Amino acid sequence homology between the C3 chain of rat prostatic steroid binding protein and human alpha 2-macroglobulin

Using 32P-labeled DNA complementary to mouse submaxillary gland renin mRNA, we probed mRNA gel blots from mouse testis and kidney tissues. Poly(A)-RNA from testis contained a hybridizable RNA species which was blotted onto nitrocellulose paper. The molecular size of testicular renin mRNA (approximately 1600 nucleotides in length) was not significantly different from tht of kidney renin mRNA. Densitometric scan revealed that the content of renin mRNA in mouse testis was approximately 5-fold lower than that in mouse kidney. These results support the proposal that mouse testicular cells synthesize renin.     

Cis- and trans-diamminedichloroplatinum(II) binding products different tertiary structural changes on SV40 DNA

The proteases secreted into culture medium by MCF-7 breast cancer cells produced both plasminogen-dependent and -independent proteolysis, as shown by casein-polyacrylamide gel electrophoresis. All of these proteases except the largest (M_r 120,000) were retained on a benzamidine-Sepharose affinity column, a characteristic of trypsinlike proteases. Among the proteases which activated plasminogen, all except a major protease of M_r 59,000 were antigenically similar to urokinase. These urokinaselike proteases (M_r 65,000 to 25,000) were isolated on a antiurokinase-Sepharose affinity column. The findings indicate that in a stable cell line derived from a human breast cancer there are two distinct types of plasminogen activators, opening the possibility that these activator types may be modulated in separate ways.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

Age related changes in lipid peroxidation in rat brain and liver

Lipid peroxidation in rat liver, unlike in brain shows wide variations with age. In liver, ascorbic acid content also undergoes wide variations and there is negative correlation between ascorbic acid content and lipid peroxidation. Heat-labile antioxidant factors are present in the cytosol fraction. There is inverse relationship between antioxidant activity and lipid peroxidation in liver.     

ADP-ribosylation of nuclear protein A24

Nuclear protein A24, which is composed of histone H2A and ubiquitin, a nonhistone protein, joined by an isopeptide linkage [Goldknopf and Busch (1977) $\text{Proc}$. $\text{Natl}$. $\text{Acad}$. $\text{Sci}$. $\text{USA}$$\text{74}$, 864–868], is found to be ADP-ribosylated in isolated rat liver nuclei.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Autophosphorylation of brain microtubule protein: evidence for endogenous protein kinase/phosphoprotein phosphatase cycling and multiple phosphorylation of a microtubule associated protein

Brain microtubule protein, prepared by two types of recycling methods, undergoes “flash” phosphorylation in the presence of [γ-³²p]ATP through sequential action of protein kinase and phosphoprotein phosphatase present in microtubule protein. SDS electrophoretic analysis indicates that MAP₁, tau protein, and tubulin are poorly phosphorylated, and MAP₂ is the major site of phosphorylation. To improve [³²P]phosphoprotein stability in the presence of the kinase/phosphatase cycle, 3′,5′-cyclicAMP, orthophosphate, or fluoride ion may be added. After separation from tubulin by phosphocellulose chromatography, the MAP fraction exhibits autophosphorylation. Finally, the maximal extent of autophosphorylation is observed with an ATP regenerating system using ADP, [³²P]acetyl-P, and bacterial acetate kinase; this results in the incorporation of 3–4 phosphoryl groups per MAP₂ subunit.     

Amino acid sequence homology between rat and human C-reactive protein.

The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], 'phosphoryl choline-binding protein' [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP.     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Highly purified preparations of Na⁺+K⁺-dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I₅₀ values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10^?4M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.     

Regulation of glucose-6-p dehydrogenase synthesis in rat epididymal fat pads.

The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA^Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K_M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

Hyaluronic acid synthesis in a cell-free system from rat fibrosarcoma

A transplantable rat fibrosarcoma (in both ascites and solid forms) has been shown to contain and synthesize large amounts of hyaluronic acid. A particulate hyaluronic acid synthesizing system has been isolated from the solid fibrosarcoma and some characteristics of the enzyme system are detailed. The enzyme complex transferred GlcUA or GlcNAc from UDP-GlcUA or UDP-GlcNAc at a rate of approximately 60 nmole/hr/mg protein to extend hyaluronic acid chains by approximately 45,000 daltons.     

Changes in the reactivity of proteins of rat liver ribosomes against [C]iodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes and on the attachment of poly-(U)

(1) The isolated mixtures of ribosomal proteins can be substituted by [¹⁴C]-iodoacetamide up to an average of about 2 equivalents per 20 000 dalton. The extent of substitution of single proteins measured after two-dimensional polyacrylamide gel electrophoresis shows that all proteins are reactive.

(2) Also in the subunits, all proteins are accessible to substitution. Compared with isolated proteins, however, the reactivity is decreased and the amount of labelling for most proteins ranges as low as 5 to 20%.

(3) Reassociation of ribosomal subunits decreases the reactivity of 12 proteins of the small subunit and that of 20 proteins of the large subunit.

(4) The presence of messenger inhibits the substitution of 10 proteins of the small subunit and of 6 proteins of the large one.

(5) Seven proteins of the small subunit and 3 proteins of the large one are influenced both by the other subunit and by messenger-RNA.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.