Showing papers in "Proceedings of the National Academy of Sciences of the United States of America in 1970"

A Protein Binding Assay for Adenosine 3′:5′-Cyclic Monophosphate

Abstract: A simple and sensitive assay for adenosine 3′:5′-cyclic monophosphate (cAMP) has been developed that is based on competition for protein binding of the nucleotide, presumably to a cAMP-dependent protein kinase. The nucleotide-protein complex is adsorbed on a cellulose ester filter. Assay conditions are such that a binding constant approaching 10-9 M is obtained, and the assay is thus sensitive to 0.05-0.10 pmol of cAMP.

Use of Dimethyl Suberimidate, a Cross-Linking Reagent, in Studying the Subunit Structure of Oligomeric Proteins

Abstract: Amidination of aldolase, glyceraldehyde-3-phosphate dehydrogenase, tryptophan synthetase B protein, L-arabinose isomerase, and the catalytic subunit of E coli aspartate transcarbamylase with the bifunctional reagent dimethyl suberimidate produces cross-linked proteins, with reaction predominating within oligomers Disc electrophoresis of a modified protein on polyacrylamide gel in the presence of sodium dodecyl sulfate resolves a set of species with molecular weights equal to integral multiples of the protomer molecular weight For oligomers composed of identical protomers, the number of principal species observed is identical to the number of protomers in the oligomer Application of the method to two proteins composed of dissimilar protomers, native aspartate transcarbamylase and tryptophan synthetase α2β2 complex of E coli, revealed differences in the reactivities of the different kinds of protomer within each oligomer

Binding of Dodecyl Sulfate to Proteins at High Binding Ratios. Possible Implications for the State of Proteins in Biological Membranes

Abstract: A wide variety of proteins have been shown to bind identical amounts of an amphiphile, sodium dodecyl sulfate, on a gram per gram basis at monomer equilibrium concentrations above 0.5 mM. The binding is independent of ionic strength and primarily hydrophobic in nature. Only the monomeric form of the amphiphile binds to proteins, not the micellar form. The application of these results to models for biological membranes and to gel electrophoresis in sodium dodecyl sulfate is discussed.

Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage.

Abstract: Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5'-AMPase (EC 3.1.3.1., 3.6.1.1, 3.6.1.3, and 3.1.3.5, respectively). Most of the acid phosphatase (EC 3.1.3.2) activity, on the other hand, was found in the cellular fractions, indicating that matrix vesicles are quite distinct from lysosomes. This appears to be the first instance of isolation of membrane-bounded extracellular particles from any normal tissue. The matrix vesicles possess enzymes that can increase the local concentration of orthophosphate and thus could lead to the formation of hydroxyapatite. The membrane-bounded matrix vesicles may also provide a mechanism for ATP-dependent transport of calcium or phosphate into the lumen of the vesicles with resultant mineralization.

Use of a Snake Venom Toxin to Characterize the Cholinergic Receptor Protein

Abstract: α-Bungarotoxin, a polypeptide of mol wt 8000 purified from the venom of Bungarus multicinctus, blocks irreversibly and specifically the excitation by cholinergic agonists on the isolated electroplax and on purified membrane fragments in vitro. The toxin also blocks the in vitro binding of decamethonium to a protein recently isolated from electric tissue. This observation strengthens our earlier conclusion that this protein is the cholinergic receptor macromolecule.

Genetic Control of the Cell-Division Cycle in Yeast, I. Detection of Mutants

Abstract: Time-lapse photomicroscopy has been utilized to detect temperature-sensitive yeast mutants that are defective in gene functions needed at specific stages of the cell-division cycle. This technique provides two types of information about a mutant: the time at which the defective gene function is normally performed, defined as the execution point, and the stage at which cells collect when the function is not performed, defined as the termination point. Mutants carrying lesions in three genes that control the cell-division cycle are described. All three genes, cdc-1, cdc-2, and cdc-3, execute early in the cell cycle at about the time of bud initiation, but differ in their termination points. Cells carrying the cdc-1 mutation terminate at the execution point, most cells ending up with a tiny bud that does not develop further. Cells carrying the cdc-2 mutation terminate at mitosis. Cells carrying the cdc-3 mutation are defective in cell separation but show no definite termination point since other processes of the cell cycle, such as bud initiation and nuclear division, continue despite the block in cell separation.

Axon growth: roles of microfilaments and microtubules

Abstract: The motile tips of elongating axons consist of growth cones from which microspikes protrude. Cytochalasin B causes retraction of microspikes, rounding-up of growth cones, and cessation of axon elongation. Drug withdrawal is followed by resumption of growth cone and microspike activity and of axon elongation. In contrast, colchicine causes shortening and retraction of axons, but it does not initially affect the tips. Growth cones and microspikes of elongating axons contain a network of 50 A microfilaments, the pattern of which is altered by cytochalasin treatment. These experiments indicate that both structural integrity of the axon and continuing function of its motile tip are essential elements in axonal elongation.

The γ-Glutamyl Cycle: A Possible Transport System for Amino Acids

Abstract: Evidence is presented that rat kidney contains enzymes that catalyze the synthesis and utilization of glutathione; these reactions, which involve the uptake and release of amino acids from γ-glutamyl linkage, constitute a cyclical process which is termed „the γ-glutamyl cycle.” The γ-glutamyl cycle has properties that fulfill the requirements of an amino acid transport system. Thus, γ-glutamyl transpeptidase may function in translocation and γ-glutamylcysteine synthetase and glutathione synthetase may catalyze energy-requiring „recovery” steps in transport. These and other considerations suggest that glutathione serves a carrier function in amino acid transport.

Regulation of Axon Formation by Clonal Lines of a Neural Tumor

Abstract: Clonal lines of neuroblastoma cells were found to extend or retract axons depending upon the concentration of serum. Neurite extension was not inhibited by cycloheximide but was sensitive to colchicine or vinblastine, suggesting that neurite formation is dependent upon the assembly of microtubules or neurofilaments from preformed protein subunits.

Elevation of Guanosine 3′,5′-Cyclic Phosphate in Rat Heart after Perfusion with Acetylcholine

Abstract: The levels of guanosine 3′,5′-cyclic phosphate (cGMP) and adenosine 3′,5′-cyclic phosphate (cAMP) were measured in rat hearts after perfusion with acetylcholine to determine if parallel or independent changes occurred in the levels of these two cyclic nucleotides. It was found that after perfusion with the cholinergic agent tissue, cGMP levels increased as much as 140%. This was accompanied by no change or slight decreases in cardiac cAMP concentrations. The increases observed in cGMP levels were found to parallel the negative inotropic but not the negative chronotropic effects of acetylcholine. Perfusion with isoproterenol led to increases in the rate and force of cardiac contractility and a lowering of cGMP levels. It was concluded that the tissue concentrations of cGMP and cAMP in the perfused rat heart can vary independently and that these two tissue cyclic nucleotides probably do not share the same metabolic or functional role in this tissue.

Specific Nucleolar and Nucleoplasmic RNA Polymerases

Abstract: The DNA-dependent RNA polymerase activity present in rat liver nuclei has been solubilized and purified from whole nuclei and from subnuclear fractions. As reported earlier (Roeder, R. G., and W. J. Rutter, Nature, 224, 234 (1969)), two major chromatographically distinct enzymatic species (I and II) are present in whole nuclei. Subfractionation of whole nuclei into nucleolar and nucleoplasmic fractions had little effect on the total recovery of activity. Purified nucleoli contain predominantly polymerase I, whereas the nucleoplasmic fraction is greatly enriched for polymerase II. A third minor peak of activity has also been resolved in the nucleoplasmic preparations. We conclude that the RNA polymerases are specifically localized within the nucleus and may, therefore, play specific roles in the regulation of genetic transcription.

Mechanism of activation of catabolite-sensitive genes: a positive control system.

Abstract: Catabolite repression is defined as the inhibition of enzyme induction by glucose or related substances. In the bacterium E. coli, the effect of glucose appears to be due to a lowering of the cyclic AMP level. A DNA-directed cell-free system for β-galactosidase synthesis has served as a model system for studying the mechanism of action of cyclic AMP. Previously, it was reported that in this system cyclic AMP is required for normal initiation of mRNA synthesis. A protein factor which acts in conjunction with the cyclic AMP has been partially purified. This protein factor has a high affinity for cyclic AMP. These and other results presented herein lead us to the conclusion that cyclic AMP and a protein factor called the catabolite gene activator protein are part of a positive control system for activating catabolite-sensitive genes.

Globoid Cell Leucodystrophy (Krabbe's Disease): Deficiency of Galactocerebroside β-Galactosidase

Abstract: Profound deficiency of a specific enzyme, galactocerebroside β-galactosidase, has been demonstrated in the brains, liver, and spleen of three patients with Krabbe's globoid cell leucodystrophy. The activity of this enzyme was normal in a variety of other cerebral diseases, including those with similarly devasted white matter. The lack of enzyme activity was not due to an inhibitor in the tissue, nor is it due to a shift in the pH optimum. The deficiency of galactocerebroside β-galactosidase as the primary enzymatic defect can account for the morphological and biochemical characteristics of this disease better than the previously reported deficiency of cerebroside-sulfatide sulfotransferase.

Unified theory for gel electrophoresis and gel filtration.

Abstract: Unified theory for gel electrophoresis and gel filtration: The behavior of macromolecules in gel filtration and gel electrophoresis may be predicted from Ogston's model for a random meshwork of fibers. This model has been generalized to apply to nonspherical molecules and to several gel types. The model provides equations for inter-relationships between mobility, partition coefficient, gel concentration, and molecular radius; it gives a non-Gaussian distribution of pore sizes as a function of gel concentration. The theory defines conditions for optimal separation and optimal resolution in gel filtration and gel electrophoresis. The difference in resolving power between the two fractionation methods is accounted for by the fact that gel filtration is a form of partition chromatography.

Ribonucleic Acid Synthesis of Vesicular Stomatitis Virus, II. An RNA Polymerase in the Virion

Abstract: The virions of vesicular stomatitis virus contain an enzyme that catalyzes the incorporation of ribonucleotides into RNA. The product of the reaction is mainly RNA complementary in base sequence to that of vesicular stomatitis virus RNA.

Surface Movements during the Growth of Single Explanted Neurons

Abstract: The processes of isolated rat sympathetic neurons growing in culture were marked with glass or carmine particles and observed with timelapse microphotography. Particles on the processes moved with the cell in relation to the dish and underwent continual small jerky movements. They did not, however, show any over-all distal motion and for long periods, during which the growth cone progressed more than 100 μ, the particles remained at about the same distance from the cell body. The most obvious explanation for this result is that new fiber surface, and perhaps the plasma membrane, is deposited in the region of the growing tip.

Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis.

Abstract: Purification of DNA polymerase from E. coli B has in two cases each time led to the isolation of two separate polymerase activities, enzyme A and enzyme B. Enzyme A was in contrast to enzyme B almost completely devoid of exonuclease activity. Each of the two enzymes yielded a single symmetrical activity peak in gel filtration chromatograms. From the elution volumes the molecular weights were estimated to be about 70,000 for enzyme A and about 150,000 for enzyme B. Treatment of enzyme B with subtilisin led to an increase of about 30 per cent of the polymerase activity while the exonuclease activity almost completely disappeared. The product of the subtilisin treatment (enzyme C) gave rise to a single symmetrical polymerase activity peak in a gel filtration chromatogram. The elution volume was identical to that obtained with enzyme A. It is concluded that enzyme A and enzyme C are formed by limited proteolysis of enzyme B.

Replication and Repair of DNA in Cells of Escherichia coli Treated with Toluene

Abstract: DNA synthesis has been studied in Escherichia coli cells made permeable to nucleotides by treatment with toluene. Replicative synthesis, as distinguished from repair synthesis, occurs at a rate comparable to that observed in vivo; it is dependent on the presence of all four deoxyribonucleoside triphosphates, but does not require exogenous DNA; and it is stimulated by ATP. Furthermore, replicative synthesis can be abolished at the restrictive temperature in DNA temperature-sensitive mutants. N-ethylmaleimide completely inhibits this type of synthesis, whereas it does not inhibit repair synthesis. Repair synthesis further differs from replicative synthesis in the following points: it does not require ATP; it persists at the restrictive temperature in DNA temperature-sensitive mutants; it can be induced by endogenous or exogenous nuclease activity; and its demonstration requires a Pol+ strain.

Isolation of Microtubule Protein from Cultured Mouse Neuroblastoma Cells

Abstract: The addition of vinblastine to high-speed supernatants derived from homogenates of cultured mouse neuroblastoma cells results in the formation of a precipitate which has been characterized as microtubule protein by the following criteria: colchicine-binding activity, molecular weight, amino acid composition, and electrophoretic mobility. The method therefore permits the rapid isolation of microtubule protein from crude supernatants of neuroblastoma cells.

Cyclic AMP Receptor Protein of E. coli: Its Role in the Synthesis of Inducible Enzymes

Abstract: A cyclic AMP binding protein has been purified over 100-fold from E. coli extracts. Protein purified from wild-type strains binds cyclic AMP with an apparent dissociation constant of 1-2 × 10-6 M. Two mutant strains that are unresponsive to exogenous cyclic AMP have altered binding activity; the protein purified from one of these mutants has a decreased affinity for cyclic AMP (apparent dissociation constant = 2 × 10-5 M). Extracts of this mutant are deficient in their ability to support β-galactosidase synthesis in vitro. The addition of purified, wild-type binding protein to these extracts restores enzyme synthesis toward normal. Because this binding protein appears to be required for cyclic AMP action, we suggest it be called the cyclic AMP receptor protein (CR protein).

Genetic Dissection of the Drosophila Nervous System by Means of Mosaics

Abstract: Given a mutant having abnormal behavior, the anatomical domain responsible for the deficit may be identified by the use of genetic mosaicism. Individuals may be produced in which a portion of the body is mutant male while the rest is normal female. In such sex mosaics, or gynandromorphs, the division line between normal and mutant parts can occur in various orientations. Mutants of five different genes (cistrons) on the X-chromosome of Drosophila melanogaster, having various abnormalities in visual function, have been tested by this method. All of these have been found to be autonomous, i.e., a mutant eye always functions abnormally, regardless of the amount of normal tissue present elsewhere, indicating that the primary causes of the behavioral deficits in these mutants are within the eye.

Correlation of in vivo and in vitro phase transitions of membrane lipids in Escherichia coli.

Abstract: A double mutant of Escherichia coli unable to synthesize or degrade unsaturated fatty acids can incorporate fatty acids with various hydrocarbon chain structures into the membrane phospholipids. The temperature characteristic of three physiological properties of cells grown with different fatty acids (growth, respiration, and efflux of thiomethylgalactoside) is compared with the physical properties of the isolated phosphatidylethanolamines in monolayers at an air-water interface. Breaks in the temperature characteristic of the properties measured in vivo correspond to phase transitions in the lipid films from a liquid-expanded to a condensed form. It is concluded that a liquid-like state of the lipid phase is required for proper membrane function.

ACTH Receptors in the Adrenal: Specific Binding of ACTH-125I and Its Relation to Adenyl Cyclase

Abstract: Pure monoiodo ACTH-(125)I was prepared that was biologically active and free of unlabeled ACTH. Extracts of adrenal cortex that contained ACTH-sensitive adenyl cyclase, bound ACTH-(125)I; extracts that lacked the ACTH-sensitive cyclase did not bind ACTH-(125)I. Unlabeled ACTH inhibited the binding of ACTH-(125)I. Five ACTH derivatives which varied widely in biological activity were tested. All inhibited the binding of ACTH-(125)I in direct proportion to their biological activity. Albumin, insulin, and four unrelated iodinated hormones were inert. The addition of excess hormone or acetic acid produced rapid dissociation of bound ACTH-(125)I. This study demonstrates directly the binding of ACTH to its biologically significant site.

Ribosomal Proteins, XII. Number of Proteins in Small and Large Ribosomal Subunits of Escherichia coli as Determined by Two-Dimensional Gel Electrophoresis

Abstract: Two-dimensional gel electrophoresis separates all of the component proteins of the ribosomal subunits of Escherichia coli. This method shows 21 proteins in the 30S, and 34 proteins in the 50S, subunit.

Contemporaneous Disequilibrium, a New Hypothesis to Explain the „Paradox of the Plankton”

Abstract: The diversity of lake phytoplankton is unexpectedly high, since the epilimnion of a lake is continuously mixing and might be expected to have only one or at most a few niches for primary producers. However, a carefully replicated series of samples from Castle Lake, Calif., showed a high degree of patchiness for many phytoplankton species, indicating that the rate of mixing is slow enough relative to the reproductive rate of the algae for many different niches to exist simultaneously. Productivity per unit biomass ratios, measured at Lake Tahoe, California-Nevada, shows that the turnover times for carbon in even this ultraoligotrophic lake are often less than 1 day. High diversity is associated with high productivity per unit biomass and high zooplankton populations in this lake. A contemporaneous disequilibrium model to explain the diversity of the lake phytoplankton is therefore highly plausible. At any one time, many patches of water exist in which one species is at a competitive advantage relative to the others. These water masses are stable enough to permit a considerable degree of patchiness to occur in phytoplankton, but are obliterated frequently enough to prevent the exclusive occupation of each niche by a single species.

Subunit Structure of Human Fibrinogen, Soluble Fibrin, and Cross-Linked Insoluble Fibrin

Abstract: The three unique polypeptide chains of human fibrinogen differ significantly in molecular weight. Cross-linkage of fibrin by fibrin-stabilizing factor results in the rapid formation of cross-links between γ-chains and a slower formation of cross-links between α-chains. β-Chains are not involved directly in the cross-linking of fibrin. Reduced, cross-linked fibrin contains uncross-linked β-chains, dimers of γ-chain, and higher polymers of α-chain. Although it is uncertain whether the γ-γ dimers are formed by chains in different molecules of fibrin, the polymers of α-chain in fibrin can only be accounted for by cross-linkage of α-chains in different molecules. The nature of cross-linkage among the subunits in fibrin can account well for the three-dimensional, covalent structure of cross-linked, insoluble fibrin.

Mechanism of Activation by Adenosine 3′:5′-Cyclic Monophosphate of a Protein Phosphokinase from Rabbit Reticulocytes

Abstract: Two protein phosphokinases (EC 2.7.1.37) were found to be present in rabbit reticulocytes. The two enzymes were separated by DEAE-cellulose chromatography and called kinases I and II. Adenosien 3′:5′-cyclic monophosphate stimulated the activity of both enzymes. However, the degree of stimulation was different and depended on the protein acceptor used. In the presence of adenosine 3′:5′-cyclic monophosphate, protein kinase I dissociated into two subunits: a subunit binding adenosine 3′:5′-cyclic monophosphate, and a catalytic subunit. The component binding the cyclic nucleotide appeared to act as an inhibitory protein, regulating the activity of the catalytic subunit. The mechanism of action of the cyclic nucleotide on kinase II appeared to be different from that of kinase I.

SPIN-STATE CHANGES IN CYTOCHROME P-450cam ON BINDING OF SPECIFIC SUBSTRATES

Abstract: The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putida were observed at temperatures from 42 to 80°K As isolated, P-450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g = 245, 226, 1915) We also detected a minor signal typical of high spin ferric heme (g = 8, 4, 18) equivalent to less than 7% of the heme at temperatures below 20°K On titration with the substrate, (+)-camphor, the low spin signal decreased and the high spin signal increased, maximally representing about 60% of the heme For reasons not thus far understood, 40% of the heme is not converted to high spin by either (+) or (-)-camphor The high spin signal has a rhombic character which is stronger than any previously observed with a heme compound (E = 033 cm-1; D = 38 cm-1; E/D = 0087) We conclude that P-450cam as isolated is equal to or more than 95% in a low spin form probably having sulfur as one of the axial ligands The binding of substrate displaces this ligand sufficiently to allow for conversion from a low to a high spin form

In vitro induction of granulocyte differentiation in hematopoietic cells from leukemic and non-leukemic patients.

Abstract: Human spleen-conditioned medium can induce the formation in vitro of large granulocyte colonies from normal human bone marrow cells. The granulocyte colonies contained cells in various stages of differentiation, from myeloblasts to mature neutrophile granulocytes. Human spleen-conditioned medium also induced colony formation with rodent bone-marrow cells, whereas rodent spleen-conditioned medium induced colony formation with rodent bone marrow but not with human cells. This in vitro system has been used to determine the potentialities for cell differentiation in bone-marrow and peripheral blood cells from patients with a block in granulocyte differentiation in vivo. The cloning efficiency, colony size, and number of mature granulocytes in bone-marrow colonies from patients with congential neutropenia, whose bone marrow contained only 1% mature granulocytes, were not less than in people whose bone marrow had the normal level of about 40% mature granulocytes. The cloning efficiency of peripheral blood cells from patients with acute myeloid leukemia was 350 times higher, with 10 times larger colonies, than the cloning efficiency of peripheral blood cells from normal people. The cytochemical properties and number of mature granulocytes in colonies from the leukemic patients were the same as in colonies from non-leukemic people. The results indicate that a block in cell differentiation in vivo, in these cases with neutropenia and acute myeloid leukemia, was overcome in vitro, in the presence of an inducer in the conditioned medium. In patients with chronic myeloid leukemia, colony formation was induced only in some of the cases. This indicates that there are blast cells with different potentialities for the development of colonies in different patients.

Controlled deamidation of peptides and proteins: an experimental hazard and a possible biological timer.

Abstract: Experiments on model peptides show that the rate of deamidation of asparaginyl residues depends strongly on the nature of neighboring residues. The natural distribution of glutaminyl and asparaginyl residues is ordered with respect to the biological lifetime of the peptides and the functional groups of the residues neighboring to glutaminyl and asparaginyl residues. The rates of deamidation of such amide peptides under physiological conditions could serve as useful timers of development and aging.