Canadian journal of biochemistry最新文献

A steroid-binding protein obtained from the supernatant of the final wash from the preparation of membrane vesicles was purified severalfold to near homogeneity. The protein binds C18 and C19 steroids but has the highest affinity for androstenedione (Kd = 1.6 x 10(-10) M). The molecular weight is 51,000 - 58,000. Binding activity is slightly inhibited by Cu2+, Ca2+, and Mg2+ and completely inhibited by Zn2+. The protein has no detectable steroid degradative activity. Analysis of androstenedione binding revealed negative cooperativity of binding for this ligand and may indicate a regulatory function for this protein. It is postulated that this protein binds the steroid after testosterone is converted to androstenedione.

Fumarate reductase, purified from the cytoplasmic membrane of Escherichia coli, has been cross-linked with the bifunctional reagent dimethylsuberimidate and shown to exist as an alpha beta dimer of polypeptides of molecular weights 69,000 and 25,000 in a 1:1 molar ratio. The protein has an s20,w of 7.67S and a D20,w of 6.5 X 10(-7) cm2/s. The purified enzyme contained 4-5 mol of nonheme iron and 4-5 mol of acid labile sulfur while the visible absorption spectrum showed a broad peak between 400 and 470 nm owing to the presence of an Fe-S centre and 8 alpha[N-3]histidyl FAD. Fumarate reductase activity was readily inhibited by the sulfhydryl reagents 5,5'-dithiobis-(2-nitrobenzoic acid), p-chloromercuribenzoate, and iodoacetamide. Using 5,5'-dithiobis-(2-nitrobenzoic acid) sulfhydryl group modification was followed as a function of enzyme activity. A single cysteine residue was shown to be required for activity and this essential sulfhydryl group was located in the 69,000 dalton subunit. The amino acid composition of E. coli fumarate reductase was similar to the succinate dehydrogenases from beef heart mitochondrion and Rhodospirillum rubrum.

Suppression, by experimental inflammation, induced by subcutaneous injection of oil of turpentine, of the usual increase in liver fatty acid synthetase (FAS) activity resulting from fat-free feeding following starvation (adaptive synthesis) was shown to result entirely from lowered hepatic content of FAS protein. Comparison of changes in the relative rate of synthesis of FAS, determined radioimmunochemically during adaptive synthesis with and without inflammation, with concomitant changes in FAS activity, revealed that inflammation partically suppressed the increased rate of synthesis characteristic of adaptive synthesis, but insufficiently to account entirely for the suppression of enzyme activity. Inflammation accelerated the relative rate of degradation of FAS, causing a 50% decrease in enzyme half-life and a corresponding increase in kd and turnover index. Levels of translatable FAS mRNA rose only fivefold after 24 h of adaptive synthesis, while the relative rate of FAS synthesis increased 12-fold indicating the operation of both transcriptional and translational control. Inflammation, induced at the start of adaptive synthesis, caused a 65% lowering of the relative rate of FAS synthesis after 24 h and a 60% decrease in mRNA translatable as FAS, but was without effect on the total translational activity of the mRNA although alterations in the size distribution of RNA species in the mRNA fraction were noted.

The heat-stable modulators of phosphoprotein phosphatase activity have been partically purified from brown adipose tissue. A nonphosphorylatable inhibitor of phosphorylase phosphatase (inhibitor 2) and an activator of phosphohistone phosphatase were similar to the corresponding modulators from muscle and liver in both their physical properties and in their relative effects upon three different preparations of phosphatase. An inhibitor of phosphorylase phosphatase that was only active when phosphorylated was eluted from DEAE-cellulose by 80 mM NaCl (inhibitor 1'). Only a small amount of inhibitor was eluted at 20 mM NaCl (inhibitor 1), which is the concentration that eluted the bulk of the phosphorylatable inhibitor in muscle and liver. Inhibitor 1 and inhibitor 1' had similar physical properties but differed in their activities towards the different phosphatases. It is suggested that they are different forms of the same protein and that inhibitor 1' more closely resembles the native inhibitor. The modulators did not compete with each other and were probably not subunits of a phosphatase complex. However, the direction and timing of the changes in their concentration in brown fat during the developmental period indicate that the inhibitors, at least, perform some useful physiological function in the tissue. The concentration of inhibitor 2 was high before birth and for 10 days after birth, when the tissue was proliferating. The concentration of the phosphorylatable inhibitor was highest immediately after birth and for the next 16 days, which is the period of greatest thermogenic activity of brown fat.

The role of humoral factors in cell-cell interactions was studied in a simple model system: the aggregation of erythrocytes into cylindrical rouleaux when suspended in normal serum preheated at 62 degree C for 20 min. The rouleaugenic activity of heated serum was associated with an increased concentration of albumin polymers. On heating above 62 degree C, albumin released ligands, such as lysophosphatidylcholine, in quantities sufficient to convert erythrocytes to acanthocytes. The latter did not participate in rouleaux formation. Thus normal serum only became rouleaugenic when heated over a narrow range of temperatures. These properties of serum were reproduced in a system consisting only of erythrocytes, heated albumin, and lysophosphatidylcholine. Rouleau formation increased as albumin polymer size increased. Unheated normal serum could also be made rouleaugenic merely by concentrating to above normal physiological concentrations. Unheated, unconcentrated, sera from patients with various diseases are known to be rouleaugenic, but polymeric albumin appears infrequently in such sera; usually there are increases in macroglobulins are large polymeric forms of smaller serum proteins. Current evidence is consistent with the hypothesis that a small shift in the concentration of one or more of these macromolecules above a critical value promotes a phase separation of erythrocytes which spontaneously aggregate to form rouleaux.

Pronase digests of cultured teratocarcinoma-derived cells (PA 1) of human origin have been previously shown to contain large-sized glycopeptides (relative mass (Mr) greater then 7400), of which 15-23% are retained by columns of concanavalin A (Con A) - Sepharose and can be eluted with 10 mM methyl alpha-D-mannopyranoside. The present data show that this fraction (A - Con A II) contains a family of glycopeptides that are degradable with anhydrous hydrazine as well as with 0.05 M NaOH - 1 M NaBH4. The cleavage products representing individual oligosaccharide chains, presumably as oligosaccharides and glycopeptides, consisted mostly of medium- (Mr 1400-6000) and small-sized (Mr less than 1400) molecules. This implies that glycopeptides bearing several oligosaccharide chains were present in A - Con A II. Most of the individual oligosaccharide chains were not bound to Con A - Sepharose, but some were retained by the lectin column in the same way as the original glycopeptides. Some of the oligosaccharides were degraded partially with endo-beta-galactosidase from Escherichia freundii suggesting the presence of GalbetaGlcNAcbeta repeats. The present findings show that A - Con A II may be different from the "embryonic" glycopeptides of mouse teratocarcinoma cells that are reportedly not cleaved by mild alkaline borohydride treatment. Instead, A - Con A II is reminiscent of the T-1 glycopeptide of glycophorin.

The rate of reduction of cytochrome c by ascorbate is decreased in the presence of anions. This decrease is due to two factors: (a) nonspecific changes in ionic strength which occur when the total ion concentration or the charge on the anion is altered and (b) "specific" binding of anions to a site on cytochrome c which directly inhibits the reaction. The reaction between cyanide ion and cytochrome c is also affected by anions: increasing the ionic strength decreases the apparent association rate constant for cyanide binding. Substitution of citrate for morpholinopropane sulphonate in isoionic buffer media increases the apparent dissociation constant for cyanide suggesting citrate stabilizes the cytochrome c heme crevice. Binding of cytochrome c to cytochrome aa3 also affects the Kd for the cyanide - cytochrome c complex indicating that cytochrome c bound to cytochrome aa3 does not react with cyanide as readily as does free cytochrome c.

A neuronal nuclear fraction (N1) and a microsomal fraction (P3) were isolated from homogenates of cerebral cortices of 15-day-old rabbits. A nuclear envelope fraction (E) was prepared from N1. To assay cholinephosphotransferase, diacylglycerols were first generated in the membranes of these subfractions using a phospholipase C (Bacillus cereus) preincubation. With levels of endogenous diacylglycerols producing maximal specific cholinephosphotransferase activities, an activity ratio of 1:1:5 was found for N1, P3, and E, respectively. An independent neuronal nuclear cholinephosphotransferase, concentrated in nuclear membranes, is indicated. With regard to changes in pH and concentrations of MgCl2 and CDP-choline, N1 and P3 activities responded in a similar manner. However, in contrast to P3, N1 activities we much more profoundly inhibited at low levels of Triton X-100 (0.01-0.02 w/v%) and N1 showed quite significant levels of cholinephosphotransferase activity in the absence of a phospholipase C preincubation. Choline phosphotransferase in N1 and P3 showed Km values for CDP-choline (0.028 and 0.031 mM, respectively) which were much lower than corresponding literature values determined using exogenous diacylglycerols as substrates for this enzyme. The presence of cholinephosphotransferase in neuronal nuclear membranes reflects a rather exceptional nuclear autonomy. This may be related to a need to maintain nuclear phospholipid in the absence of a well-developed endoplasmic reticulum at early stages of neuronal development or to synthesize phospholipid in response to functions unique to the nucleus.

A mitochondrial endonuclease from Saccharomyces cerevisiae was previously shown to cut both strands of native DNA at opposite or nearby sites. The present studied demonstrate that the endonuclease activity is dependent on the strength of the hydrogen bonds between the DNA strands; the activity was measured at different ionic strengths, with substrates of different base compositions and also with DNA in which the double helix has been locally destabilized by ultraviolet irradiation, by depurination, and by single-stranded nicks. The activity is 30% greater with mitochondrial DNA (mt-DNA) than with nuclear DNA. At 0.08 ionic strength, the relative activities with double-stranded polydeoxyribonucleotides are 2.4:1:0.6 for poly(dA).poly(dU) : poly(dA).poly(dT) : poly(dG). poly(dC). Increasing ionic strength decreases similarly the activity with poly(dA).poly(dU) and poly(dA).poly(dT), but has little effect with poly(dG).poly(dC). The greater activity with poly(dA).poly(dU) than with poly(dA).poly(dT) was confirmed with nick-translated mt-DNA and with DNA synthesized in isolated mitochondria using [3H]TTP and [3H]dUTP in both cases. The endonuclease cuts modified DNA preferentially in the thymine dimer regions, at the apurinic sites, and at sites opposite to nicks. The possible involvement of this endonuclease in the degradation of mitochondrial DNA during "petite" induction is discussed.