Analysis of chromatin repeat units in logarithmically and stationary growing cells of Paramecium aurelia and Tetrahymena pyriformis.

We have used micrococcal nuclease as a probe of the repeating structure of chromatin isolated from the macronuclei of logarithmically and stationary grown $\text{Paramecium aurelia}$ and $\text{Tetrahymena pyriformis}$. For both these lower eukaryotes, the monomer size is shown to vary depending on the stage in the growth cycle. $\text{P. aurelia}$ exhibits a monomer size of 153±7 bp and 178±6 bp and $\text{T. pyriformis}$ 207±10 bp and 230±10 bp in logarithmic and stationary cells, respectively. Both exhibit a nucleosome size of 140 bp. We discuss the possible association of these changes with histone content and nuclear activity changes, and also a possible reason for the divergence from the size pattern of monomer repeats seen in lower eukaryotes by $\text{T. pyriformis}$.     

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.     

Evidence for a novel circulating alpha 1 protein in tumour-bearing rats. Differentiation from acute phase alpha 1 proteins and from alpha 1 fetoprotein

It has been found in this study that the serum from rats bearing a transplanted dibenzanthracene-induced tumour ($\text{RD}\text{3}$), has a high concentration of alpha₁ proteins compared with normal rat serum. These alpha₁ proteins have been isolated by an immunoabsorption method and have been compared by immunological methods with the acute phase alpha₁ proteins isolated by the same method from the serum of rats presenting an inflammatory reaction. It has been found that the isolated $\text{RD}\text{3}$ alpha₁ proteins were composed of two major proteins: one of these corresponded to an inflammatory protein, the alpha₁-AP-globulin. The other may be a new protein, as it is absent from the serum of rats with an acute phase inflammatory reaction and nor does it correspond to alpha₁ feto-protein, a carcino-embryonic protein presenting the same electrophoretic mobility.     

Translation of AKR-murine leukemia viral RNA in an E. coli cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an $\text{E. coli}$ cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized $\text{in vitro}$ in response to AKR-MuLV RNA in the $\text{E. coli}$ cell-free system.     

The metabolic fate of 14C-labeled immunoadjuvant peptidoglycan monomer: II. In vitro studies

Peptidoglycan monomer (GlcNAc-MurNac-L-Ala-D-isoglutamine-meso-diaminopimelic acid-D-Ala-D-Ala), labeled with ¹⁴C both in the disaccharide and pentapeptide portions, was incubated with slices of mouse liver, kidney or spleen as well as with mouse and human blood cells, plasma and serum. Peptidoglycan monomer was isolated unchanged after incubations with mouse organs and blood cells. However, upon incubation with mouse or human blood, 10–50% of the peptidoglycan monomer underwent hydrolysis to the corresponding disaccharide and pentapeptide. After incubations with plasma and serum more than 90% of the [^14C]peptidoglycan monomer was metabolized: about 50% of the administered radioactive dose was recovered in the disaccharide unit and about 35% in the pentapeptide part. These results suggest that in blood, plasma and serum of mouse and man, an $\text{N-}\text{acetylmuramoyl-}\text{L}\text{-alanine}$ amidase (mucopeptide amidohydrolase, EC 3.5.1.28) exists which splits the amide bond between the lactyl carboxyl group of the muramyl residue and the amino group of the peptide moiety in the peptidoglycan molecule.     

The study of multiple polymerization equilibria by glass bead exclusion chromatography with allowance for thermodynamic non-ideality effects

Two related aspects are explored of the frontal exclusion chromatography of proteins employing controlled-pore glass beads as the stationary phase. First, it is shown theoretically that, despite the absence of osmotic shrinkage effects previously encountered with Sephadex matrices, the experimentally measurable partition coefficient of a single non-associating solute will be dependent on its concentration due to the differing ratios of activity coefficients in mobile and stationary phases at different total concentrations. The effect is demonstrated with results obtained using ovalbumin in phosphate buffer of pH 7.4, and is Shown to be consistent (up to a solute concentration of 5 $\text{g}\text{litre}$) with theoretical prediction formulated in terms of a single virial coefficient. Secondly, it is shown for self-associating systems that it is possible to determine the monomer concentration as a function of total concentration, provided the stationary phase is selected to ensure exclusion of all oligomeric species except monomer: the relation derived for this purpose accounts for the concentrationdependence of the partition coefficient of monomer, again as a first approximation involving one virial coefficient. Such information on the monomer concentration permits elucidation of the polymerization characteristics of the system in terms of the types of species present and the relevant equilibrium constants. The feasibility of the method, its likely sources of error and the relative contribution of the non-ideality effect are investigated using bovine glutamate dehydrogenase (up to a total concentration of 5.4 $\text{g}\text{litre}$) in phosphate buffer of pH 6.9. This system was selected since comparison was possible with results obtained by other methods, which have established the enzyme polymerization pattern as an isodesmic indefinite self-association. The isodesmic equilibrium constant of 1.5 ± 0.3 $\text{litre}\text{g}$ found in this work is in reasonable agreement with previous findings.     

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.     

The amino acid sequence of desulforedoxin, a new type of non heme iron protein from Desulfovibrio gigas

A new type of non heme iron protein called desulforedoxin has been isolated from the sulfate reducing bacterium, $\text{Desulfovibrio gigas}$. The complete amino acid sequence has been established. The 36 amino acid residues of the sequence are aligned with the aid of peptides obtained by cyanogen bromide cleavage and by hydrolysis with a peptidase isolated from $\text{Staphylococcus aureus}$. Desulforedoxin has been described as a non heme iron protein of molecular weight 7,600 with 2 iron atoms linked to eight cysteine residues. In fact, sequence elucidation shows that it consists of a dimer of a peptide containing 36 aminoacids. We do not know whether if each monomer contains 1 iron atom linked to 4 cysteine residues or whether the two iron cross link the two monomers. Additional studies on the elucidation of the structure of this new cluster are presently under study.     

Origin of the labile sulfide in the iron-sulfur proteins of Escherichiacoli

A method has been devised for measuring the abundance of sulfur-34 in the hydrogen sulfide released upon the acidification of $\text{Escherichia}\text{coli}$ cells. Evidence is presented, based on the rate at which the hydrogen sulfide is released from the cells as well as the total amount released, that this hydrogen sulfide originates from the iron-sulfur proteins present in the cells. The sulfur-34 abundance in this hydrogen sulfide which was isolated from cells grown with [sulfane-³⁴S]thiocystine, a compound which can differentially label $\text{in}\text{vivo}$ the sulfur-34 abundance of cysteine and hydrogen sulfide, shows cysteine sulfur and not hydrogen sulfide to be the origin of the sulfide sulfur of iron-sulfur proteins in aerobically grown $\text{E.}\text{coli}$     

Immunological properties of membrane fractions from wild type and dnaA mutants of Escherichia coli

Membrane vesicles from Escherichia coli wild type and an otherwise isogenic $\text{dna}\text{A}$ mutant were used to immunize rabbits. In addition, a membrane protein fraction, containing the material found deficient in $\text{dna}$A mutants, was purified by preparative polyacrylamide gel electrophoresis in sodium dodecylsulfate, and used for immunization. The antisera produced were analyzed by immunoelectrophoresis and immunofluorescence microscopy. The antisera obtained by immunization with membrane vesicles from either wild type or $\text{dna}\text{A}$ mutant membrane preparations were qualitatively similar in the precipitin bands seen after immunoelectrophoresis. The antisera obtained by immunization with the purified protein fraction contained a subset of the antibodies seen when whole vesicles were used for immunization. In a semiquantitative precipitin assay, the antisera prepared against whole membrane vesicles or the isolated protein fraction both caused the precipitation of more protein from sodium dodecylsulfate-solubilized membranes of wild type than of $\text{dna}\text{A}$ mutants. No difference was seen by immunoelectrophoresis between the protein composition of wild type or $\text{dna}\text{A}$ membrane preparations. Thus, the $\text{dna}\text{A}$ mutant appears to differ from the wild type in the quantitative composition of its membrane proteins, whereas no qualitative differences were detected.Fluorescein-conjugated antiserum preparations were employed to assess the reactivity of intact cells, spheroplasts and membrane vesicles with the antisera studied above. Wild type cells of E. coli have a barrier to reaction with the antisera; this barrier is removed when the cells are converted to spheroplasts or to membrane vesicle. Similarly, a highly permeable mutant of E. coli permits reaction of the antisera with unaltered cells. Antisera to both whole membrane vesicles and to the isolated protein fraction react identically with the cellular and subcellular preparations. Thus, antisera prepared from membrane proteins isolated after sodium dodecylsulfate-polyacrylamide gel electrophoresis can still recognize some antigens present in membrane vesicle preparations.     

Disposition of membrane proteins as affected by changes in the electrochemical gradient across mycoplasma membranes

The effect of ionophores on the exposure of $\text{Acholeplasma}$$\text{laidlawii}$ membrane proteins to the aqueous surroundings was studied by the lactoperoxidase-mediated radio-iodination procedure. The iodination values of intact cells pre-treated with valinomycin and/or carbamylcyanide m-chlorophenylhydrazone (CCCP) were much lower than those of untreated cells. The iodination values of isolated membranes from treated or untreated cells were, however, the same. Our results suggest that membrane proteins are less exposed to the aqueous external surroundings when electrical gradients of ions across the cell membrane are collapsed.     

Uptake of ribosomal proteins by isolate HeLa nuclei.

The uptake of radioactive ribosomal proteins by isolated HeLa cell nuclei has been studied. Ribosomal proteins are taken up by nuclei $\text{in vitro}$ more rapidly than are cytosol proteins, suggesting that the uptake is selective. In addition, the ribosomal proteins are found associated with the nucleolus to a greater extent than are the cytosol proteins.     

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.     

Preliminary crystallographic data on buffalo beta-lactoglobulin.

Lactoglobulin isolated from Italian buffalo milk has been crystallized in two different forms. Crystals of type I have been grown at pH 3.5 and belong to space group P6₃; the unit cell dimensions are $\text{a = b = 67}\text{A}\text{?}\text{, c = 142}\text{A}\text{?}$, and accordingly the asymmetric unit contains one dimer of molecular weight 36,000. Type II crystals, grown at pH 8.5, have only one monomer per asymmetric unit; the space group is P3₁21 (or enantiomorph) and the unit cell edges are: $\text{a = b = 54.4}\text{A}\text{?}\text{, c = 113.2}\text{A}\text{?}$. The crystals of the trigonal form show low radiation damage and are suitable for a structural investigation.     

In vitro synthesis of photosynthetic membranes: I. Development of photosystem I activity and cyclic photophosphorylation

First successful $\text{in vitro}$ synthesis of functional photosynthetic phosphorylating membrane is reported. Etioplasts, highly enriched in cytoplasmic and plastid proteins, isolated from etiolated Cucumber cotyledons pretreated with kinetin and gibberellic acid, and illuminated in a cofactor fortified medium showed commencement of chlorophyll (Chl) synthesis immediately after illumination from exogenous δ-aminolevulinic acid, while photosystem I (PS I) activity commenced 15 min after the onset of illumination. When cotyledons pretreated with kinetin and gibberellic acid were illuminated directly, there was a lag phase of 30 min before the commencement of Chl synthesis and PS I activity developed after 1 h of illumination. In plastids developed both $\text{in vivo}$ and $\text{in vitro}$, the electron flow from dichlorophenolindophenol to methyl-viologen was coupled to phosphorylation as observed by an increase in the electron transport rate on the addition of uncouplers. Analysis of polypeptide profiles of the greening plastids $\text{in vitro}$ showed the disappearance of many higher molecular weight proteins during greening. Polypeptides of molecular weight 32, 20.5, 19.5 K absent in etioplasts appeared as distinct bands after 4 h of greening $\text{in vitro}$.     

Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles

Phagocytic vacuoles containing lgG coated latex particles were isolated from human neutrophils by floatation. The absorbance spectrum of the cytochrome $\text{b}$ was associated with the vacuoles within 10 sec of particle uptake and the vacuolar concentration increased little thereafter. In contrast, the cytoplasmic granule proteins myeloperoxidase and vitamin B₁₂ binding protein associate with the vacuoles more slowly. The addition of dithionite to intact cells rapidly reduces most of the cytochrome $\text{b}$, whereas only a small proportion of the myeloperoxidase, which is located intracellularly, is reduced in the absence of detergent. Most of the cytochrome $\text{b}$ appears to be localised in the neutrophil plasma membrane.     

Isolation of the light-harvesting chlorophyll a/b--protein complex from thylakoid membranes of barley by adsorption chromatography on controlled-pore glass.

The light-harvesting chlorophyll $\text{a}\text{b}$-protein complex has been isolated from barley thylakoids by a rapid, single-step procedure involving adsorption chromatography on controlled-pore glass columns. The Triton X-100-solubilized complex contains a polypeptide of apparent molecular weight, 26,000; the 0.25% Triton X-100 light-harvesting chlorophyll $\text{a}\text{b}$-protein has spectral characteristics consistent with its assumed in vivo state. On the same column free chlorophyll and carotenoids have been separated from chlorophyll-protein complex 1, but this complex contained many polypeptides other than those associated with chlorophyll. This method is potentially suitable for the isolation of other thylakoid membrane proteins. It may also be generally applicable for fractionation of intrinsic membrane proteins from other sources and for separation of mixed Triton X-100-lipid micelles.     

Electron microscopic and renaturation kinetic analysis of mitochondrial DNA of cytoplasmic petite mutants of Saccharomyces cerevisiae

Mitochondrial DNA isolated from a series of nine petite yeast strains and from the parent grande strain was characterized by electron microscopic and renaturation kinetic analysis. The mtDNA² from all strains contained a variety of branched molecules which may be intermediates of replication or recombination. Although no circles were observed in the grande mtDNA, all the petites contained circular mtDNA molecules. The size distribution of the circles conformed to an oligomeric series that was characteristic for each strain. In seven petites, the length series could be related to a single circle monomer size, ranging from 0.13 μm to 5.5 μm; and in two petites to two or more circular monomer lengths. In contrast to circular mtDNA, linear molecules showed no unique size distribution. Circle monomer lengths were linearly related to the kinetic complexity (κ₂ or $\text{C}{}_0\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of sheared total mtDNA in the seven petite strains that contained a predominant single series of circle lengths. Thus in each of these petite strains the circle monomer length defined the same DNA sequence present in the linear DNA molecules of non-unique length.     

Pyrene fluorescence fine structure as a polarity probe of hydrophobic regions: Behavior in model solvents

The vibrational fine structure superimposed on pyrene monomer fluorescence is solvent dependent at room temperature. The fine structure pattern of pyrene monomer fluorescence was found to be independent of excitation conditions or collisional quenching, but highly dependent on solvent polarity. Variation in pyrene fluorescence fine structure was investigated in alkane and alkanol solvents and solvent mixtures. Although changes in the fine structure pattern were detected across the monomer fluorescence spectrum, the greatest difference occurred at the 373- and 384-nm peaks. In nonpolar environments, the 384-nm peak dominated the pyrene spectrum, whereas at higher polarity, the 373-nm peak was the most intense. By expressing the relative emission intensities at 373- and 384-nm peaks as a ratio, a sensitive parameter of solvent polarity was obtained. The $\text{373}\text{384}$ peak ratio results yielded a curvilinear relationship as a function of dielectric constant. This effect can be applied to monitor changes in polarity of pyrene microenvironments in proteins and membranes.