Mucin degradation in the intestine

Rat intestinal mucin was labelled biologically by intraperitoneal injection of radioactive amino acids and monosaccharides 3–6 h prior to killing, followed by isolation and purification of the mucin from mucosal scrapings. The labelled product was then introduced into intestinal segments of rats under ether anesthesia for periods up to 3 h, removed by washing and assessed for evidence of degradation. In segments containing the pancreatic ducts the total mucin precipitable by cetyltrimethylammonium bromide fell from 80% to 5% in 3 h. At 3 h, chromotography on Sephadex G-100 and Sepharose 4B revealed multiple products, including very small molecular weight fragments deficient in carbohydrate label. With the introduction of neomycin sulfate into the segments to reduce bacterial growth, only two products were found, one corresponding in size to the original mucin and one somewhat smaller, although still in excess of 200 000 daltons. These products occurred independently of the presence of the pancreatic ducts in the segments, and in chronically pancreatectomized rats. The smaller product could not be produced by incubation with trypsin or elastase. Both products were altered antigenically as compared with the original mucin. Both products also retained the same ratio of carbohydrate and protein label as the original. It is concluded that mucins undergo early degradative changes in the intestine which do not involve deglycosylation but which involve partial loss of antigenicity and a fall in molecular weight. The pancreas is not responsible for these changes.     

Differential expression and positioning of chemotaxis methylation proteins in Caulobacter

Proteins involved in chemotaxis methylation reactions have been identified in Caulobacter crescentus and their activities, times of synthesis and cellular positions have been determined. The methyl-accepting chemotaxis proteins, the methyl-transferase and the methylesterase were all shown to be active in the flagella-bearing swarmer cell, but all three activities were lost after the swarmer cells shed their flagellum and differentiated into a stalked cell. The membrane methyl-accepting chemotaxis proteins were shown to be synthesized before cell division, coincident with the synthesis of the components of the flagellum, and to be specifically localized in the membrane of the incipient swarmer cell portion of the predivisional cell. The cytoplasmic methylesterase was also found to be differentially synthesized coincident with the period of flagellar biogenesis. Furthermore, methyltransferase activity, present in the predivisional cell, was detected only in the swarmer cell upon cell division. These results demonstrate that the chemotaxis methylation machinery is positionally biased toward one portion of the predivisional cell, and that the time of expression of a set of fla and che genes is correlated with the positioning of their gene products within the cell.     

Studies on the oligomeric structure of yeast phosphofructo-kinase by means of cross-linking diimidoesters.

Intracellular cross-linking of yeast phosphofructokinase with a series of diimidoesters of different chain length resulted in the appearance of tetramers as largest cross-linked product of the enzyme subunits. The native enzyme is evidently composed of eight subunits being arranged in two tetramers α₄β₄. In the tetramers the monomers are probably assembled in tetrahedral geometry.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.     

Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure

A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure.     

A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction

Methods are presented for the complete removal of dodecyl sulfate from proteins. Themethods utilize the extraction of dodecyl sulfate anions as ion pairs with triethylammonium or tributylammonium cations into an organic solvent. The protein is insoluble in the organic solvent and is recovered as a precipitate. The methods are applicable to microgram as well as milligram amounts of protein. In all cases studied, the recovery of protein ranges from 70 to 100%. The recovered protein is suitable for N-terminal Edman degradation, tryptic peptide mapping, and amino acid analysis and can be renatured to regain enzymatic activity and antigenicity.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

Identification of a major extracellular non-collagenous glycoprotein synthesised by human skin fibroblasts in culture

The major protein released into the medium by human skin fibroblasts in culture has been shown to be a fucosylated glycoprotein (designated MFGP). Analysis by gel filtration chromatography and polyacrylamide gel electrophoresis demonstrated that under reducing conditions MFGP has a molecular weight of approx. 250,000, but occurs as a disulphide-linked aggregate in the medium. Three lines of evidence are presented to establish that MFGP is a non-collagenous molecule.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase 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.     

Chemical analysis of stalk components of Dictyostelium discoideum

Structural components of the stalks of mature fruiting bodies of Dictyostelium discoideum have been isolated and characterized after solubilizing non-structural components with urea and sodium dodecyl sulfate. The urea/sodium dodecyl sulfate-insoluble stalks are composed of about 52% cellulose, 15% proteins and 3% of a non-cellulosic heteropolymer in a covalently bound matrix. Non-covalently bound fatty acid containing material was also found. The composition and structural interrelationships of these components are essentially identical to that of the urea/sodium dodecyl sulfate-insoluble surface sheath which is produced earlier in development before culmination. These results suggest that the same components are involved in making structural elements which differ substantially in their functional role in the developmental sequence as well as in their spatial and temporal localization and morphological appearance.     

Trypsin-catalyzed activation of aspartase

Aspartase [EC 4.3.1.1] of Escherichia coli is several-fold activated by treatment with trypsin. The activation requires a few minutes to attain a maximal level, and hereafter the enzyme activity gradually decreases resulting in a complete inactivation in about 4 hours. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. No appreciable change is detected in the molecular weight of the subunits upon trypsin-mediated activation as judged from dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the structural alteration of the enzyme associated with the activation is a minor one. Kinetic properties of aspartase are also compared before and after the trypsin-activation.     

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.     

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.     

Synthesis of a photoaffinity probe for the beta-adrenergic receptor.

The synthesis and characterization of a beta-adrenergic photo-affinity label, N-(-2-hydroxy-3-naphthoxypropyl)-N′ (-2-nitro-5-azidophenyl ethylenediamine, (NAP-propranolol) is described. The inhibition constants (Ki) for the NAP-propranolol inhibition of ³H-dihydroalprenolol binding and the inhibition of (?)-isoproterenol-stimulated adenylate cyclase in turkey erythrocytes are 100 nM and 19 nM respectively.     

Properties of iron-sulfur protein isolated from Pseudomonas ovalis.

Mutants of $\text{Escherichia coli}$ C were selected for resistance towards a set of cell wall LPS core specific bacteriophages, including øX174. Increasingly deficient LPS's from wt and mutant $\text{E. coli}$ C were tested for inactivation of øX174, and the core oligosaccharides were subjected to structural analysis by methylation/g.l.c./m.s. Loss of the terminal galactose in the following basic structure of the $\text{E. coli}$ C wt core was found to lead to adsorption resistance towards øX174:

     

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.     

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.