Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles

A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote $\text{Physarum polycephalum}$, purified, and found to contain a substantial amount of the unusual amino acid N^G, N^G-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in $\text{Physarum polycephalum}$ and the possible significance of the N^G, N^G-dimethylarginine are discussed.     

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.     

Complete amino acid sequence of the coat protein of the Pseudomonas, aeruginosa RNA bacteriophage PP7

As part of a project intending to assess the evolutionary kinship between the RNA coliphages and RNA bacteriophages of other bacterial genera, we have sequenced the coat protein of $\text{Pseudomonas}\text{,}\text{aeruginosa}$ RNA phage PP7. Like the coat proteins of coliphages MS2 and Qβ and of the broad host range RNA phage PRR1, PP7 coat protein (127 residues) is highly hydrophobic, and contains a cluster of basic residues between positions 40 to 60. Minimal mutation distance values were calculated for comparison of PP7 coat protein with each MS2, Qβ and PRR1 coat proteins. Application of the Moore-Goodman criterion to those values, shows that these four RNA bacteriophage coat proteins very likely descent from a common ancestor.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Tunicamycin inhibits glycosylation of precursor polyprotein encoded by env gene of Rauscher murine leukemia virus.

The molecular weight of the precursor polyprotein to the envelope proteins of Rauscher murine leukemia virus is reduced from 85,000 to 68,000 daltons when synthesized in the presence of tunicamycin, a specific inhibitor of the synthesis of oligosaccharides that attach to glycoproteins via asparagine residues. The unglycosylated precursor protein (Pr68^$\text{env}$) is synthesized at a rate comparable to that of the normal carbohydrate-containing envelope precursor (gPr85^$\text{env}$). Pr68^$\text{env}$ is not proteolytically processed and remains undegraded in the cell. Thus, most if not all of the carbohydrate content of gPr85^$\text{env}$ is N-linked, and glycosylation appears to be necessary for normal processing of precursor proteins into viral particles.     

Identification of ubiquinone binding proteins in ubiquinol-cytochrome c reductase by arylazido ubiquinone derivative

A functionally active $\text{arylazido-1-[}{}^{14}\text{C]-β-alanine}$ ubiquinone derivative has been synthesized for the identification of the ubiquinone binding protein in ubiquinol-cytochrome $\text{c}$ reductase. After photolysis, the ¹⁴C activity was found to be specifically associated to proteins with mobilities relative to cytochrome $\text{c}$ of 0.841 and 0.475 in the sodium dodecylsulfate polyacrylamide gel electrophoresis of the Weber and Osborn system. These two proteins have previously been identified as $\text{b}$ cytochromes. The ¹⁴C activity distribution pattern was observed to be identical in the presence or absence of phospholipids during the photolysis. Antimycin A also produces no change in the ¹⁴C activity distribution among the proteins of this enzyme complex.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

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.     

Replacement of the B protein requirement of the E. coli quinolinate synthetase system by chemically-generated iminoaspartate

Two proteins (A and B) from $\text{Escherichia}\text{,}\text{coli}$ are required for $\text{in}\text{,}\text{vitro}$ synthesis of the NAD⁺ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with $\text{N-}\text{ethyl}\text{[1?}{}^{14}\text{C}\text{]}\text{maleimide}$, phenyl[²⁰³Hg]mercuric acetate and $\text{p-}\text{chloro}\text{[}{}^{203}\text{Hg}\text{]}\text{mercuribenzoate}$ showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with ¹⁴C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

Endonuclease of high specific activity in a purified mouse interferon preparation

We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 10⁹ NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA₂^Gln species from $\text{E. coli}$, or tRNA^Val species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of $\text{in vitro}$-synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established.     

Experimental studies on supercooling in two Antarctic micro-arthropods

Samples of mites and Collembola which had been acclimated at 5°C and provided with natural foods were cooled at four constant cooling rates: 1, $\text{1}\text{2}$, $\text{1}\text{4}$, $\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$ and ca 20 deg min^?1, and their individual supercooling points measured. Frequency distributions of supercooling points comprised not less than 84 (Alaskozetes antarcticus) and 96 (Cryptopygus antarcticus) individuals in each case. Two modal groups were displayed in these distributions, which were widely separated in temperature and termed low group and high group. In Alaskozetes a trough between ?3 and ?4°C was present in the high-group distribution, which may be due to a lack of a certain class of nucleators. The highest temperatures at which animals froze occurred at the slowest cooling rate ($\text{1}\text{8}\text{deg min}{}^{\mathrm{?1}}$), whereas rapid cooling removed the trough to form a single high-group peak. In Cryptopygus, the high groups were narrow and peaked (<2 deg wide) at all cooling rates, with a downward shift of ca 1 deg between the rates $\text{1}\text{8}$ and $\text{1}\text{2}\text{deg min}{}^{\mathrm{?1}}$. Both species showed a trend towards a lower mean low-group supercooling point at faster rates of cooling, but these were not significant. Regressions of cooling rate on individual low-group supercooling points (≥?20°C) for both species showed a significant negative correlation, which did not differ between species. The distribution of the deviations about each rate-defined mean in the low group for each species was skewed to the right, with 88% occurring between ±2 deg of the means. It is suggested that minor deviations (e.g. halving or doubling of the cooling rate) do not affect the resultant supercooling points at non-constant cooling rates, but a rate of 1 deg min^?1 is to be preferred.     

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [¹⁴C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[¹⁴C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the $\text{sn}$-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-$\text{O}$-alkyl-2-arachidonoyl-$\text{sn}$-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-$\text{O}$-alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.     

Phosphorylation and acetylation of nonhistone chromosomal proteins of the brain of rats of various ages and their modulation by calcium and estradiol.

$\text{In}\text{vitro}$ phosphorylation and acetylation of nonhistone chromosomal (NHC) proteins and their modulation by Ca⁺⁺ and estradiol were studied by incubating slices of cerebral cortex of 2-, 15- and 84-week female rats with ³²Pi and ¹⁴C-Na-acetate. Phosphorylation pattern of NHC proteins is unique for each age. Ca⁺⁺ and estradiol stimulate phosphorylation of different NHC proteins which is also age-specific. Acetylation of NHC proteins decreases precipitously with age. No unique NHC protein is acetylated preferentially at any age, nor does Ca⁺⁺ stimulate acetylation. Estradiol, however, stimulates acetylation of a few NHC proteins. It is suggested that phosphorylation of NHC proteins and its modulation by effectors may be more important for gene expression than their acetylation.     

An F-actin- and calmodulin-binding protein from isolated intestinal brush borders has a morphology related to spectrin

A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW $\text{260}\text{240}$), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW $\text{260}\text{240}$ is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW $\text{260}\text{240}$ is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW $\text{260}\text{240}$ indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW $\text{260}\text{240}$ with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW $\text{260}\text{240}$ and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW $\text{260}\text{240}$ (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.     

Transferrin acquisition of catabolized erythrocyte iron in the rat

Rat plasma contains two isotransferrins rather than the single iron-binding protein found in plasma of other species, and it was recently proposd that differences between the biological behavior of each isotransferrin accounted for observations previously attributed to behavioral differences between each of the two transferrin iron-binding sites. The two isotransferrins were isolated from rat plasma by DEAE-Sephadex ion-exchange chromatography and isoelectric focusing. The pH-dependent iron-dissociating and reticulocyte iron-donating properties of each isotransferrin were investigated and found to be indistinguishable. Like human transferrin, one iron-binding site retains its affinity for iron below pH 6 and this property was used to investigate the $\text{in}$$\text{vivo}$ acquisition of catabolic iron in order to determine whether the process occurs at one specific or both binding sites. Plasma radioactive iron, derived from injected ⁵⁹Fe-labelled heat denatured erythrocytes was bound with high specificity to the transferrin iron-binding site that was most resistant to acidic dissociation. This finding supports Fletcher and Huehns' hypothesis that each of the two rat transferrin iron-binding sites is endowed with a separate functional role.     

Separate identities of ligandin and the h-protein,a major protein to which carcinogenic hydrocarbons are covalently bound

There is a protein moiety in the C3H mouse liver cytosol which gives a line of identity with rat liver ligandin one of three azo dye binding proteins of the liver using anti-rat ligandin. This mouse liver protein has been termed mouse ligandin and is not the $\text{h}$-protein, the major target protein in the mouse liver of methylcholanthrene and its metabolites. Mouse ligandin is identical to a minor methylcholanthrene binding protein species that was found previously to consist of basic proteins II and III. Both mouse ligandin and mouse $\text{h}$-protein contain glutathione S-transferase activity with different substrate specificitles.     

Comparison of developmentally regulated lectins from three species of cellular slime mold

Extracts of the cohesive forms of the cellular slime molds Dictyostelium discoideum, Dictyostelium mucoroides and Dictyostelium purpureum contain lectin activity, assayed as hemagglutination activity. The lectin activity from each species binds quantitatively to Sepharose 4B and can be eluted with d-galactose. The resultant purified lectins are abundant proteins representing, in the case of D. purpureum, up to 5% of the total soluble protein of cohesive cells. The preparations from each species are similar but distinct in amino acid composition and other properties. Each purified preparation gives rise to two protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the major band representing as little at 77% (D. purpureum) and as much as 96% (D. mucoroides) of the total protein in the two bands. The molecular weights of the pair of bands were different for each species, ranging between about 23 000 and 26 000. The two bands are believed to represent subunits of lectins made up of either one or a combination of these two proteins. The apparent molecular weights of the purified lectin activities determined by sucrose density gradient centrifugation were all in the range of 100 000. $\text{N-}\text{Acetyl-}\text{d}\text{-galactosamine}$ was a potent inhibitor of the hemagglutination activity of each preparation; but there were some differences in the relative inhibitory potency of a number of other saccharides. Antiserum raised against each preparation, as well as univalent antibody fragments derived from these antisera, reacted best with the antigens to which they were raised; but showed some cross reaction measured both by precipitin reactions and by inhibition of hemagglutination activity of the purified lectins. The differences between the lectins from the different species could be trivial; but they also could be important for defining specific properties of these three species which reliably segregate into colonies of a single species when grown in mixed culture.     

Thin-layer mapping of peptides labeled with 3H or 14C via reductive methylation.

Two dimensional tryptic peptide maps have been obtained from 2 μg (40 pmol) of protein digest following labeling with ³H or ¹⁴C via reductive methylation. A simple labeling procedure is complete within 1 h; autoradiographs of ¹⁴C-labeled maps and fluorographs of ³H-labeled maps are obtained in 72 and 24 h, respectively. Tryptic peptide maps of ${}^{14}\text{C}{}^3\text{H}$ methylated α- and β-tubulin, and rabbit muscle, chick muscle, and chick brain actins show approximately the expected number of peptides. Methylation does not appear to measurably alter the map positions of the peptides relative to unmethylated peptides in the solvent systems used for either electrophoresis or chromatography.