The effect of metyrapone on the 17,20-lyase from rat testis microsomes

A new and simple method was presented to isolate purified holoenzyme of $\text{E. coli}$ RNA polymerase. When a purified enzyme preparation was chromatographed on a DNA-cellulose column equilibrated with a buffer containing 10mM MgCl₂, holoenzyme was separated from core enzyme. Thus holoenzyme was eluted at 0.15M KCl and core enzyme at 0.25M KCl.     

Hydrodynamic properties of 5.8S rRNA,salt and temperature effects

Sedimentation coefficients and apparent molecular masses of 5.8S rRNA from rat liver and yeast (Saccharomyces cerevisiae) depend considerably on the ionic strength and the kind of ions in solution. At 20°C the sedimentation coefficient of 5.8S rRNA in 10 mm sodium cacodylate, pH 7.0, amounts to 5.1 ± 0.2 S. By addition of NaCl up to 1.1 m the data increase reversibly to 6.1 ± 0.2 S (rat liver) or 5.4 ± 0.1 S (yeast) without significant changes of the molar mass (52 000 ± 2000) g/mol. Similar effects but with different extent were obtained using KCl or LiCl. These results can be explained by counterion effects on the conformation and changing of the water shell surrounding the RNA molecule. Short heat incubation (5 min at 65°C) and immediate cooling of rat liver 5.8S rRNA lead to dimer or oligomer formation. Its portions depend strongly on RNA concentration and are enhanced also with increasing NaCl concentration and incubation temperature as can be seen fro higher sedimentation coefficients and molecular masses as well as from additional bands in the electrophoretic pattern. At 20°C MgCl₂ provokes, in concentrations up to 1.5 mm, a reversible increase of sedimentation coefficients of rat liver 5.8S rRNA to 6.65 ± 0.1 S whereas the molecular mass remains unchanged indicating strong Mg⁺⁺ effects on conformation and/or water shell of the 5.8S rRNA. A further increase of sedimentation coefficients up to 8.2 ± 0.1 S combined with higher apparent molar masses up to 90 000 g/mol was observed in the presence of 30 to 50 mm MgCl₂. In this concentration range of Mg⁺⁺ the association constants of 5.8S rRNA dimerization increase from about $\text{10}{}^5\text{to}\text{3 × 10}{}^7\text{m}{}^{\mathrm{?1}}$. After removal of free Mg⁺⁺ by addition of EDTA the 5.8S rRNA dimers dissociate if no incubation step at higher temperature in involved. The Mg⁺⁺ induced 5.8S rRNA dimers differ in their stability from those formed by incubation at 65°C in the presence of higher concentrations of monovalent ions.     

Cryopreservation of Plasmodium chabaudi I: Protection by glycerol and dimethyl sulfoxide during cooling and by glucose following thawing

Two additives, glycerol and dimethyl sulfoxide (Me₂SO), were investigated for toxic and protective effects for the intraerythrocytic stages of Plasmodium chabaudi. After incubation for 15 min, at 0 °C in Me₂SO and at 37 °C in glycerol, with various concentrations of these additives, half the blood from each treatment was cryopreserved in glass capillary tubes cooled at approximately 3600 °C min^?1 by plunging into liquid nitrogen. Warming was rapid, approximately 12000 °C min^?1, produced by agitation in a water bath at 40 °C for 1 min. The effect of dilution in phosphate-buffered saline (PBS) supplemented with various concentrations (5 to 25% $\text{v}\text{v}$) of glucose was also investigated in conjunction with the two cryoprotectants. Survival of both the frozen and the unfrozen control parasites was assayed by the mean time taken for the parasitemia in groups of five mice to reach a level of 2% following intraperitoneal injection of 10⁶ parasitized erythrocytes into each mouse. Glycerol was toxic at concentrations above 10% $\text{v}\text{v}$ and Me₂SO above approximately 15%. The use of glucose in the recovery medium resulted in a substantial improvement in the survival of frozen and unfrozen parasites previously incubated in either cryoprotectant. The amount of glucose required varied with the concentration of additive used, and optimum survival of cryopreserved parasites was obtaind with 10% $\text{v}\text{v}$ glycerol or 15% $\text{v}\text{v}$ Me₂SO and with 15% $\text{w}\text{v}$ glucose in the diluent medium.     

Glycerol fomation inDunaliella cells under non-growing conditions with hypertonic lithium or magnesium media

Glycerol formation ofDunaliella cells in non-growing media was investigated.Dunaliella tertiolecta andD. bioculata grew well in a NaCl medium but not at all in a LiCl or a MgCl₂ medium. When the cells originally suspended in a medium containing 0.5 M NaCl were transferred to media which contained one of 1 M NaCl, 1 M LiCl or 0.7 M MgCl₂, the intracellular glycerol content increased.D. tertiolecta cultured in either a 1 M LiCl or a 0.7 M MgCl₂ medium did not multiply, but maintained abilities to evolve O₂ in the light and absorb O₂ in thedark even after about a 5 day culture. From these results, it can be concluded that the halotolerance ofDunaliella to different kinds of salts is not directly related to osmoregulation by the glycerol formation.     

Viral nucleocapsid melting to measure protein: RNA interactions

Thermal denaturation of nucleocapsids of wild type (WT) vesicular stomatitis virus (VSV), containing only the nucleocapsid protein (N) and viral RNA, caused a “melting” that resulted in an A_260nm absorbance increase of 140%. The nucleocapsids of two temperature-sensitive ($\text{ts}$) VSV mutants, $\text{ts}$ G31BP and $\text{ts}$ G22, both underwent larger absorbance increases of 251% and 177% respectively, suggesting these nucleocapsids are complexed by weaker N protein: RNA interactions than the WT-VSV. Two other mutants, $\text{ts}$ G31 and $\text{ts}$ G41 underwent A_260nm increases either similar to, or smaller than, that measured with WT-VSV nucleocapsids. RNA synthesis by $\text{ts}$ G31BP in infected cells was also found to be decreased at elevated temperatures. This temperature sensitive defect in viral RNA metabolism in $\text{ts}$ G31BP may be the result of weaker protein:RNA interactions associated with the nucleocapsid.     

Control of puffing activity in three chromosomal segments of explanted salivary gland cells of Chironomus thummi by variation in extracellular Na+,K+ and Mg2+

Salivary glands of Chironomus thummi larvae were incubated in media composed of those $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{or}\text{MgCl}{}_2\text{KCl}$ combinations and at concentrations which they tolerated without visible damage. Resulting changes in puffing activity were recorded for three chromosomal segments. Within certain combinatorial ranges $\text{NaCl}\text{KCl}\text{,}\text{MgCl}{}_2\text{NaCl}\text{and}\text{MgCl}{}_2\text{KCl}$ induced puffs in the three segments. Each inducing range is depicted as a ‘puff-inducing field’ for extracellular ion concentrations (IF^e) in a two-dimensional lattice. The IF^e_s are coherent, distinctly delineated and highly overlapping. At most places a transition from 0 to 100 % puff induction (induction of respective puff in each nucleus) depends on changes in media composition of 10–20 mM. Ion sensitivities of the three chromosomal segments were computed for NaCl/KCl/MgCl₂ combinations and were found to conform to actual puff-inducing capacities of selected NaCl/KCl/MgCl₂ media.     

Isolation of ACTH1-39,ACTH1-38 and CLIP from the calf anterior pituitary

Calf anterior pituitaries were defatted and homogenized and peptides were adsorbed from the homogenate supernatant onto octadecylsilyl-silica. After elution, the resulting extract was subjected to gradient elution reversed-phase high pressure liquid chromatography (RP-HPLC) using aqueous acetonitrile containing 0.1% ($\text{v}\text{v}$) trifluoroacetic acid (TFA). Radioimmunoassay of column fractions for corticotropin (ACTH) revealed three major areas of immunoreactivity. Each was purified to homogeneity by gradient elution RP-HPLC employing aqueous acetonitrile containing either 0.13% heptafluorobutyric acid ($\text{v}\text{v}$) or 0.1% TFA ($\text{v}\text{v}$). Amino acid analysis and exopeptidase and trypsin digestions revealed the three forms of corticotropin to be ACTH_1–38, corticotropin-like intermediary lobe peptide, (CLIP, ACTH_18–39) and ACTH_1–39. ³H-labeled ACTH_1–39 did not give rise to either ³H-ACTH_1–38 or ³H-CLIP during isolation.     

The NAD-linked α-glycerophosphate dehydrogenase of trypanosomes

NAD-linked α-glycerophosphate dehydrogenase plays a key role in the α-glycerophosphate cycle of $\text{Trypanosoma brucei}$. The activity in cell lysates was ample for this role. The enzyme was activated by salts (e.g. MgCl₂ or NaCl); it had a broad pH-optimum for the reduction of dihydroxyacetone phosphate centred at pH 7.4, with an apparent K_m of 0.5 mM; and it was weakly bound to particulate components of cell lysates. The enzyme from $\text{T. vivax}$ was similar to that of $\text{T. brucei}$. These trypanosomal enzymes resemble that of the trypanosomatid $\text{Crithidia fasciculata}$, but are rather different from the enzymes of mammals, birds and insects.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

Immunochemical detection of multiple conformations within a 36 base pair oligonucleotide.

A 36 base pair chimeric oligonucleotide containing a central core of DNA duplex flanked by RNA/DNA hybrid at each end was synthesized. These distinct regions of the oligonucleotide adopt different conformations which were detected with antibody probes. Enzyme linked immunosorbent assays (ELISA) and a gel electrophoresis retardation assay were used to demonstrate the binding of antibodies which recognize B-DNA, Z-DNA and RNA/DNA hybrid. The DNA duplex core of this oligonucleotide adopts the B-conformation in 0.14 M NaCl. In high salt solution (4 M NaCl) the DNA core adopts the Z-conformation. The RNA/DNA hybrid at the ends of the oligomer adopt a conformation which is distinct from both B-DNA and A-RNA.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.     

Picosecond time-resolved study of MgCl2-induced chlorophyll fluorescence yield changes from chloroplasts

The MgCl₂-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp $\text{?At}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, where A was found to be 0.052 $\text{ps}{}^{\mathrm{<mtext>?1</mtext><mtext>2</mtext>}}$, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl₂ produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.     

Kinetics of active and passive components of water exchange between the air and a mite, Dermatophagoides farinae

Female North American house dust mites were found to exchange water with the ambient air from two compartments. At humidities above the critical equilibrium activity (CEA), transpiration out of a single large compartment was observed using HTO as a tracer for water. Total sorption into this compartment was also observed by following changes in the specific radioactivity. The sorption data required that an active process or pump be present. The water in this pump is the second compartment above the CEA. Below the CEA the large compartment could be identified as a compartment characterized by a small transpiration rate constant. The pump below the CEA becomes a rapidly transpiring fast compartment. By separating the water pool into two compartments, it was possible to relate a_v to k and $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$. The major effect of a_v on k was related to its effect on the permeability of the cuticle. The influence of a_v on $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was different for active and passive sorption. Above the CEA the pump operated at full capacity and active $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was directly proportional to a_v. Passive sorption was influenced by a_v in two ways. The driving force for $\text{m}\text{?}{}_{\mathrm{<mtext>S</mtext>}}$ was further reduced below saturation by the effect of a_v on the permeability of the exchange surface.     

The presence of polyadenylate sequences in the ribonucleic acid of a higher plant

Rice callus tissue in suspension culture was labeled $\text{in}$$\text{vivo}$ with ³²PO₄^?3. Approximately 4% of the total RNA extracted from the tissue could be bound to nitrocellulose filters in the presence of 0.5 M KCl. The base composition of this material was: A, 30.6; U, 32.7; G, 18.0; and C, 18.6%. If the RNA was digested with both T₁ and pancreatic ribonucleases prior to binding, the base composition of the bound material was: A, 97.8; U, 0.0; G, 1.6; and C, 0.5%. About 14% of the RNA isolated from purified polyribosomes could be bound to filters in 0.5 M KCl. It is concluded that higher plant RNA contains polyadenylate sequences.     

Expression and subcellular distribution of mouse cytochrome P1-450 mRNA as determined by molecular hybridization with cloned P1-450 DNA

Cytochrome P₁-450 (P₁-450) is defined as that cytochrome most closely associated with 3-methylcholanthrene (MC)-induced aryl hydrocarbon hydroxylase (AHH) activity. Recently a cloned DNA sequence (clone 46) was shown to represent a portion of the P₁-450 structural gene [Negishi $\text{et}\text{al.}\text{,}\text{Proc. Nat. Acad. Sci. U.S.A.}\text{78}$: 800–804 (1981)]. Poly(A⁺)-enriched RNA was isolated from total liver homogenate, membrane-bound polysomes and from free polysomes at various times after MC treatment of $\text{Ah}$-responsive $\text{C57BL}\text{6N}$ (B6) and $\text{Ah}$-nonresponsive $\text{DBA}\text{2N}$ (D2) inbred mice. The poly(A⁺)-enriched RNA was separated by methylmercury-agarose gel electrophoresis and hybridized to nick-translated [³²P]DNA from clone 46. By means of this RNA-DNA hybridization, only 6% of total polysomal P₁-450 mRNA exists in free polysomes after 24 h of MC treatment. The data indicate that the endoplasmic reticulum is the principal site of synthesis for this integral microsomal protein.Studies of induction kinetics following MC treatment provided the evidence of the rapid increase of total liver and membrane bound P₁-450 mRNA preceding the synthesis of apo-P₁-450 and the increase of AHH activity.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

A hemoprotein from azotobacter containing non-heme iron: isolation and crystallization

A new type of hemoprotein was isolated from extracts of $\text{Azotobacter vinelandii}$ using a heat step, application of the protamine-cellulose phosphate procedure, and precipitation with MgCl₂. Crystallization was achieved using low concentration of MgCl₂. The protein was shown to be a unique cytochrome of the b type containing up to 20% non-heme iron but no “acid labile sulfur”. Both the heme and non-heme iron appear to be functional redox components.