Saccharomyces cerevisiae killer expression mutant kex2 has altered secretory proteins and glycoproteins.

The extracellular proteins and glycoproteins of a yeast mutant $\text{kex}\text{2–15}$ defective in killer toxin expression were separated by one and two dimensional polyacyylamide gel electrophoresis. Many mutant extracellular proteins and glycoproteins show both altered electrophoretic mobility and isoelectric points when compared with the parent strain. Altered proteins and glycoproteins from $\text{kex}\text{2–15}$ were identified with their parental counterparts by peptide mapping. The observed alterations co-segregated with the $\text{kex}\text{2}$ nuclear mutation in genetic crosses.     

Acidic proteins from Saccharomyces cerevisiae ribosomes.

Two dimensional gel electrophoresis of ribosomal proteins from $\text{Saccharomyces}\text{cerevisiae}$ reveals the presence of three spots in the region corresponding to proteins of high acidic character. Washing the ribosomes with 0.4 M NH₄Cl and 50% ethanol, followed by chromatography of the extracted proteins on DEAE-cellulose, indicated the presence of two fractions of acidic proteins; (A and Ax), having very similar molecular weights (12.000–13.000), but phosphorylated to different extents. Fractions A and Ax are immunologically distinct and their immunologic properties differ from acidic proteins found in $\text{Escherichia}\text{coli}$, rat liver, and $\text{Artemia}\text{salina}$ ribosomes.Protein A can be resolved into two bands by electrofocusing, and two dimensional gel electrophoresis. The two components correspond to proteins L44 and L45 according to the standard nomenclature. Proteins Ax seems to correspond to the spot that moves above and to the left of L44 and L45 and is at least three times more phosphorylated than these two proteins.     

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 $\text{L}\text{7}\text{L}\text{12}$.     

Ribosomal proteins L1, L17 and L27 from Escherichia coli localized at single sites on the large subunit by immune electron microscopy

Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “$\text{L}\text{7}\text{L}\text{12}$ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the $\text{L}\text{7}\text{L}\text{12}$ side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the $\text{L}\text{7}\text{L}\text{12}$ stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins.     

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.     

Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range

The effects of nonsaturating amounts (5–60 nmol/mg membrane protein) of $\text{p-}\text{chloromercuribenzoate}$ on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher $\text{p-}\text{chloromercuribenzoate}$ concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled $\text{p-}\text{chloromercuribenzoate}$. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to $\text{p-}\text{chloromercuribenzoate}$. Other reagents including $\text{N-}\text{ethylmaleimide}$, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.     

Synthesis and decay of lambda DNA replication proteins in minicells

The coliphage λ DNA replication proteins, the $\text{O}$- and $\text{P}$-gene products, have been identified by infection of nonpermissive $\text{Escherichia coli}$ minicells with the appropriate λ amber mutants as proteins of a molecular weight of about 34000 and 23000, respectively. Proteins of exactly the same size were found in minicells harbouring the plasmid $\text{λ}\text{dv}$. Both proteins seem to be synthesized at the same rate. In λ-infected minicells, as well as in $\text{lambda;}\text{dv}$-harbouring minicells the pulse-and-chase experiments have shown an exceptionally rapid decay of the O-protein.     

Mass spectrometric identification of various molecular forms of dynorphin in bovine adrenal medulla

Deliberate miscompartmentalization of liver outer mitochondrial membrane (OMM) proteins and liver mitochondrial proteins has been achieved by polyethylene-glycol mediated OMM vesicle-hepatocyte or mitochondrial-hepatocyte fusion. Reductively methylated OMM and mitochondrial proteins (³H) are destroyed at rates remarkably similar to those for OMM ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 60–70 h) or mitochondrial proteins ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, 84–104 h) in liver $\text{in vivo}$ when studied over 4–5 days in hepatocyte monolayers cultured in conditions giving stabilized endogenous protein catabolic rates mimicking endogenous $\text{in vivo}$ rates. Destruction of transplanted OMM proteins is partially sensitive to chloroquine, supporting some lysosomally mediated autophagic destruction of long-lived transplanted OMM proteins in hepatocyte monolayers.     

An endodextranase inhibitor from batch cultures of Streptococcus,mutans

An inhibitor of $\text{Streptococcus}$,$\text{mutans}$ endodextranase was detected in proteins prepared from batch cultures of $\text{S.}$,$\text{mutans}$ strains representing serotypes $\text{a}$ through $\text{g}$. Affinity chromatography of strain 6715-49 proteins, which apparently were free of endodextranase activity, yielded an active endodextranase and, in a separate peak, the endodextranase inhibitor. The presence of the inhibitor in culture fluids accounts for the absence of endodextranase activity in batch-grown cultures of $\text{S.}$,$\text{mutans}$ known to produce this enzyme.     

Calcium-dependent regulation of NAD kinase.

An activator protein of NAD kinase from the pea, $\text{Pisum}\text{satavum}$ L., has been shown to be Ca²⁺-dependent. This plant activator protein also stimulates the activity of modulator protein dependent-cyclic nucleotide phosphodiesterase from porcine brain. This stimulation is similar to that observed with modulator protein isolated from animal sources. Furthermore, Ca²⁺-dependent modulator proteins isolated from porcine brain, bovine brain, and the coelenterate, $\text{Renilla}$, will regulate the NAD kinase activity of peas. Other common properties of the plant activator protein and animal modulator proteins are their acidic nature, heat stabilities, similar Stokes' radii, and their interactions with troponin I.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.     

Ionophore A23187 raises cyclic AMP levels in macrophages by stimulating prostaglandin E formation.

A class of non-histone chromatin proteins that were bound tightly to DNA and could not be dissociated from the chromatin by high salt and urea was isolated from HeLa cell nuclei and separated from DNA by DNase digestion. These ‘tight’ proteins retained their ability to bind to single- and double-stranded DNA as assayed by nitrocellulose filter binding. Polyacrylamide gel electrophoresis showed that the most prominent proteins possessed molecular weights of about 60 000 D. In asynchronously growing HeLa cell cultures about $\text{1}\text{3}$ of the cell nuclei were immunoreactive to fluorescein-labeled antinucleoside antibodies. The immunoreactive cells were the fraction in S phase. Cycloheximide treatment of the cultures raised the fraction of immunoreactive nuclei to over $\text{sol2}\text{3}$. Exposure of the fixed cycloheximide-treated cell to tight proteins prior to staining with the antibody reduced the fraction of immunoreactive cells to the normal S phase level. Immuno-reactivity induced by X-irradiation or by the intercalating mutagen hycanthone was also suppressed by tight proteins. Cycloheximide treatment preferentially reduced the cellular content of tight proteins, suggesting that these proteins undergo a metabolic turnover with a half-life of about 5 h.     

Amino acid sequence homology between “α” subunits from Torpedo and Electrophorus acetylcholine receptor

The amino terminal amino acid sequence of the 41,000 dalton subunit of $\text{Electrophorus}\text{electricus}$ acetylcholine receptor has been determined for 35 cycles by automated sequencing procedures. Comparison of the unique polypeptide sequence obtained for this molecule with that of the major subunit of $\text{Torpedo}\text{californica}$ acetylcholine receptor reveals extensive primary structural homology between the two proteins.     

The sites of synthesis and the subsequent migration of newly synthesized protein in retina

Radioautographs of rabbit retinas fixed immediately after a 1 or 2 min exposure in vitro to ³H leucine revealed high rates of protein synthesis in receptor cell inner segments, perikarya of ganglion cells, and cells of the inner nuclear layer. If these brieflly labelled retinas were returned to unlabelled medium for periods of up to 6 hr, the radioautographs revealed a progressive dispersion of the labelled proteins from their sites of synthesis. This was largely completed by $\text{1}\text{1}\text{2}$ hr and appeared, in one instance at least, to involve processes other than simple diffusion. Superimposed on the dispersive phenomenon was a process of concentration of the newly formed proteins at two sites quite distant from their synthesis, that was apparent after $\text{1}\text{1}\text{2}$hr. One of these sites was the receptor cell outer segments, as has been previously described, the other was the outer plexiform layer.     

Involvement of 50S ribosomal proteins L6 and L10 in the ribosome dependent GTPase activity of elongation factor G

CsCI-prepared 50S cores in the presence of groups of individual split proteins were tested for their capacity to support EF-G dependent GTP hydrolysis. The activity of cores prepared at 40 mM Mg²⁺ could be restored by adding $\text{L7}\text{L12}$ and L10 together, each in an amount of two copies per 50S particle, which abolishes the difference in activity between L7 and L12. In the range of 20-2 mM Mg²⁺, 50S cores lose the protein L6, which is also required for GTP hydrolysis. L10 cannot replace L6, or $\text{vice versa}$.     

The presence of multiple cytochrome b proteins in succinate-cytochrome c reductase.

Two cytochrome $\text{b}$ proteins were isolated from succinate-cytochrome $\text{c}$ reductase and the cytochrome $\text{b-c}{}_1$ complex. Their molecular weights were determined to be 37,000 and 17,000 daltons by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Spectral properties and amino acid composition of these two proteins are reported in the paper.     

DNA-binding proteins of Xenopus laevis: synthesis during oogenesis-ovulation and early embryogenesis

DNA-binding proteins of Xenopus laevis synthesized during two periods of early development (oogenesis-ovulation and early embryogenesis) were co-chromatographed on DNA-cellulose. Proteins with an affinity for DNA were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most of the proteins eluted from DNA-cellulose with 0.6 M NaCl had mol. wts less than 40 000; some of these proteins were synthesized to a greater extent by developing embryos than by oocytes. A DNA-binding protein or group of proteins with a mol. wt of approx. 70 000 was synthesized during oogenesis-ovulation but not during embryogenesis. Differential labeling of developing embryos with $\text{[}{}^3\text{H}\text{]}\text{tryptophan}$ and $\text{[}{}^{14}\text{C}\text{]}\text{lysine}$ indicated that some of the low mol. wt DNA-binding proteins are histones. Some of these proteins also incorporated monosodium $\text{[}{}^{32}\text{P}\text{]}\text{phosphate}$. A greater fraction of the proteins synthesized by oocytes and developing embryos were bound to DNA-histone-cellulose than to DNA-cellulose. A group of low mol. wt proteins made during oogenesis-ovulation were bound more to DNA-histone-cellulose than were proteins with similar mol. wts made by developing embryos.     

Localization of the lactose permease protein(s) in the E. coli envelope.

The amino acid double labeling technique was used to identify and localize membrane-bound lactose operon proteins in $\text{E.}\text{coli}$. Both the “M” protein, thought to be the $\text{y}$ gene product, and a polypeptide of MW ～15,000 appeared in the membrane following $\text{lac}$ operon induction. The amounts of these two proteins were approximately equal.The inner and outer membrane layers of the cell envelope were separated by sucrose density gradient centrifugation or by selective solubilization of inner membranes with the detergent Sarkosyl. When gentle lysis conditions were employed to prepare membrane vesicles, both $\text{lac}$ induced proteins fractionated with the inner membrane. However, the “M” protein was more easily randomized in the envelope structure by sonication than the 15,000 dalton component or an inner membrane marker enzyme.     

Structural polypeptides of primate derived type C RNA tumor viruses

Proteins of gibbon ape lymphosarcoma virus (GaLV) and woolly monkey sarcoma virus, type 1, together with its associated virus ($\text{SSV-1}\text{SSAV-1}$) were analyzed by guanidine-agarose chromatography and the separation patterns were compared with those of mouse and feline type C viruses. GaLV contained five major proteins, including two glycoproteins, whereas lower mammalian viruses contained six major proteins, including two glycoproteins. The molecular weights of the five GaLV proteins closely resembled the molecular weights of the five equivalent lower mammalian viral proteins. $\text{SSV-1}\text{SSAV-1}$ showed a separation pattern similar to GaLV except it contained a low but detectable amount of an additional glycoprotein. Both GaLV and $\text{SSV-1}\text{SSAV-1}$ were deficient in a protein of molecular weight about 15,000 daltons which is found in all known type C viruses of avian, reptilian and lower mammalian species.     

Flash spectroscopic studies of cyclic electron flow in intact chloroplasts

The kinetic behaviours of cytochrome $\text{b}$-563 and cytochrome $\text{f}$ are shown to be consistent with their participation in coupled cyclic electron flow in intact chloroplasts. Electron transfer between cytochromes $\text{b}$-563 and cytochrome $\text{f}$ is antimycin sensitive. Fluorescence induction studies indicate that plastoquinone may function in a coupled step between the cytochromes.