The binding of core histone proteins to DNA, measured as a function of [NaCl[ is a reversible process. Dissociation and reassociation occurs in two stages. Between 0.7 and 1.2 M NaCl H2a H2b bind non-cooperatively as an equimolar complex with deltaGo = 1.6 Kcals/mole at 4 degree C and 1.0 M NaCl. Between 1.2 and 2.0 M NaCl H3 and H4 bind cooperatively as an equimolar complex with delta Go = 7.4 Kcal/mole at 4 degree C and 1.0 M NaCl. The proper binding of H2a and H2b requires the presence of bound H3 and H4. Nuclease digestion of the H3-H4 DNA produces a tetramer of H3-H4 bound to fragments of DNA 145, 125 and 104 base pairs long. Thus an H3-H4 tetramer can protect fragments of DNA as long as those found in complete core particles and must therefore span the nucleosome core particle.
The circular dichroism of calf thymus deoxyribonucleohistone was compared with that of samples partially depleted of histone in solutions of sodium chloride. Differences in the near ultraviolet circular dichroism of DNA and deoxyribonucleohistone were associated with the presence of the f2b, f2a2 and f 3 histone fractions in the complex. The near ultraviolet circular dichroic spectra of both DNA and deoxyribonucleohistone were very sensitive to the solvent environment. Thus differences in the circular dichroic spectra of DNA and deoxyribonucleohistone cannot necessarily be associated with long-range effects, such as supercoiling, but may be due to short-range effects, such as might be caused by histone-DNA interactions.The absorbance and the circular dichroic melting profiles of deoxyribonucleohistone were biphasic. A new dichroic species was formed during the melting of the first of these steps and evidence is presented that this is a new histone-DNA complex produced by the migration of some or all of the fractions f2b, f2a2 and f 3 from denatured t o native regions of the DNA during the melting process. Thus the biphasic melting profile of deoxyribonucleohistone does not necessarily imply that these fractions are arranged heterogeneously on the DNA.Studies on the hydrodynamic properties of nucleohistone have shown that certain histone fractions maintain nucleohistone in a more compact conformation than DNA [l]. It has been suggested that this conformation is a supercoil [2]. Marked differences are also found in the optical rotatory dispersion and circular dichroic spectra of DNA and nucleohistone [3-51. I n order to see whether the histone fractions reponsible for the characteristic difference in hydrodynamic properties were also responsible for the difference in optical properties, we have investigated the circular dichroic spectra of nucleohistone partially depleted of specific histone fractions by salt.The histone fractions which are implicated in the supercoiling of nucleohistone also produce biphasic melting profiles in the complex [1,6]. Therefore we have studied the circular dichroism of native and partially depleted nucleohistone as a function of temperature in order to gain further information about the contribution of specific histone fractions to the conformation of the native nucleoprotein. Apreliminary account of this work has been published [7]. EXPERIMENTAL PROCEDUREThe preparation, characterisation and experimental methods, other than circular dichroism, have been described previously [11. The solvent used in all cases was 0.7 mM sodium phosphate, p H 6.8, except where otherwise statcd. Deoxyribonucleohistone samples that have been partially depleted of histone will be identified by the molarity of the sodium chloride solution used for the depletion. Thus a sample depleted in 0.7 M NaCl is designated 0.7 deoxyribonucleohistone. The extinction coefficients for the heat-treated samples were obtained by phosphorus analysis [S]. Circular dichroic spectra were measured in a Jouan Dicrographe Model C...
The binding curves of histones H1 and H5 to chromatin in nuclei have been determined by a novel method which utilises the differential properties of free and bound histones on cross-linking with formaldehyde. The dissociation is thermodynamically reversible as a function of [NaCl]. The binding curves are independent of temperature over the range 4 degrees - 37 degrees C and independent of pH over the range 5.0 to 9.0. The curves are sigmoid, indicating co-operative dissociation with NaCl. The standard free energy of dissociation in 1 M NaCl for H1 is 0.5 Kcals/mole and for H5 is 3.5 Kcals/mole.
Rabbit skeletal-muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis-Menten with respect to fructose 6-phosphate, ATP and manganous ion concentrations ; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.Studies on the rates of water-proton longitudinal relaxation a t pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase-manganese system shows low relaxation enhancements (&b m 2). On titrating with ATP a t fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data a t fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn-ATP sites with dissociation constant 240 pM and an enhancement for the ternary complex Et, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line-broadening studies on the H-2 and H-8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton-manganese distances in Mn-ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn-ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.The kinetic activity of phosphofructokinase from a great variety of sources has been studied with particular regard to its role in the control of glycolysis. Studies on the enzyme from mammalian skeletal muscle have been mainly involved with its characterisation [l], subunit structure [2], the regulatory properties [3], the effects of chemical modification [4,5] and the reaction mechanism [6]. All the studies on activity have used magnesium as the essential divalent cation. The metal ion is thought to bind to the enzyme in the form of a complex with ATP which is the true substrate.A preliminary observation made in this laboratory [7] showed that the enzyme was highly active when manganese replaces magnesium as the essential diAbbreviations. NMR, nuclear magnetic resonance; ESR, electron spin resonance; Fru-6-P, fructose 6-phosphate.Enzymes. Phosphofructokinase (EC 2.7.1.11) ; creatine kinase (EC 2.7.3.2) ; pyruvate kinase(EC 2.7.2.40) ; aldolase orketose-l-phosphate aldehyde-lyase (EC 4.1.2.7) ; triosephosphate isomerase (EC 5.3.1.1); glycerol-l-phosphate dehydrogenase (EC 1.1.1.8).valent cation. This opened up the possibil...
Photodynamic therapy (PDT) is a treatment combining a photosensitiser, molecular oxygen and visible light of characteristic wavelength to produce cytotoxic reactive oxygen species (ROS). Within our centre, a series of phenothiazinium salts were synthesised and initial characterisation studies performed to determine any potential use for PDT. All photosensitisers within the series were shown to have useful spectral properties for PDT, with absorbance lambdamax above 667 nm. The Log P values of the compounds were shown to range from -0.9 to > +2.0. Furthermore, Log P values were shown to be important in determining the site of subcellular localisation and as such the site of photooxidative damage. Derivatives with a Log P value of greater than +1.0 were shown to initially localise to the lysosomes then relocalise throughout the cytoplasm following illumination, whereas compounds with intermediate Log P values (-0.7 to +1.0) all remained lysosomal. Only methylene blue (Log P-0.9) was shown to redistribute to the nucleus upon illumination. Following treatment of RIF-1 cells with each phenothiazinium salt for 1 h and subsequent exposure to 665 nm laser light at a fluence rate of 10 mW cm(-2)(18 J cm(-2)), it was determined that the most potent photosensitiser was 260-fold more potent than methylene blue. Furthermore, the PDT efficacy of the photosensitisers was shown to be related to the level of mitochondrial damage induced directly following illumination.
Increasing treatment specificity is one of the major aims of cancer research. Photodynamic therapy is a clinically proven treatment for some cancers and certain other diseases. Photosensitisers generally have little intrinsic selectivity for tumours and any accumulation is dependent upon the type of tumour involved. Increasing tumour selective accumulation could improve the efficacy of PDT and reduce any risk of side effects caused by photosensitiser accumulation in non-target tissue. In order to target photosensitisers to tumours, a cyclic peptide, cRGDfK (arginine-glycine-aspartic acid-phenylalanine-lysine) has been synthesised using solid phase peptide chemistry and conjugated to the porphyrin photosensitiser, protoporphyrin IX. The arginine-glycine-aspartic acid (RGD) motif has been shown to specifically bind alphavbeta3 integrins, heterodimeric glycoproteins upregulated on the surface of proliferating endothelial cells such as those in tumour neovasculature. This study reports the synthesis, in vitro and in vivo characterisation of this novel compound and compares its properties to the free photosensitiser. The individual components in our system, protoporphyrin IX and cRGDfK retain their respective photodynamic and integrin binding activity following the coupling step and produce a conjugate of high purity. The PpIX:cRGDfK conjugate is shown to be a good photosensitiser in vitro in the integrin positive human SiHa cell line and in vivo in a mouse CaNT tumour model. Moreover, pharmacokinetic analysis of PpIX:cRGDfK treated mice shows significant retention and accumulation of photosensitiser in tumour tissue with higher tumour : normal tissue ratios than the free photosensitiser. However, although the conjugate shows this higher accumulation and improved tumour : non-target tissue ratios, the overall in vivo PDT effect, between dose-light intervals of 0 and 6 h, is not significantly better than for free protoporphyrin IX This is possibly due to differences in the target environment or in the subcellular localisation of the compounds.
The sedimentation pattern of phosphofructokinase indicates that the enzyme undergoes a self‐association reaction. Alkaline pH and high ionic strength prevented the reaction. At pH 10.5 fructose 1, 6‐bisphosphate stabilised the “monomer” of the self‐association, which had s020,w= 11.9 ± 0.4 S, and D020,w= 3.22 ± 0.33 × 10−7 cm/sec giving a molecular weight of 340000. Sedimentation equilibrium gave a molecular weight of 330000 ± 12000. The Z‐average molecular weight, measured at pH 8 by sedimentation equilibrium, increased in a sigmoid fashion with concentration from 340000 at 0.5 mg/ml to 2000000 at 7 mg/ml. By comparing the experimental curve for the variation of molecular weight versus concentration with calculated curves for open and closed polymerisations, it is suggested that the monomer associates to give a closed hexamer probably via an intermediate dimer or trimer which is present in very small amounts. The apparent association constant at pH 8.0 and 20°C was 6 × 1023 M−1 for the monomer‐hexamer reaction. The minimum molecular weight obtained in 6 M guanidine hydrochloride, 0.1 M 2‐mercaptoethanol was 85000 ± 10000 measured by a sedimentation velocity method and 76000 ± 5000 measured by sedimentation equilibrium. This implies that the “monomer” is composed of four polypeptide chains of approximately equal molecular weight.
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