bluuebunny/crossref_metadata_2025 huggingface dataset:
pH dependence of free and immobilized yeast alcohol dehydrogenase kinetics
Authors: Mazid M. A. , Laidler K. J.
Published: December 2009

A study was made of the influence of pH on the reaction between NAD and ethanol, catalyzed by yeast alcohol dehydrogenase, both in free solution and attached to the inner surface of a nylon tube. A new least-squares analysis of the results has been devised; it is simpler to apply and is more realistic than those previously employed. Analysis of the results for the free enzyme indicated that the free enzyme has two active ionizing groups having pK values of about 6.6 and 8.8. These pK values undergo only small changes when the enzyme is bound to NAD and when it is bound to both NAD and ethanol.With the immobilized enzyme and saturating concentrations of ethanol the rates went through a maximum as the pH was varied from 6.5 to 10.0. With saturating concentrations of NAD there was a steady increase in rate, with no falling off at pH 10. Immobilization generally brought about an increase in the pK values. These increases are attributed partly to a residual negative surface charge which attracts the leaving H+ ions. They are also attributed partly to the formation in the reaction of H+ ions, which cause the local pH to be lower than that in the bulk solution. This effect is more important with saturating NAD ions, since the buffer anions will then be less mobile and less able to mediate the movement of protons.
The purification and characterization of a phospholipase A2 activity from the 106 000 × g pellet (microsomal fraction) of bovine brain acting on phosphatidylinositol
Authors: Gray N. C. C. , Strickland K. P.
Published: December 2009

A phospholipase A2 acting on phosphatidylinositol (PI) has been purified from the 106 000 × g pellet (microsomal fraction) of bovine grey matter. The purification steps included extraction with Triton X-100 (0.05%), ammonium sulfate fractionation (20–50% fraction), consecutive column chromatographic runs on Sephadex G-200 and DEAE-Sephacel, and preparative gel electrophoresis (on 10.5% polyacrylamide gel). These steps achieved a purification of 1614 times. The purified enzyme ran as a single band on sodium dodecyl sulfate (SDS) gel electrophoresis. Molecular weight estimations gave values of 18 300 by SDS gel electrophoresis and 18 521 based on amino acid analysis. Amino acid analysis showed the presence of 173 residues with aspartic acid (46), glutamic acid (26) and glycine (21) being the most abundant. Single residues of cysteine, tyrosine, and arginine were measured. The remaining 11 amino acids were present in amounts ranging from 3 to 11 residues.The purified enzyme had a pH optimum of 7.4, was heat stable (to 70 °C), and was activated by Ca2+ (5 mM). Other divalent cations were either slightly inhibitory (Mg2+ and Mn2+) or strongly inhibitory (Zn2+). The nonionic detergents, Triton X-100 (0.02 to 0.03%) and octyl glucoside (30 mM) showed 70 and 25% stimulations, respectively. Other detergents showed no effect (Cutscum), slight inhibition (G3634A), or strong inhibition (cetyltrimethylammonium bromide). Determination of the apparent Km and Vmax by an Eisthenal–Cornish-Bowden plot gave values of 0.52 mM and 1440 nmol [1-14C]oleic acid min −1∙mg protein −1, respectively, for 1-acyl-2-[1-,14C]oleoyl-sn glycerol-3-phosphoinositol as substrate. The above plot confirmed the presence of a strong inhibition by substrate (i.e., PI) beyond 0.4 mM. The properties of this enzyme and its location (microsomal) make it uniquely different from other phospholipase A2 activities reported for brain. The microsomal location and preference for PI shown by this enzyme lend support to the view that it may function to form lyso-PI in a deacylation–reacylation cycle for altering the fatty acid distribution in PI.
A method for the specific Inhibition of poly[d(A-T)] synthesis using the A-T specific quinoxaline antibiotic TANDEM
Authors: Evans D. H. , Lee J. S. , Morgan A. R. , Olsen R. K.
Published: December 2009

A serious problem in the replication of repeating-sequence DNA polymers using Escherichia coli DNA polymerase I arises from the fact that this polymerase has a very strong preference for the replication of poly[d(A-T)]. Thus reactions primed with DNA containing small amounts of contaminating poly[d(A-T)] will eventually result in complete domination of the synthesis by poly[d(A-T)]. This problem can be overcome by the addition to the reaction mixture of the synthetic quinoxaline antibiotic TANDEM which binds specifically to poly[d(A-T)] completely inhibiting its replication. Using thermal denaturation experiments it can be shown that TANDEM does not bind to most other synthetic DNA polymers (e.g., poly(dA)∙poly(dT) and poly[d(A-T-C)]∙poly[d(G-A-T)]) and therefore their replication is not inhibited. The only exception we have encountered is poly[d(T-A-C)]∙poly[d(G-T-A)] which does bind TANDEM and therefore the drug cannot be used during the synthesis of this polymer. The fact that poly[d(T-A-C)]-poly[d(G-T-A)] does bind TANDEM while poly [d(A-T-C)]-poly[d(G-A-T)] does not, suggests that the drug recognizes T-A rather than A-T sequences.
Genetic and biochemical consequences of thymidylate stress
Authors: Barclay B. J. , Kunz B. A. , Little J. G. , Haynes R. H.
Published: December 2009

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.
Vitellogenin mRNA in locust fat body: identification, isolation, and quantitative changes induced by juvenile hormone
Authors: Chinzei Y. , White B. N. , Wyatt G. R.
Published: December 2009

Changes in RNA during the juvenile hormone (JH) stimulated synthesis of vitellogenin (Vg) in the fat body of adult female Locusta migratoria have been studied. Total RNA from mature females, but not that from males or prereproductive females, shows a 6300 nucleotide component, which has been isolated by binding to oligo(dT)-cellulose and sucrose gradient centrifugation, and identified as Vg mRNA by translation in Xenopus oocytes. It has been assayed quantitatively by photometric scanning after electrophoresis. During a gonotrophic cycle, Vg mRNA increased rapidly from 0 up to about 1% of the total fat body RNA, or more than 106 copies per cell. After destruction of the corpora allata (the source of JH) by treatment with ethoxyprecocene, Vg synthesis was stimulated by injection of 150 μg of the JH analog, methoprene. In primary stimulation, Vg mRNA was first detected at 24 h and showed a marked lag in accumulation; in secondary stimulation by methoprene after decay of the primary effect, Vg mRNA was detected after only 12 h and accumulation was much more rapid. Both in the natural cycle and in experimental stimulation, Vg mRNA did not disappear in correlation with declining Vg synthesis, which suggests conservation of mRNA in untranslated form. Total (chiefly ribosomal) RNA showed a different pattern, accumulating markedly during primary and only slightly during secondary stimulation. The data indicate that JH acts selectively (though not necessarily directly) on transcription of the Vg genes.
Rat liver ribonucleotide reductase: separation, purification, and properties of two nonidentical subunits
Authors: Youdale T. , MacManus J. P. , Whitfield J. F.
Published: December 2009

Two nonidentical subunits of mammalian ribonucleotide reductase, L1 and L2, from regenerating rat liver have been extensively purified for the first time. They were separated by dATP-Sepharose affinity chromatography. Subunit L1, which bound to dATP-Sepharose, was eluted with 50 mM ATP and purified to homogeneity (as demonstrated by sodium dodecyl sulfate (SDS) – polyacrylamide gel electrophoresis) by molecular exclusion high-pressure liquid chromatography (HPLC). This subunit had an apparent relative mass (Mr) of 45 000 and a Km of 0.9 × 10−4 for CDP. Subunit L2, which did not bind to dATP-Sepharose, was purified by pH 5.2 precipitation followed by chromatography on CM-Sephadex, molecular exclusion HPLC, and DEAE-cellulose. This subunit contained iron and had an apparent Mr of 120 000 by HPLC molecular exclusion chromatography, but showed two bands (Mr 75 000 and Mr 47 000) on SDS–polyacrylamide gel electrophoresis. Neither L1 nor L2 separately had any enzyme activity but when combined they reduced CDP to dCDP.
Study of the evolution of the genetic code by comparing the structural and catalytic properties of the aminoacyl-tRNA synthetases
Authors: Lapointe Jacques
Published: December 2009

Proteins of the primitive cells probably contained only a few of the 20 amino acid species presently incorporated into proteins. The 20 corresponding species of aminoacyl-tRNA synthetases found in most cells could result from the divergent evolution of the genes of one or a few primitive aminoacyl-tRNA synthetases. A comparison of the structural and catalytic properties of the 20 aminoacyl-tRNA synthetases should help to determine the steps of this evolution, which is also that of the genetic code. First, the very diverse quaternary structures of these synthetases are not generally correlated with the evolutionary linkages suggested by many theories of the evolution of the genetic code. The results discussed here indicate that these quaternary structures are more related to the various regulatory functions of the aminoacyl-tRNA synthetases than to their basic function which is the specific aminoacylation of tRNAs. Secondly, a comparison of the catalytic peculiarities of these enzymes appears to be useful for understanding the evolution of the genetic code; for instance, the requirement for the presence of tRNA in the activation of glutamate, glutamine, and arginine by the corresponding aminoacyl-tRNA synthetases, and the absence, in some Gram-positive bacteria, of a glutaminyl-tRNA synthetase, which is replaced by a less specific glutamyl-tRNA synthetase and an amido-transferase of Glu-tRNAGln, suggest that these three aminoacyl-tRNA synthetases specific for amino acids of the glutamate family have a common ancestor. Finally, the comparison of the sequences of the structural genes of these enzymes is a promising approach for studying their evolution and that of the genetic code.
Comparative sequence analysis as an approach to evaluating structure, function, and evolution of 5S and 5.8S ribosomal RNAs
Authors: MacKay Ron M. , Spencer David F. , Schnare Murray N. , Doolittle W. Ford , Gray Michael W.
Published: December 2009

Nucleotide sequences of nine eukaryotic and nine eubacterial 5S rRNAs have been selected for their diversity and subjected to analysis of primary and potential secondary structure. This analysis has allowed the quantitative confirmation of several previously made observations concerning 5S rRNA structure: (i) these two 5S rRNAs are derived from a common ancestor and probably perform essentially the same function in protein synthesis; (ii) one domain of 5S rRNA has undergone considerable divergence of structure (and presumably function) since the separation of the eukaryotic and eubacterial lineages; and (iii) single-stranded regions are more highly conserved than double-stranded regions. In addition, this analysis leads us to propose that (i) some of the highly conserved nucleotide residues in single-stranded regions interact in a specific manner with protein components of the translational apparatus, and (ii) repetitive folding and unfolding of helical regions occurs in two regions of eukaryotic 5S rRNA and one region of eubacterial 5S rRNA. In the context of these observations and propositions we also consider the potential secondary structure of plant mitochondrial 5S rRNA.Nucleotide sequences of 5.8S rRNAs have yielded less information about secondary structure and possible functional interactions. However, we have identified highly conserved and variable regions within this molecule and we show (in contrast to the situation with 5S rRNA) that these do not correlate well with proposed single-stranded and helical regions in a current model of 5.8S secondary structure.
Role of thyroid hormone in cold-induced changes in rat brown adipose tissue mitochondria
Authors: Triandafillou Joan , Gwilliam Cynthia , Himms-Hagen Jean
Published: December 2009

The role of thyroid gland in cold-induced growth of brown adipose tissue and in cold-induced adaptive changes in brown adipose tissue mitochondria was investigated. Interscapular brown adipose tissue of thyroidectomized rats maintained at 28 °C was of normal size (protein, cytochrome oxidase content) and its mitochondria were normal, as judged from the level of GDP binding and the polypeptide composition. (GDP binds to a 32 000 dalton polypeptide component of the inner mitochondrial membrane. This component is part of the thermogenic proton conductance pathway. The level of GDP binding is a sensitive indicator of the thermogenic state of the tissue.) Brown adipose tissue mitochondria of thyroidectomized rats exposed to 4 °C for 15 h did not show the usual increase in GDP binding, an indication of a lack of a thermogenic response; the response was restored in thyroxine-treated thyroidectomized animals. In thyroidectomized rats treated with a low maintenance dose of thyroxine (25 μg/kg, s.c., three times per week) and acclimated to cold (4 °C) for 2 weeks, a normal growth of brown adipose (increase in protein and cytochrome oxidase) and normal changes in mitochondria (increase in GDP binding and an increase in the proportion of polypeptides of molecular weight 31 200 – 34 400) occurred. Treatment of intact rats with a large dose of thyroxine (1000 μg/kg, s. c., per day) resulted in a large increase in wet weight, mainly due to lipid accumulation since only small increases in protein and cytochrome oxidase and no change in DNA content occurred; mitochondrial GDP binding was decreased and polypeptide composition unchanged. It is concluded that thyroid hormone exerts a permissive effect on the cold-induced, noradrenaline-mediated, unmasking of GDP-binding sites in brown adipose tissue. The failure of the thyroidectomized rat to survive at 4 °C is probably primarily due to its inability to activate thermogenesis in its brown adipose tissue. Thyroid hormone does not appear to be involved, other than in a permissive way, in long-term cold-induced growth and mitochondrial changes in brown adipose tissue, since these occur normally in the presence of only small amounts of thyroxine in thyroidectomized rats and do not occur in intact rats treated with large amounts of thyroxine.
Plasma methyl sterol sulfates in familial hypercholesterolemia after partial ileal bypass
Authors: Massé R. , Huang Y. S. , Eid K. , Laliberté C. , Davignon J.
Published: December 2009

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 β-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-dihydrolanosteroi, 4,4-dimethyl-5α-cholest-8-en-3β-ol, 4,4-dimethyl-5α-cholest-9(11)-en-3β-ol, 4α-methyl-5α-cholest-7-en-3β-ol, and 4α-methyl-5α-cholest-8-en-3β-ol. Their free forms are known to be biosynthetic intermediates in the transformation of lanosterol into cholesterol.
Influence of heat on the conformational stability of a human IgG cryoglobulin
Authors: Chowdhury Parimal , Saha Anil
Published: December 2009

Temperature-induced conformational changes of a human immunoglobulin G cryoglobulin (cryoIgG) (IgG) (γ1:λ, Gm4) was investigated and compared with a human myeloma IgG (γ1:λ, X, Gm4) employing spectrofluorimetric and immunochemical methods. Fluorescence measurements revealed the major changes in protein conformation of both proteins at a temperature of 62 °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 to 25 °C, indicated that both proteins underwent progressively irreversible conformational changes beyond 65 °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 temperature of 68 °C than Fd or λ-chain antigenic determinant sites. The Fd determinant sites of myeloma IgG were, however, found to be more heat labile than those of cryoIgG.
Inhibition of cathepsin L and B by haptoglobin, the haptoglobin–hemoglobin complex, and asialohaptoglobin. "In vitro" studies in the rat
Authors: Pagano M. , Nicola M. A. , Engler R.
Published: December 2009

In broadening our research on the inhibition of cathepsin B (EC 3.4.22.1) by rat haptoglobin, we have used the haptoglobin–hemoglobin complex and asialohaptoglobin. The inhibition of cathepsin L (EC 3.4.22.15), another lysosomal thiol proteinase, by haptoglobin and its related molecules has also been investigated.With azocasein as substrate, both enzymes were inhibited by both haptoglobin and its related molecules. When azocasein was used as a substrate, the apparent Michaelis constant (Km, app.) for cathepsin L was 1 × 10−5 ± 0.4 × 10−5 M. When haptoglobin was added, the apparent inhibition constant (Ki, app.) was 3 × 10−8 ± 2.5 × 10−8 M.The results suggest that rat haptoglobin specifically inhibits lysosomal thiol proteinases and that it has a regulatory role in tissue proteolysis associated with the inflammatory reaction. On the other hand, these properties would seem to be peculiar to the systems rat haptoglobin – rat liver cathepsin B or L.
Formation of erythrocyte rouleaux in preheated normal serum: roles of albumin polymers and lysophosphatidylcholine
Authors: Forsdyke D. R. , Palfree R. G. E. , Takeda A.
Published: December 2009

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 °C for 20 min. The rouleaugenic activity of heated serum was associated with an increased concentration of albumin polymers. On heating above 62 °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 or 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.
A microassay for UDP-N-acetylglucosamine pyrophosphorylase
Authors: Gopal Pramod K. , Sullivan Patrick A. , Shepherd Maxwell G.
Published: December 2009

A rapid and sensitive radiochemical microassay for UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) activity was developed in which the rate of production of UDP-N-acetylglucosamine was monitored. In this direction the assay was suitable for measuring the amount of pyrophosphorylase activity in cellular extracts in the presence of pyrophosphatase. The Km values for UTP and N-acetylglucosamine 1-phosphate in extracts prepared from Candida albicans are 0.8 and 2.1 mM, respectively.
[3H]Fucose incorporation by healing skin wounds and the effect of transglutaminase inhibitors
Authors: Bowness J. Michael
Published: December 2009

Punch wounds (3 mm) were made in the skin of rats and the animals were killed after 1 or 3 days. Plugs (4 mm) of wounded and unwounded skin were incubated in vitro with [3H]fucose. The labelled plugs were homogenized and subjected to sequential extraction with buffered salt solutions, ethanol–ether, and 8 M urea – 50 mM dithiothreitol (DTT). Nondialysable counts in the extracts and insoluble residue were determined and the incorporation of label by wounded and unwounded skin plugs was compared. Wound plugs showed a greater total incorporation of [3H]fucose. In addition, a greater proportion of [3H]fucose was found in the urea–DTT extracts. The highest specific activity (disintegrations per minute t [3H]fucose per milligram dry weight) was found in a finely dispersed precipitate, sedimenting at 10 000 × g but not at 1000 × g. The transglutaminase inhibitors aminoacetonitrile and dansyl cadaverine were found to increase the extractability of a portion of the material which incorporated [3H]fucose without affecting the total incorporation. These results show that healing wounds have an increased biosynthetic capacity for an insoluble fucosylated glycoprotein fraction and they suggest that transglutaminase is necessary to make this fraction fully insoluble.
Association of the polyene antibiotic amphotericin B with phospholipid vesicles: perturbation by temperature changes
Authors: Bolard Jacques , Cheron Monique
Published: December 2009

Conformational changes of amphotericin B in the presence of cholesterol as well as in the presence of bilayer vesicles of phosphatidylcholine with saturated fatty acid chains of various lengths (14 < n < 22) have been monitored by circular dichroism (CD).It has been shown that the observed species are not only dependent on such parameters as the cholesterol content of the vesicles, the vesicles' physical state, and the number of amphotericin B molecules per vesicle, but also on the time elapsed after mixing and the thermal treatment of the system, which may create irreversible changes. In particular, heating through the transition temperature (Tc) vesicles containing cholesterol and loaded with amphotericin below Tc leads to the expulsion into the aqueous medium of a cholesterol–amphotericin complex, a phenomenon which affords an explanation for some of the electron paramagnetic resonance and resonance Raman results. It has also been shown by gel filtration, ultracentrifugation, and Tc determination that interaction of amphotericin B with vesicles in the gel state induces fusion or aggregation of the vesicles, which is not the case (or at least weakly) when the vesicles are in the liquid crystalline state. This aggregation is the more rapid the nearer the temperature of the reaction is to Tc.This study confirms the great complexity of events which may occur during interaction of amphotericin B with model membranes and presents some results which complement those of studies performed with other spectroscopic methods.
Comparison of complexes containing lysyl-tRNA synthetase from normal and virus-transformed cells
Authors: Thomas Kent , Scheets Kay , Allen Sharon , Hedgcoth Charles
Published: December 2009

We compared the lysyl-tRNA synthetases from normal (Balb/3T3) and murine sarcoma virus-transformed (KA31) mouse fibroblasts. In agreement with several other reports of mammalian systems, the lysyl-tRNA synthetases from these cells occurred in very large postmicrosomal complexes as determined by gel filtration on agarose columns. Arginyl-, isoleucyl-, methionyl-, phenylalanyl-, and tyrosyl-tRNA synthetases also occurred as part of a large complex or complexes. Activity of glycyl- or leucyl-tRNA synthetase was not detected in a complex. The specific activities of arginyl- and methionyl-tRNA synthetases were three- and five-fold higher, respectively, in a complex from KA31 as compared with a complex from Balb/3T3. In contrast, the specific activity of lysyl-tRNA synthetase from the Balb/3T3 complex was 50% higher than that of the KA31 complex. tRNALys obtained from the complexes of Balb/3T3 and KA31 was fractionated into isoacceptors on columns of RPC-5. The relative amounts of lysine isoacceptors in total preparations of tRNA from normal whole cells and in tRNA obtained from the normal enzyme complex were the same. However, two isoacceptors were present in greater amounts and two were present in lesser amounts in the KA31 enzyme complex as compared with lysine isoacceptors in a total preparation of tRNA from KA31 cells.
Interaction of lipids with intestinal brush border membrane preparations
Authors: Proulx P. , McNeil J. , Brglez I. , Williamson D. G.
Published: December 2009

Conditions for uptake of lipids by rabbit intestinal brush border membrane preparations were investigated. A variety of lipids were found to be incorporated, including choline and ethanolamine phosphatides as well as cholesterol, diglyceride, and fatty acid. The incorporation of those lipids tested was enhanced by Ca2+ and other divalent cations but not by monovalent cations. The optimal Ca2+ concentration was approximately 10 mM. The uptake varied with lipid and membrane protein concentration and proceeded at rates which were too rapid to measure under several assay conditions tried. Incorporations were decreased substantially outside the pH range of 6.5–8.0. The effect of one lipid, phosphatidylcholine, on the structural appearance of the membrane fraction was examined by electron microscopy. No free or surface-bound lipid structures could be detected and the membrane fractions appeared to be unchanged after uptake.
In situ enzymatic removal of orthophosphate by the nucleoside phosphorylase catalyzed phosphorolysis of nicotinamide riboside
Authors: Shriver John W. , Sykes Brian D.
Published: December 2009

An enzymatic orthophosphate removal system is described which can be effectively used to continuously remove orthophosphate from biochemical samples. The phosphorolysis of nicotinamide riboside is catalyzed by calf spleen nucleoside phosphorylase to give ribose-1-PO4 and nicotinamide along with a proton. At pH 8 the production of ribose-1-PO4 from orthophosphate is essentially quantitative. This reaction can be monitored optically or by 31P nuclear magnetic resonance (NMR). Equations are given for determining the time required to remove a given amount of phosphate from a typical NMR sample with a known amount of nucleoside phosphorylase. The effects of a competing orthophosphate-producing reaction are considered.
Characterization of the human placental receptor for basic somatomedin
Authors: Bhaumick B. , Armstrong G. D. , Hollenberg M. D. , Bala R. M.
Published: December 2009

We have determined optimal conditions for the solubilization of the basic somatomedin (SM) receptor from human plancental membranes and for the measurement of the binding of basic SM to the solubilized receptor. Further, we have developed conditions under which the basic SM receptor, in the presence of equivalent amounts of insulin receptor, can be selectively and specifically affinity-labeled with 125I-labeled basic SM, using the cross-linking reagent disuccinimidyl suberate (DSS). Our results with these developed methods indicate that the properties of the soluble basic SM receptor (pH optimum for ligand binding, pH 7 to 9; adsorption to lectin-agarose derivatives; sedimentation coefficient in detergent–sucrose solutions, 11S) closely parallel data previously reported for the insulin receptor. Based on the sedimentation coefficient and the previously estimated Stokes radius of the soluble receptor (7.2 nm), a molecular weight of 402 000 can be calculated for the detergent–receptor complex. Electrophoretic analysis of the basic SM receptor, selectively cross-linked to 125I-labeled basic SM with DSS in the presence of excess unlabeled insulin revealed, under reducing conditions, a major labeled constituent of 140 kdaltons, substantiating our previous work employing a photoaffinity labeling reagent. DSS cross-linking also demonstrated the presence of less intensely labeled components with apparent molecular weights of 54 000, 43 000 and 35 000 but failed to reveal a distinct 90- to 100-kdalton species visualized in parallel experiments with insulin. The 53-kdalton species was not detected in similar experiments with insulin. A specifically labeled basic SM receptor component of 300 kdaltons was also observed under reducing conditions; in the absence of β-mercaptoethanol, all labeled components migrated in the 300-kdalton range. In comparison, selective DSS labeling of the insulin receptor in the presence of excess basic SM revealed components which, upon electrophoresis under reducing conditions, exhibited apparent molecular weights of 300 000, 140 000, 90 000–100 000, 43 000 and 35 000. The major insulin-labeled component (140 000) comigrated with the major constituent (140 000) selectively labeled with basic SM. Chymotryptic digestion of the receptors selectively DSS labeled with either 125I-labeled insulin or 125I-labeled basic SM yielded quite similar, but distinctive, gel electrophoretic maps. We conclude that the receptors for basic SM and insulin are highly homologous structures, particularly with respect to their glycoprotein nature, their hydrodynamic properties, their disulphide cross-linked composition, and with respect to the size of the major constituent detected by selective affinity labeling. Nonetheless, the detection of electrophoretically distinct labeled receptor substituents upon analysis of specifically labeled material, both before and after chymotryptic cleavage, points to subtle differences between the polypeptide compositions of the two receptors.
Glycosylation of plasma proteins with [U-14C]gIucose in vivo
Authors: Vrba Rudolf , Adams Stephen P.
Published: December 2009

Within 1–12 h after subcutaneous injection of [U-14C]glucose, significant amounts of 14C attached by stable (probably covalent) bonds were found in plasma proteins of rats. More than 30% of 14C of [U-14C]glucose injected per 1 g body weight was incorporated into proteins contained in 1 mL of plasma. Less than 5% of 14C of the [14C]glucose-labelled plasma proteins was soluble in cold dilute perchloric acid, whereas more than 80% of 14C in the [14C]glucose-labelled plasma proteins was soluble in 50% saturated solutions of ammonium sulfate. From [U-14C]glucose-labelled plasma proteins, approximately 50% of the incorporated 14C was recovered in carbohydrate moieties (sialic acid, 8–12%; mannose and galactose, 15–31%; hexosamines, 8–14%) and the rest of the 14C (42–64%) was recovered from protein residue. Gel-filtration and electrophoresis profiles of distribution of 14C in [U-14C]leucine-labelled plasma proteins were very similar to those of [U-14C]glucose-labelled plasma proteins; a relatively high specific radioactivity was observed in fractions having an apparent molecular weight of 105 × 103 or its multiples (220 × 103 and 520 × 103). However, about 99% of the incorporated 14C was recovered from the protein residue of [14C]leucine-labelled plasma proteins, whereas only traces of 14C were found in the carbohydrate moieties. [U-14C]Alanine is also a relatively poor precursor of 14C for incorporation into carbohydrate moieties of plasma proteins as compared with [U-14C]glucose.
Nuclear membrane-bound UDPglucuronosyltransferase of rat liver
Authors: Zaleski Jan , Bansal Surendra K. , Gessner Teresa
Published: December 2009

Some properties of rat liver nuclear membrane-bound UDPglucuronosyltransferase were compared with those of the endoplasmic reticulum bound enzyme. The activity of nuclear membrane-bound UDPglucuronosyltransferase was stimulated only about 1.5-fold by Lubrol WX. Under the same conditions microsomal UDPglucuronosyltransferase was, as usual, highly activated (up to 10-fold), when 4-nitrophenol was the acceptor of glucuronic acid. Specific activities of the detergent-activated enzyme were similar in microsomal and nuclear membrane preparations, when the following aglycone substrates were used: 4-methylumbelliferone, 4-nitrophenol, 1-naphthol, phenolphthalein, and testosterone. Apparent Km values for UDP-glucuronic acid ranged between 0.15–0.25 mM for glucuronidation of 4-nitrophenol and 1-naphthol, by either Lubrol WX activated or non-activated, nuclear membrane-bound UDPglucuronosyltransferase. These values were comparable to those found for detergent activated microsomal enzyme. The results show a similarity in behavior of detergent-activated UDPglucuronosyltransferase regardless of subcellular membrane source and, therefore, suggest the association of the same glucuronosyltransferase with nuclear membrane and endoplasmic reticulum. A possible significance of the presence of high activity of this enzyme in nuclear membrane is discussed.
Adenoassociated virus has a unique chromatin structure
Authors: Brown Martha , Weber Joseph
Published: December 2009

The organization of intranuclear adenoassociated virus DNA (AAV) was examined following micrococcal nuclease digestion of nuclei prepared from cells coinfected with AAV type 2 (AAV-2) and adenovirus type 2 (Ad2). Blot-hybridization analysis of the DNA with AAV-2, Ad2, and cellular DNA probes revealed that AAV-2 chromatin has a unique structure, which upon nuclease digestion gives rise to a smear of oligomeric DNA fragments from 600–2200 base pairs in length with only a very faint band about 160 base pairs and no discrete multimers. This structure was similar to, but distinguishable from, Ad2 chromatin and completely unrelated to eukaryotic chromatin.
Hyper(ADP-ribosyl)ation of histone H1
Authors: Aubin R. J. , Dam V. T. , Miclette J. , Brousseau Y. , Huletsky A. , Poirier G. G.
Published: December 2009

Nucleosomal chains of various repeat unit lengths were generated by a mild micrococcal nuclease digestion of purified pancreatic nuclei. Maximal nucleosome associated poly(ADP-ribose) polymerase activity was recovered in trimeric to tetrameric chromatin fragments, after which the enzyme activity gradually decreased and stabilized towards oligomeric periodicities of 11 to 16 nucleosomes. Electrophoresis of [32P] ADP-ribosylated histones on first-dimension acid–urea or acid–urea–Triton gels and on second-dimension acid – urea – cetyltriammonium bromide gels revealed that, of all histones, only histone H1 could be significantly poly(ADP-ribosyl)ated while only minimal modification could be recovered with histone H10. Furthermore, the extent of ADP-ribosylation present on pancreatic histone H1 is shown to proportionally retard this protein's electrophoretic mobility in all gel systems and to consist of a distinct series of at least 12 modification intermediates which can be evidenced, in nuclei or nucleosomes, and fully recovered along with histone H1 upon its selective extraction with 5% perchloric acid. The generation of these increasingly ADP-ribosylated forms of histone H1 is also demonstrated to be time dependent and the more complex ADP-ribosylated forms of this histone are favored at high NAD+ concentrations. Moreover, the electrophoretic mobilities of all intermediates are unaffected by the presence of the nonionic detergent Triton X-100.
Glycerol-induced adenine nucleotide catabolism in rat liver cells
Authors: Rosiers Christine Des , Lalanne Marcel , Willemot Joan
Published: December 2009

Prelabeled adenine nucleotides were rapidly degraded in freshly isolated rat liver cells incubated with glycerol at concentrations greater than 0.25 mM. Concomitantly, the [14C]ATP/[14C]ADP and [14C]ATP/[14C]AMP ratios were decreased. There was a transient increase in radioactive IMP, inosine, and hypoxanthine and a constant accumulation of allantoin. Radioactive adenosine also accumulated transiently under certain conditions. The intracellular Pi; concentration was decreased. The magnitude of most of these changes was dependent upon the concentration of glycerol. The effects of glycerol were similar to those of fructose. The extent of endogenous adenine nucleotide catabolism was smaller at high Pi concentration, whereas the extent of glycerol-induced catabolism was unaffected. However, less inosine and more hypoxanthine accumulated during glycerol-induced catabolism at high Pi concentration.
Phosphorylated and unphosphorylated forms of cardiac tropomyosin
Authors: Segura Magdalena , Palmer Elizabeth , Saborío José L.
Published: December 2009

Cardiac tropomyosin from 20-day-old chick embryos is composed of three different polypeptides with the same molecular weight but different isoelectric points. These polypeptides, which are designated as α1, α2, and α3, have identical peptide maps. In vitro, however, only polypeptide α1 is synthesized in a reticulocyte lysate programmed with cardiac RNA. These results, together with the observations indicating that tropomyosin α2 corresponds to a phosphorylated polypeptide, suggest that only tropomyosin α1 corresponds to a primary translational product and that forms α2 and α3 are derived from α1 as a consequence of posttranslational modifications.
2-Fluoro-ATP, a fluorinated ATP analog. 19F nuclear magnetic resonance studies of the 2-fluoro-ADP∙myosin subfragment-1 complex
Authors: Baldo John H. , Hansen Poul E. , Shriver John W. , Sykes Brian D.
Published: December 2009

The synthesis of a fluorinated ATP analog, 2-fluoro-ATP (2-flATP), is described. This analog is designed for 19F nuclear magnetic resonance (NMR) studies of large enzymes and proteins which bind adenine nucleotides. 2-flATP is shown to be active as an ATP analog in a number of enzyme systems, and its 19F-NMR properties are determined. Specifically the principal elements of the 19F-NMR chemical shift tensor are shown to be 104, 12, and −116 ppm. The complex between 2-flADP and the myosin subfragment-1 ATPase is studied by 19FNMR, comparing the normal Michaelis complex and 2-flADP "trapped" on subfragment-1. These complexes are shown to be indistinguishable from the standpoint of the environment and mobility of the adenine ring.
Immunoadsorbent isolation of pregnancy-specific β1-glycoprotein from maternal serum
Authors: Griffiths Bertram W. , Godard André
Published: December 2009

A three-step procedure for the purification of pregnancy-specific β1-glycoprotein (PSβ1G) on a milligram scale from maternal serum has been developed. The principal purification was achieved by the use of an immunoadsorbent and the remaining impurities were removed by hydroxylapatite chromatography and negative affinity chromatography. The overall procedure resulted in the purification of approximately 10 mg of PSβ1G which represented about 21% of PSβ1G in 300 mL of serum. The PSβ1G was of high purity as shown by analytical polyacrylamide gel electrophoresis, sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and immunochemical tests. Experiments by immunoelectrophoresis and gel chromatography indicate that the electrophoretic mobility and relative mass of the purified PSβ1G are very similar to those of the native serum protein. Structural analysis of PSβ1G suggests that it is composed of two identical subunit chains bonded noncovalently. However, a trimeric structure for PSβ1G cannot be ruled out based on the uncertainty of relative mass estimates by gel chromatography in nondenaturing solvent. The anomalous characteristics of a previous purified polymeric form of PSβ1G (PSβ1G-I) are discussed in relation to the new findings presented here.
Mechanism of a long-chain fatty aldehyde dehydrogenase induced during the development of bioluminescence in Beneckea harveyi
Authors: Bognar Andrew , Meighen Edward
Published: December 2009

The kinetic mechanism of a long-chain aldehyde dehydrogenase that is induced during the development of bioluminescence in Beneckea harveyi has been investigated. Parallel lines were obtained in Lineweaver–Burk plots with NAD+ and long-chain aldehydes (heptaldehyde, nonylaldehyde). However, product and dead-end inhibitor studies, substrate protection (NAD+, aldehyde) against inactivation with N-ethylmaleimide, and in particular, a noncompetitive substrate inhibition pattern with aldehyde at high concentrations showed that aldehyde dehydrogenase had a sequential mechanism. The data were consistent with a nonrapid equilibrium random mechanism with a preferred order of addition of substrates (NAD+, aldehyde) and an ordered release of products with NADH release being the last and rate-limiting step in the reaction, a mechanism very similar to that found for short-chain mammalian aldehyde dehydrogenases. The present experiments emphasize the caution that must be taken in interpreting parallel patterns in initial velocity experiments, as well as the difficulty in classifying sequential enzyme mechanisms as either strictly ordered or random.
Multiple hormonal control of pars intermedia cell activity
Authors: Meunier H. , Labrie F.
Published: December 2009

In rat pars intermedia cells, the rate of α-melanocyte-stimulating hormone (α-MSH) secretion was so far known to result from a balance between the stimulatory effect of β-adrenergic agonists and the inhibitory influence of dopaminergic substances. Recently, we have identified a second stimulatory substance, namely corticotropin-releasing factor (CRF). CRF is a potent stimulator of pars intermedia adenylate cyclase activity, cAMP accumulation and α-MSH release. A requirement for calcium ions was observed on basal as well as on CRF-induced α-MSH secretion. The β-adrenergic and CRF effects on adenylate cyclase activity, as well as the dopamine inhibition of adenylate cyclase activity, are potentiated by guanine nucleotides (GTP). Stimulation of the β-adrenergic receptor with isoproterenol causes a rapid loss in cAMP responsiveness, which can be completely blocked by β-adrenergic antagonists and partially prevented by dopamine. These findings suggest that CRF should now be considered, in addition to β-adrenergic agents, as a stimulator of the activity of pars intermedia cells and that cAMP is also involved as mediator of its action. Changes of receptor sensitivity, as well as interaction of the two stimulatory receptors with the inhibitory dopaminergic receptor, are involved in the fine control of pars intermedia cell activity. All three receptors appear to exert their action through a common pathway, namely changes of adenylate cyclase activity.
Evidence for the existence of a C25-sesterterpene pathway of steroidogenesis not involving cholesterol as an obligatory intermediate
Authors: Bhavnani Bhagu R. , Wong Tony
Published: December 2009

Previous in vitro studies had indicated the possibility of steroidogenesis through a C25-sesterterpene pathway in which squalene and cholesterol are not required as obligatory intermediates. To investigate whether such a pathway exists in vivo, the precursor role of [7-3H]23,24-dinor-5-cholene-3β,20-diol in the in vivo formation of cortisol by the guinea pigs was studied. The [3H]23,24-dinor-5-cholene-3β,20-diol was synthesized by reacting [3H]pregnenolone acetate with a Grignard reagent. The product was purified by chromatography and its radiochemical purity was established by the isotope dilution technique. In the first experiment a total of 134 × 106 dpm of [3H]23,24-dinor-5-cholene-3β,20-diol was injected subcutaneously into three guinea pigs. Urine was collected for 8 days and was pooled. Only 12% of the administered dose was excreted in the urine. The urine was extracted and a neutral extract (3 × 106 dpm) was obtained. From this extract 2.3 mg of cortisol containing 2.9 × 104 dpm was isolated. Radiochemical purity of the isolated cortisol was established by the isotope dilution technique. Radiochemical purity was further confirmed by conversion to cortisol 21-acetate and subsequently to 11β-hydroxyandrost-4-ene-3,17-dione and recrystallization to constant specific activity. The results of this experiment were confirmed by repeating the experiment with a higher specific activity [3H]23,24-dinor-5-cholene-3β,20-diol. These results indicate that the C25-sesterterpene pathway is a possible in vivo alternate pathway of steroidogenesis, not involving either squalene or cholesterol as obligatory intermediates.
Progesterone C21 hydroxylation and steroid carboxylic acid biosynthesis in the rabbit. In vitro studies with endocrine, metabolic, and potential target tissues
Authors: Senciall I. R. , Bullock G. , Rahal S.
Published: December 2009

Progesterone C21-hydroxylase activity has been demonstrated with rabbit kidney microsomes for the first time and the formation of 21-hydroxy-4-pregnen-3,20-dione (DOC) by rabbit liver and kidney microsomes has been quantitated. Considerable intraspecies variability in enzyme activity occurred with both tissues. The liver enzyme (Vmax = 1.28–38.0 nmol/mg protein per 30 min of incubation) was significantly more active than the kidney enzyme (Vmax = 0.028–0.28 nmol/mg protein per 30 min of incubation). Apparent KM values were 1.39 and 0.8 μM, respectively. Cytochrome c (10−5 M), potassium ferricyanide (10−3 M), and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone; 10−3 M) were strongly inhibitory with both tissues, whereas the liver microsomal system was less sensitive than the kidney to CO–air (90:10 v/v) inhibition. Metabolism of [14C]DOC to 4-pregnen-3,20-dione-21-oic (pregnenoic) and 4-androsten-3-one-17β-carboxylic (etienic) acids by liver microsomes and adrenal and ovary homogenates was differentially affected by several factors. CO–air (90:10 v/v), cytochrome c (10−5 M), and metyrapone (10−3 M) inhibited pregnenoic acid synthesis to a greater extent than etienic acid. Sodium cyanide had a stimulatory effect on the synthesis of pregnenoic acid by the liver but less consistent effects with other tissues. These results suggest that one or more cytochrome P-450 systems may be involved in the oxidation of progesterone through to pregnenoic acid by rabbit tissues.