetetraacetate (EDTA) on the mitogen response of porcine lymphocytes and the role of metal ions in reversal of the inhibitory effect of EDTA were determined. Porcine lymphocyte responses to mitogens were totally suppressed when serum used to supplement Ca²⁺, Mg²⁺-free minimum essential medium (MEM) was dialyzed against saline or saline with 0.2 or 0.60 mM EDTA, but the responses were only partially reduced when the same serum was added to RPMI-1640 medium. The inhibition observed in MEM could be reversed by adding 1×10⁻³ M Ca²⁺ and 1×10⁻³ M Mg²⁺ to the dialyzed serum. Serum treated directly with 0.60 mM EDTA completely suppressed blastogenesis in lymphocyte cultures maintained in RPMI-1640 or Ca²⁺, Mg²⁺ free MEM. The inhibitory effect of EDTA-treated serum could be completely reversed by adding Zn²⁺ or a combination of Zn²⁺ with other cationic ions, or partially reversed by adding Ni²⁺ or Fe³⁺. Zn²⁺ was the most effective ion, in that it was the only ion that, when alone added to the serum, could completely restore lymphocyte responses to phytohemagglutinin (PHA) or pokeweed mitogen (PWM).     

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity

The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylimidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 X 10(-6) M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.     

Studies on the luminescent response of the Ca2+-activated photoprotein,obelin

1. The kinetics and stoicheiometry of the Ca²⁺-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K⁺, Na⁺, Ca²⁺, Mg²⁺ and H⁺. 2. The results suggest Ca²⁺-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca²⁺] and indicate that changes in [Ca²⁺] over the range 1 · 10^?6?3 · 10^?4 M are followed significantly faster by the obelin response (approx. 3 ms delay at 20°C) than by the aequorin response (approx. 10 ms delay at 20°C). 3. Obelin was found to emit low-intensity light (less than 10^?6 of the maximum Ca²⁺-activated response), which was independent of Ca²⁺ at concentrations below about 10^?7 M. The level of this Ca²⁺-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increases in the concentration of K⁺, Na⁺, Mg²⁺ and H⁺ generally shift the Ca²⁺ activation curve for obelin toward higher Ca²⁺ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca²⁺ nor the quantum yield were modified by these substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca²⁺ movements, had little effect on the Ca²⁺-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca²⁺ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin respones under a variety of ionic conditions at room temperature.     

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca²⁺ or Mg²⁺ and was not affected by F-actin and ouabain. V_max was 2.8 and 10.0 μmol P_i/mg protein per h for Ca²⁺- and Mg²⁺-dependent activity, respectively, and the corresponding K_m was 4.8·10^?4 M and 3.2·10^?4 M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Effects of human choriogonadotropin on mitochondrial and microsomal cytochrome P-450 levels in mouse testes

The mitochondrial and microsomal cytochrome P-450 contents of $\text{C57B1}\text{6}$ mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis.     

Cooperative binding of calcium to glycerinated skeletal muscle fibers

The binding of ⁴⁵Ca²⁺ to glycerinated rabbit psoas fibers was measured by means of a double isotope technique. With 5 mM Mg²⁺ (no ATP) binding was half-maximal at 1.4 · 10^?6M Ca²⁺ and the maximal amount bound was 1.6 μmol/g protein. At < 50% saturation, the Scatchard plot had a positive slope and the Hill coefficient was 2.2. At greater than 50% saturation, the Scatchard plot was linear with a negative slope (K′ = 0.8 · 10⁶ M^?1) and the Hill coefficient was 1.0. In the absence of Mg²⁺, binding was half-maximal at 3 · 10^?7 M Ca²⁺ and the maximal amount bound was 2.9 μmol/g protein. The Scatchard plot indicated two classes of sites with K′ values of about 2 · 10⁷ and 2 · 10⁶ M^?1. The Hill coefficient in the mid-saturation range was approx. 0.6. The data indicate that in the presence of Mg²⁺ binding to about half of the total Ca²⁺ binding sites is suppressed and there is a strong positive cooperativity involving half of the remaining sites.     

Photosynthetic Prokaryotes. Cell Differentiation and Functions : Edited by G.C. Papageorgiou and L. Packer Elsevier Biomedical; New York, 1983 405 pages. $65.00

A protein (designated as luffin) with an app. M_r of 26000, which inhibits protein synthesis in rabbit reticulocyte lysate, was purified to homogeneity from the seeds of Luffa cylindria roem by extraction with 20 mM Na phosphate buffer (pH 7.2) containing 0.2 M NaCl, ammonium sulfate fractionation, and chromatography on Sephacryl S-200 and CM-Sephadex C-25. Luffin exhibited 10-times as strong inhibitory activity against protein synthesis in rabbit reticulocyte lysate (IC₅₀, 0.42 ng/ml) as that of ricin A-chain, but it showed only a weak cytotoxicity against murine leukemia L1210 cells, an activity of 1/10⁶ to 1/10⁵ that of ricin.     

Energy-dependent endocytosis in erythrocyte ghosts. IV. Effects of Ca2+, Na+ + K+, and 5'-adenylylimidodiphosphate

The requirement of actual splitting of ATP for endocytosis in erythrocyte ghosts has been confirmed by use of the ATP analog, 5'-adenylylimidodiphosphate. (AMP-P(NH)P. This compound, in which the oxygen connecting the β and γ phosphorus atoms was replaced by an NH group, did not cause endocytosis nor was it a substrate for ATPase activity. AMP-P(NH)P was a competitive inhibitor both for the endocytosis and the Mg²⁺-ATPase activities. The $\text{K}{}_{\mathrm{<mtext>1</mtext>}}$ of AMP-P(NH)P for Mg²⁺-ATPase activity was 2.0 · 10^?4 M and, while the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of ATP for this activity was also 2.0 · 10^?4 M indicating nearly identical affinities of ATP and AMP-P(NH)P for the active site. ADP, or ADP plus orthophosphate, did not cause endocytosis, showing that endocytosis was not due to binding of the products of ATP hydrolysis. Sodium or potassium ion or ouabain had no effect on endocytosis, which eliminated the possibility of involvement of the Na⁺, K⁺ ATPase in the endocytosis process. Calcium could not be substituted for magnesium; rather it inhibited endocytosis at the concentration of 1 · 10^?3 M. EGTA relieved the inhibitory effect of Ca, which indicated that the binding of calcium to the membrane was reversible. These experimental results reaffirm the conclusion that ATP must be split to engender endocytosis under these conditions. Some characteristic parameters of the hemoglobin-fre porcine erythrocyte ghosts were studied in order to characterize the system more adequately.     

DNA distortion and specificity in a sequence-specific endonuclease

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca²⁺, Mg²⁺, and Mn²⁺) are presented. While previous structures were produced from soaking Ca²⁺ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca²⁺, Mg²⁺, or Mn²⁺. The Mn²⁺-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca²⁺ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.     

Apparent nuclear localization of unoccupied receptors for 1,25-dihydroxyvitamin D3

Previous studies have demonstrated that unoccupied 1,25-dihydroxyvitamin D₃ receptors are associated with crude chromatin under hypotonic conditions $\text{in}\text{vitro}$. The data presented herein show that unoccupied 1,25-dihydroxyvitamin D₃ receptors appear to be associated with chromatin prior to solubilization by dilution/homogenization in both high and low salt buffers. Additionally the unoccupied receptors are recovered nearly quantitatively from purified nuclei. These results suggest that unoccupied 1,25-dihydroxyitamin D₃ receptors may be localized within nuclei $\text{in}\text{vivo}$.     

Vasopressin-sensitive kidney adenylate cyclase: Modulation of the hormonal response

Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg²⁺, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg²⁺, and GMPPNP, respectively. Mg^2+·ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg²⁺ interacts with an enzyme regulatory site. Finally, Mg²⁺ can modulate the hormonal response, with Mg²⁺ions affecting the coupling function–that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg²⁺ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg²⁺ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30° C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15° C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10^?8 M to 10^?5 M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg²⁺ concentrations except for the increase in velocity when raising Mg²⁺ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms.