Canadian journal of biochemistry最新文献

Rabbit reticulocyte membrane-bound ribosomes liberated by deoxycholate treatment contain degraded forms of ribosomal and messenger RNA. This degradation occurs after the liberation of the ribosomes from the membranes by the detergent because intact birosomal and messenger RNA can be extracted from washed membranes by phenol treatment. Increasing the ionic strength of the detergent buffer prevents this RNA degradation and allows the recovery of membrane-bound ribosomes capable of protein synthesis. Comparison of the proteins synthesized in vitro by the polyribosomes shows that the main protein produced by both free and membrane-bound ribosomes is globin. However, the two types of polyribosomes could be distinguished by the nonglobin proteins they produce.

3-Hydroxy-3-methylglutarylCoA reductase activity was found to be low in the microsomal fraction of the small intestine, liver, and brown fat in suckling rats. It was high perinatally. Treatment with phosphatase of the microsomal fraction increased activity considerably in gut, somewhat in liver, and not al all in brown fat of infant rats. Activity in the mitochondrial fraction of the small intestine showed no developmental changes. injections of infant rats with triiodothyronine increased and with cortisone decreased hepatic activity but had no effect on gut or brown fat. It is concluded that even though activity decreases in all three tissues after birth, it is regulated differently in liver than in gut and brown fat.

We demonstrate, in this study, the presence of several unidentified components in the sterol sulfate fraction of familial hypercholesterolemia patients treated with partial ileal bypass surgery. The sterols obtained after solvolysis and derivatization of this fraction had a retention time, on gas-liquid chromatography, intermediate between cholesterol and beta-sitosterol. They were not present in the sterol sulfate fraction obtained from normal subjects, hypercholesterolemic patients, or ileal bypass subjects before surgery or after reanastomosis. The substances isolated from the sterol sulfate fraction were identified by combined gas-liquid chromatography and mass spectrometry to be 24,25-dihydrolanosterol, 4,4-dimethyl-5alpha-cholest-8-en-3beta-ol, 4,4-dimethyl-5alpha-cholest-9(11)-en-3beta-ol, 4alpha-methyl-5alpha-cholest-7-en-3beta-ol, and 4alpha-methyl-5alpha-cholest-8-en-3beta-ol. Their free forms are known to be biosynthetic intermediates in the transformation of lanosterol into cholesterol.

Dipalmitoyl phosphatidylcholine (DPPC) and egg phosphatidylcholine (egg PC) are not completely miscible at all temperatures. Their phase diagram was determined by differential scanning calorimetry (DSC) of aqueous mixtures of the two. From the integrated DSX curves we obtained the enthalpy of solution of DPPC in egg PC delta hs, as a function of the mole fraction of DPPC, X, and using the empirical relationship between delta hs and X, the solubility Xsat as a function of temperature, T. The latter could be described by the semiempirical relationship: R1nXsat = a + blnT - c/T, where a = 6.57 X 10(-2) kcal.mol-1. degree -1 and c = 20.5 kcal. mol-1 (1 cal = 4.1868 J); the coefficient b was very small and could be ignored. The quantity delta hs can be given as XdeltahDPPC + deltah mix, where deltah DPPC is the gel - liquid crystalline transition enthalpy of DPPC (8.74 kcal.mol-1) and deltah mix is the enthalpy of mixing the two liquid crystalline lipids. Deltahmix depends on X in approximately a parabolic fashion, having a maximal value of 4.8 kcal.mol-1 at X = 0.6. It was shown that both the solubility and mixing enthalpy data can be described by the theory of regular solutions (RST). In RST, the activity coefficient of the solute (component 2) of a binary solution is given by RTIngamma 2 = (1 - Theta2)2deltaU, while the mixing enthalpy is given by delta hmix = Theta1 Theta2 delta U/v2, where Theta1 and Theta2 are the volume fractions of solvent and solute (egg PC and DPPC, respectively), v2 is the partial molar volume of DPPC, and deltaU is the energy change per mole on interchanging a DPPC and an egg PC molecule between their respective liquid crystalline phases. The thermodynamic data are accurately described by RST, the molar volume of DPPC being found to be about half that of egg PC solution and the interchange energy deltaU having a value of 10-11 kcal.mol-1. There was some evidence that deltaU may be an increasing function of temperature. The large value of the deltaU accounts for the pronounced temperature dependence of the solubility Xsat, which decreases from 0.35 at 35 degrees C to 0.02 at 10 degrees C. The presence of cholesterol in the mixtures decreases both the transition enthalpy of DPPC and the mixing enthalpy in a linear fashion, so that deltahs is zero at Xcholesterol greater than or equal to 0.2. The results are consistent with recent data including the formation of a PC-cholesterol complex oc stoichiometry approximately 4:1.

Temperature-induced conformational changes of a human immunoglobulin G cryoglobulin (cryoIgG) (IgG) (gamma 1:lambda, Gm4) was investigated and compared with a human myeloma IgG (gamma 1:lambda, Gm4) employing spectrofluorimetric and immunochemical methods. Fluorescence measurements revealed the major changes in protein conformation of both proteins at a temperature of 62 degrees C and above, the measurements being carried out with excitation wavelengths at 278 and 295 nm, respectively. Studies on both cryoIgG and myeloma IgG, which were heat denatured at high temperatures and subsequently cooled at 25 degress C, indicated that both proteins underwent progressively irreversible conformational changes beyond 65 degress C and cryoIgG appeared to be more temperature sensitive than myeloma IgG. Evaluation of the changes on specific antigenic determinant sites using antigen-antibody interaction revealed that the Fc determinant sites of both the proteins were disorganized to a greater extent at a temperture of 68 degress C than Fd or lambda-chain antigenic determinant sites. The Fd determinant sites of myeloma IgG were, however, found to be more heat labile than those of cryoIgG.

Subjecting 5-day-old plumules of corn (Zea mays L.) to elevated temperatures for brief periods of time causes the pattern of protein synthesis to shift from the production of a broad spectrum of proteins to the new and (or) enhanced synthesis of a small number of heat-shock polypeptides (HSPs). Most notable is the depressed synthesis of a major polypeptide (relative mass (Mr) = 93 000 and isoelectric point = 8.0) normally made at 27 degrees C and the enhanced and (or) new synthesis of polypeptides with MrS of 108 000, 89 000, 84 000, 76 000, 73 000, and 18 000, following 1 h of heat shock. These six HSPs is observed within 120 min following heat shock. Recovery from heat shock is rapid; after 6 to 8 h at 27 degrees C following heat shock, the polypeptide pattern is indistinguishable from the control. Extracts from individual heat-shocked shoots produced polypeptide synthetic patterns identical to those from extracts from 20 shoots, regardless of whether single shoots were intact or excised during labelling. Single 5-day-old primary roots exhibited polypeptide synthetic patterns and responded to heat shock in a manner similar to shoots. This is the first demonstration of the induction of heat-shock polypeptides in a whole, intact higher plant.

Inorganic pyrophosphatase has been purified from the soluble fraction of Streptococcus salivarius by protamine sulfate treatment, ammonium sulfate fractionation, and chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was purified approximately 500-fold with a 33% yield. The purified enzyme was homogeneous since it showed a single band when examined by nondenaturing polyacrylamide gel electrophoresis. It was rich in acidic (glutamic and aspartic) amino acids, as well as serine and glycine. The enzyme was devoid of sulfur-containing amino acids. The purified enzyme was specific for the hydrolysis of inorganic pyrophosphate and did not hydrolyze any other phosphate-ester compound examined. Inorganic pyrophosphatase activity was completely dependent on a divalent cation. Activity was maximum in the presence of Mg2+ while activity in the presence of Mn2+ and Co2+ was significantly lower. In the presence of Mg2+, a number of divalent cations, however, inhibited the enzyme activity. The true substrates for S. salivarius inorganic pyrophosphatase were magnesium-pyrophosphate complexes, i.e., MgPPi and Mg2PPi, while free Mg2+ had no effect on the enzyme activity and free PPi inhibited the hydrolysis of inorganic pyrophosphate. Km value for magnesium-pyrophosphate complexes was 16.4 microM. Km value for total Mg2+ was similar ranging between 14.4 and 20 microM. Analysis of data by Hill plots indicated one binding site for Mg2+ and two for PPi. Among various nucleotides and glycolytic intermediates examined, GDP, GMP, and fructose-1, 6-P2 showed significant inhibitory effect on enzyme activity.

Extensive sequence data on mitochondrial (mt) tRNAs give for the first time an opportunity to evaluate tRNA gene evolution in this organelle. Deductions from these gene structures relate to the evolution of tRNA genes in other cellular systems and to the origin of the genetic code. Mt tRNAs, in contrast to the prokaryotic nature of chloroplastic tRNA structure, can not at the present time be definitely related to either prokaryotic or eukaryotic tRNAs, probably because of a higher mutation rate in mitochondria. Fungal mt tRNAs having the same anticodon and function are generally similar enough to be considered homologous. Comparisons af all mt tRNA sequences contained in the same mitochondrion indicate that some tRNAs originated by duplication of a prototypic gene which, after divergence, led to tRNAs having different amino acid specificities. The deviant mt genetic code, although admittedly permitting a simpler decoding mechanism, is not useful in determining whether the origin of mitochondria had preceded or was derived from prokaryotes or eukaryotes, since the genetic code is variable even among mitochondria. Variants of the mt genetic code lead to speculation on the nature of the primordial code and its relation to the present "universal" code.