Gel electrophoretic studies on ribosomal proteins L7L12 and the Escherichia coli 50 s subunit

Three forms of the 50 S ribosomal subunit of Escherichia coli have been separated by agarose/acrylamide gel electrophoresis. The slowest migrating form, S-50 S, corresponded to native 50 S subunits and contained four copies of proteins $\text{L}\text{7}\text{L}\text{12}$. Removal of the four copies of this protein produced a more rapidly migrating form, M-50 S. The M-50 S form was then converted to the fastest migrating form, F-50 S, by removal of additional proteins, including L10 and L11. A one-step removal of a pentameric complex of four copies of $\text{L}\text{7}\text{L}\text{12}$ plus L10 converted the S-50 S subunit directly to the F-50 S subunit. These proteins recombined specifically with the appropriate protein-deficient 50 S subunit at 3 °C to reform the S-50 S subunit, i.e. the M-50 S subunit was converted back to the S-50 S form by the addition of purified proteins $\text{L}\text{7}\text{L}\text{12}$; and the F-50 S subunit bound the pentameric complex of $\text{L}\text{7}\text{L}\text{12}$ and L10 to form S-50 S. The binding of the pentameric complex, isolated by glycerol gradient centrifugation, supports the model that all four copies of proteins $\text{L}\text{7}\text{L}\text{12}$ are together in one part of the ribosome called the “$\text{L}\text{7}\text{L}\text{12}$ stalk”. Only the four copies of $\text{L}\text{7}\text{L}\text{12}$ were removed from the 50 S subunit in low salt (0.125 m-NH₄Cl) plus 50% ethanol at 0 °C. These ribosomes (in the M-50 S form) had less than 5% of the peptide-synthesizing activity of untreated control ribosomes as measured by a poly(U) translation system in vitro. Peptide-synthesizing activity was restored, upon addition of $\text{L}\text{7}\text{L}\text{12}$, back to the treated ribosomes to give 50 S subunits (S-50 S) with a full complement of four copies of $\text{L}\text{7}\text{L}\text{12}$. Antibody to proteins $\text{L}\text{7}\text{L}\text{12}$ bound only to the S-50 S subunits, producing four new bands separated by gel electrophoresis. The bands represented complexes of one, two, three and four antibodies bound to a 50 S subunit. This result was obtained using either 50 S subunits or 70 S tight couples and indicated that all four copies of $\text{L}\text{7}\text{L}\text{12}$ are either located at a single site in the $\text{L}\text{7}\text{L}\text{12}$ stalk or, much less likely, are divided between two symmetrical sites. Proteins $\text{L}\text{7}\text{L}\text{12}$ were not only accessible to their specific antibody but could also be removed from 70 S ribosomes and polyribosomes without causing their dissociation into subunits. The ribosomes and polyribosomes had an increased gel electrophoretic mobility which was reversed by addition of proteins $\text{L}\text{7}\text{L}\text{12}$.     

Evidence for two types of binding of 3H-gaba and 3H-muscimol in rat cerebral cortex and cerebellum.

The binding of n$\text{M}$ concentrations of ³HGABA and ³Hmuscimol to synaptosomal membrane preparations from different areas of rat brain were studied by a radioreceptor assay. The characteristics of binding, with respect to kinetic parameters and inhibition of binding by nonradioactive GABA, before and after detergent treatment, suggest the presence of at least two types of binding at putative GABA receptor sites.     

The occurrence of glutamate synthase in algae.

The presence of glutamate synthase in the green algae $\text{Chlorella fusca}$ var. $\text{vacuolata}$ has been demonstrated using a whole cell assay as well as cell free extracts. The assay is complicated by the presence of glutamine (amino): α-oxoglutarate transaminase, but this enzyme can be inhibited by amino oxyacetate. The rates of glutamate synthase activity are sufficient to account for the known rates of nitrate assimilation to occur via the glutamine synthetase/glutamate synthase pathway.     

Prey capture by the cuttlefish (Sepia officinalis L): An experimental study of two strategies

This study shows that the size of the prey ($\text{Carcinus maenas}$) relative to the predator ($\text{Sepia officinalis}$) is of importance in the choice between two types of attack: either capture by ejection of the two extensible tentacles, or capture by jumping on the prey. Small crabs are preferentially captured by the first method and large crabs by the second. Other factors which may explain the observed variations, include previous experience of the predator and the behaviour of the prey.     

Ribosomal proteins L7/L12 localized at a single region of the large subunit by immune electron microscopy.

Ribosomal proteins $\text{L}\text{7}\text{L}\text{12}$ have been mapped by immune electron microscopy. These multiple copy proteins are located at a single region extending from the large subunit, known as the $\text{L}\text{7}\text{L}\text{12}$ stalk. The $\text{L}\text{7}\text{L}\text{12}$ stalk is approximately 100 Å long, about 40 Å wide and extends at an angle of approximately 50 ° from one side of the central protuberance of the large subunit. In the monomeric 70 S ribosome, the portion of the $\text{L}\text{7}\text{L}\text{12}$ stalk proximal to the 50 S subunit is located in the vicinity of the 30 S-50 S interface.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibody binding to the stalk was shown to be solely dependent upon the presence of $\text{L}\text{7}\text{L}\text{12}$ by the following experiments. Sucrose gradient analysis was used to demonstrate that large subunits depleted of $\text{L}\text{7}\text{L}\text{12}$ were unable to bind anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ restored the ability of $\text{L}\text{7}\text{L}\text{12}$-depleted cores to react with anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies. Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies pre-absorbed with $\text{L}\text{7}\text{L}\text{12}$ did not react with 50 S subunits.Anti-$\text{L}\text{7}\text{L}\text{12}$ antibodies used in these experiments reacted only with the $\text{L}\text{7}\text{L}\text{12}$ stalk and with no other region of the subunit. This was shown by electron microscopy and by immune electron microscopy in the following ways. Electron microscopy of 50 S subunits, $\text{L}\text{7}\text{L}\text{12}$-depleted 50 S cores, and reconstituted 50 S subunits was used to demonstrate that stripping removes the $\text{L}\text{7}\text{L}\text{12}$ stalk from more than 95% of the subunits, and that re-incorporation of $\text{L}\text{7}\text{L}\text{12}$ into depleted cores restores the $\text{L}\text{7}\text{L}\text{12}$ stalk. Double-labelling experiments, using monomeric subunits with two or more attached anti-$\text{L}\text{7}\text{L}\text{12}$ immunoglobulins, were used to demonstrate, independently of 50 S subunit morphology, that $\text{L}\text{7}\text{L}\text{12}$ are located only on the $\text{L}\text{7}\text{L}\text{12}$ stalk.     

Catechol-O-methyltransferase from rat liver: two forms having different meta:para methylation ratios.

Cytoplasmic catechol-O-methyltransferase activity from rat liver was resolved by gel filtration into two enzymes: a major form having an estimated molecular weight of 23,000 and a minor one of 45,500. The relative abundance of these forms in liver is about 5:1, respectively. Microsomal catechol-O-methyltransferase constituted only 2% of the total liver activity. After solubilization by sonication most of the microsomal enzyme showed a molecular weight in excess of 100,000, but some 23,000 - enzyme was also released. The bound enzyme thus may represent an aggregate form of the soluble activity. The two cytoplasmic enzymes differ in several properties, including pH optima and thermal stability. The two forms also differ in the extent of methylation of the $\text{para}$ hydroxyl group, the larger enzyme having a $\text{meta}$:$\text{para}$ methylation ratio twice that obtained with the smaller form.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.     

A comparison of DNA content between two substrains of Escherichia coli B/r.

The average DNA content per cell was measured in steady-state cultures of two substrains of $\text{E. coli}\text{B}\text{r}$ growing at various rates at 37°C. The DNA content of substrain $\text{B}\text{r}$ F was consistently lower than that of substrain $\text{B}\text{r}$ A. It is suggested that the differences in DNA contents are consequences of strain-specific differences in the relationship between chromosome replication and the division cycle of $\text{E. coli.}$     

Characterization of two new crystal forms of uteroglobin.

Two new crystal forms of oxidized uteroglobin have been obtained. An orthorhombic one ($\text{P2}{}_1\text{2}{}_1\text{2, Z = 2, a = 44.48 (5)}\text{A}\text{?}\text{, b = 36.93 (5)}\text{A}\text{?}\text{, c = 32·34 (5)}\text{A}\text{?}$) and a monoclinic one ($\text{P2}{}_1\text{, Z = 2, a = 44.56 (5)}\text{A}\text{?}\text{, b = 46.06 (5)}\text{A}\text{?}\text{, c = 37.43 (4)}\text{A}\text{?}\text{, β = 120.92 ° (5)}$). Both were grown at pH ~7.0 and diffract to a resolution of 2·1 to 2·2 Å. Data collections for native crystals have been recorded with an automatic four-circle diffractometer.     

Identification of ortho-octopamine and meta-octopamine in mammalian adrenal and salivary gland.

$\text{Ortho}$- and $\text{meta}$- octopamine have been identified in beef and rat adrenal gland and in rat salivary gland by means of gas chromatography-mass spectrometry. The tritrifluoroacetyl derivatives of $\text{ortho}$-, $\text{meta}$- and $\text{para}$- octopamine were resolved by gas chromatography and shown to produce two characteristic ions at $\text{m/e}$ 315 and $\text{m/e}$ 328. The di-O-trimethylsilyl-N-trifluoroacetyl derivatives of these three isomers were also resolved by gas chromatography and shown to produce a characteristic ion at $\text{m/e}$ 267. Biological samples were homogenized in formic acid:acetone, subjected to ion-exchange chromatography and then derivatized. When the derivatized biological extracts were examined for each characteristic ion, peaks were observed at the exact retention times of the standards. The three isomers are present in adrenal gland in concentrations of ～1 μg g^?1 and in rat salivary gland in concentrations of ～0.1 μg g^?1. This evidence confirms a previous report of the presence of $\text{m}$-octopamine in rat salivary gland measured by a radiochemical enzyme assay and is the first report of the presence of $\text{o}$-octopamine in biological tissue.     

The effect of ionising radiation and chemical methylation upon the activity and accuracy of E. COLI DNA polymerase I

The activity of $\text{E. coli}$ DNA polymerase I decreases on treatment with γ-rays, methylnitrosourea or dimethyl sulphate. In the case of the first two agents the decrease in activity is accompanied by a decrease in the accuracy of the enzyme in an $\text{in vitro}$ assay. There is no detectable change in the ratio of DNA polymerase activity to 3′→5′ exonuclease activity on treatment.     

The foundation of two distinct cell lineages within the mouse morula

The division of single cells, isolated from an 8-cell mouse embryo, to give $\text{2 ×}\text{1}\text{16}$ cells has been studied by sampling cells for analysis at defined stages during and after the division. Cells were analyzed for evidence of polarity in their surface organization as assessed by fluorescent ligand binding and distribution of microvilli. Individual $\text{1}\text{8}$ cells are polarized. At division, most (82%) divide such that both the pole of ligand binding and the pole of microvilli are distributed to only one of the two daughter cells. A couplet is thereby formed with a large polar cell and a small apolar cell. Some $\text{case1}\text{8}$ cells divide through the pole, generating a couplet of two polar cells, the poles being contiguous at the midbody. Elements of the surface polarity observed in the $\text{1}\text{8}$ cells can be found at all stages throughout division. Analysis of couplets of cells derived from newly formed 16-cell morulae also reveals that most consist of a polar:apolar pair and some consist of a polar:polar couplet in which the poles are contiguous at the midbody.The results indicate that two distinct cell populations are generated at division. These cells are known to occupy different positions within the morula, the polar cells being peripheral and the apolar cells being central. Since peripheral and central cells give rise to trophectoderm and inner cell mass in the blastocyst, we therefore suggest that the foundation of the trophectoderm and inner cell mass lineages may occur by a process of differential inheritance. This conclusion supports the recently proposed polarization hypothesis, which is discussed.     

Resistance to (L)-azetidin-2-carboxylic acid in Bacillus subtilis.

A spontaneous mutant of $\text{Bacillus subtilis}$ resistant to (L)-azetidin-2-carboxylic acid, a structural analogue of (L)-proline, has been isolated and characterized. Data have been obtained which indicate that $\text{in vitro}$ binding of [¹⁴C]-proline to tRNAs from the resistant strain is reduced only in part by (L)-azetidin-2-carboxylic acid, while a complete inhibition of binding occurs using tRNAs from parental strain. The mutation conferring resistance to the analogue and a mutation for proline auxotrophy have been mapped.     

Interaction between thymopoietin and facteur thymique serique in the rosette inhibition assay.

In the rosette inhibition assay, thymopoietin was active and its activity was enhanced in the presence of high concentrations of ubiquitin. Facteur thymique serique (FTS) was active in the same assay, but its activity was completely inhibited by high concentrations of ubiquitin. Mixtures of thymopoietin and FTS showed activity both in the presence or absence of ubiquitin depending on the concentration of thymopoietin or FTS in the mixtures. There seemed to be an important biologic interaction of thymopoietin and FTS when presented as a mixture, suggesting that thymopoietin and FTS might interact $\text{in}\text{vitro}$ and possibly $\text{in}\text{vivo}$.     

Suppression of iron uptake deficiency in Escherichia coli K-12 by loss of two major outer membrane proteins.

A novel iron uptake system was observed in pseudorevertants of $\text{Escherichia}$,$\text{coli}$ strains defective in ferrienterochelin transport. The new system is unique in that it is an active transport system that does not utilize any known siderophore. Acquisition of the new uptake system occurs concomitantly with the loss of two major outer membrane proteins (b and c) believed to function as structural components of transmembrane pores.     

Chloramphenicol damages bacterial DNA.

L(+)-$\text{threo}$-chloramphenicol induces reversion of His^?$\text{Salmonella typhimurium}$ strains TA100 and TA1535 in the conventional Ames' assay without microsomal activation. Any mutagenicity of D(?)-$\text{threo}$-chloramphenicol was masked by toxicity. Similarly, a sensitive fluctuation test showed mutagenesis with L(+)-$\text{threo}$-chloramphenicol at concentrations of 0.5 μM and above but the D(?) isomer proved to be toxic even at these low levels. The L(+) isomer caused single strand breaks in the DNA of $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ strains TA1535, TA100 and TA1976. The D(?) isomer caused breaks in $\text{Escherichia coli}\text{B}\text{r}$ and $\text{Salmonella typhimurium}$ TA1976 although it was less effective and it did not produce DNA breaks in TA1535 or TA100.     

Improving utilization of crop residues. Evaluation of two chemicals used to improve digestibility of crop residues

Two chemical treatments involving chelating metal caustic swelling (CMCS) and sodium hydroxide (NaOH) were evaluated for their ability to affect in vitro dry matter and in vitro cellulose disappearance (IVDMD and IVCD, respectively), dry matter disappearance in sacco and the chemical composition of two low-quality crop residues, cornstalks (CS) and soya bean residue (SBR). At $\text{chemical}\text{substrate}$ ratios ranging from 0.25:1 to 5:1, linear increases in IVCD of both roughages were noted. Improvements in IVCD of CS and SBR were noted at 1:1, 3:1 and 5:1$\text{chemical}\text{substrate}$ ratios, regardless of whether the treated roughages were used immediately after treatment (fresh basis) or preserved by drying or freezing. Decreased concentrations of neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL) and crude protein (CP) resulted from chemical treatment. No effects of the water component of the solvent solutions were observed. Dry matter disappearance in sacco of CMCS- and NaOH-treated roughages was increased at all $\text{chemical}\text{substrate}$ ratios tested. Treatment of either roughage with CMCS resulted in greater digestibility in sacco than did treatment with NaOH. Treatment of CS with CMCS resulted in higher rates of digestion than did treatment with NaOH. No differences in rates of digestion of SBR were noted between chemicals. Both chemicals were more effective in improving digestibility of CS than of SBR.     

The exchange and maximal net flux of glucose across the human erythrocyte. II. The effect of two sulphydryl enzyme inhibitors,chlormerodrin and p-chloromercuribenzene sulfonic acid

The exchange and maximal net fluxes of [¹⁴C]glucose across the membrane of the human red cell were measured. The effects of $\text{p-}\text{chloromercuribenzene}$ sulfonic acid and chlormerodrin on these two parameters of glucose transport were determined. At low concentrations $\text{p-}\text{chloromercuribenzene}$ sulfonic acid (a non-penetrating organic mercurial) was found to readily inhibit the exchange flux but not the net efflux. At extremely high concentrations of $\text{p-}\text{chloromercuribenzne}$ sulfonic acid the maximal net efflux showed some degree of inhibition. Chlormerodrin (a penetrating organic mercurial) inhibited both the exchange and net fluxes in the same manner. The addition of insulin in certain instances reduced the degree of inhibition caused by the organic mercurials. Insulin had no effect on the amount of either $\text{p-}\text{chloromercuribenzene}$ sulfonic acid or chlormerodrin which bound to the red cell. From the results obtained, it is suggested that there exist glucose-reactive sites on both the outer and inner surfaces of the membrane. The results also suggest a carrier system possessing different sites or molecular arrangements for glucose egrees and for glucose entry.     

The nature of the pituitary gonadotropins and their role in ovulation in a urodele amphibian (Ambystoma tigrinum).

Gonadotropic hormones were fractionated from pituitaries of the urodele amphibian $\text{Ambystoma}$$\text{tigrinum}$ (tiger salamander) by methods previously employed to separate these hormones in other species of tetrapod vertebrates. These procedures yielded two distinct fractions that resembled the FSH and LH from other species in regard to their biological profiles in several nonmammalian bioassays and by their chromatographic behavior. The $\text{Ambystoma}$-FSH was free of LH activity (< 0.1%). The major $\text{Ambystoma}$-LH was highly potent in ovulation assays for LH; it also had a high activity in the $\text{Anolis}$ lizard assay, but it is not clear whether this reflects high intrinsic activity or incomplete separation of FSH.$\text{In vitro}$ studies with these and other (frog, turtle, mammalian) gonadotropins indicate that the induction of ovulation in $\text{Ambystoma}$, as in anurans, is highly specific for LH, independent of the source of the gonadotropins. These data support the view that two separate gonadotropins existed early in tetrapod evolution.     

Structural investigations of mode of action of drugs. I. Molecular structure of mitomycin C.

The crystal and molecular structure of the antitumor antibiotic mitomycin C has been determined by X-ray diffraction. The space group is monoclinic P2₁ with cell dimensions $\text{a}\text{=77.988(3)}$, $\text{b}\text{=20.355(7)}$, $\text{c}\text{=9.679(3)}\text{A}\text{?}$, β=95.99°(1) and Z=4. The structure was solved by direct methods. There are two independent molecules per asymmetric unit. The structure was refined to an R value of 0.049 for 2151 observed reflections measured on diffractometer. The benzoquinone ring is slightly deviated from planarity. The N4 in the indole ring behaves like an amide nitrogen owing to its participation in the conjugated benzoquinoid system. The five membered ring through C1, C2, C3, N4 and C9a adopts an envelope conformation. The molecules are held together in crystal by the hydrogen bonds. All nitrogens except N4 are involved in the hydrogen bonding. Studies with models of drug and DNA indicate two different kinds of mechanism for crosslinking.