bluuebunny/crossref_metadata_2025 huggingface dataset:
Germin: compartmentation of two forms of the protein by washing growing wheat embryos
Authors: Lane Byron G. , Grzelczak Zbyszko F. , Kennedy Theresa D. , Kajioka Robert , Orr Jeanne , D'Agostino Sandra , Jaikaran Anna
Published: December 2009

(1) The electrophoretic band patterns (sodium dodecyl sulfate (SDS) – polyacrylamide) for either isotopically labeled or dye-stained proteins in a wash fraction obtained by rinsing germinated wheat embryos with any of a variety of buffers are different from those observed for proteins in either the soluble or pellet fractions of embryo homogenates. Interestingly, the wash fraction is enriched with respect to germin, a marker protein for the onset of growth in germinating wheat embryos.(2) There is selective accumulation of a novel form of germin in the wash fraction. This novel form migrates just ahead of the more usual form of germin during electrophoresis in SDS–polyacrylamide gel, being about 5% smaller, assuming a linear relation between gel mobility and log (relative mass), but it yields the same methionine-labeled peptides when denatured and digested by Staphylococcus aureus protease.(3) Quasi-quantitative analyses of dye-stained proteins suggest that, in the case of embyros cultured in 5% sucrose, which show greatest growth, 50% or more of the total germin is present, in the novel form, in the wash fraction, which contains less than 5% of the total proteins. By way of contrast, germin is barely detected in the wash fraction of embryos excised from germinated grains, unless the excised embryos are incubated in water or 5% sucrose for an additional 24 h.(4) Allied studies of (possibly epiphytic) bacteria, found in wash fractions prepared following germination of preisolated embryos or grains, are described.(5) The significance of the present findings for our continuing efforts to define a phyiological role for germin are discussed at some length.
The roles of cAMP and cAMP-dependent protein kinase in the regulation of adrenocortical functions: analysis using DNA-mediated gene transfer
Authors: Wong Margaret , O'brien Donalee , Schimmer Bernard P.
Published: December 2009

DNA-mediated gene transfer was used to evaluate the cause and effect relationship between mutations in cAMP-dependent protein kinase activity and cellular resistance of adrenocortical tumor cells to ACTH and cAMP. Protein kinase defective, Kin 8 adrenocortical tumor cells were transformed with genomic DNA from an ACTH- and cAMP-responsive adrenocortical cell line and screened for the recovery of morphological responses to the cAMP analog 8-bromo-cAMP (8BrcAMP). 8BrcAMP-responsive transformants were recovered with a frequency of approximately 0.5 per 103 transformation-competent cells. These transformants recovered the ability to round up in the presence of ACTH and were able to respond to both ACTH and 8BrcAMP with increased steroidogenesis. They also recovered cAMP-dependent protein kinase activity. The transformants, however, were unstable and concomitantly lost cAMP-dependent protein kinase activity and steroidogenic and morphological responses to ACTH and 8BrcAMP. These observations suggest that a single gene, probably the gene encoding the regulatory subunit of cAMP-dependent protein kinase, is responsible for the resistance of the Kin 8 mutant to ACTH and cAMP.
Modulation by phenobarbital of the differentiation of 3T3 preadipocytes
Authors: Parkes Joel G. , Hussain Roshan A. , Goldberg David M.
Published: December 2009

The effect of phenobarbital upon the differentiation of two preadipocyte cell lines, 3T3 F442A and 3T3 L-1, was examined by measuring the synthesis and secretion of lipoprotein lipase. Extracellular enzyme was measured by treating intact cells with heparin, and the intracellular enzyme was subsequently assayed in cell homogenates. When confluent cultures of 3T3 F442A cells were treated with insulin, the cells underwent differentiation as indicated by increased activity of lipoprotein lipase within 6 days, followed in turn by increased levels of protein and triglyceride. Addition of phenobarbital with insulin enhanced total lipoprotein lipase, protein, and triglyceride content. The activity of lipoprotein lipase accumulated in the heparin-releasable fraction during differentiation was increased 2- to 3-fold and the intracellular enzyme was enhanced 15- to 20-fold by the addition of phenobarbital. The ability of phenobarbital to modulate differentiation was dependent upon the time of addition. When added early in the postconfluent period, there was a greater increase in lipoprotein lipase activity than when the drug was added at later times. Phenobarbital also stimulated lipoprotein lipase in differentiating 3T3 L-1 cells in the presence of insulin, although lipoprotein lipase activity was moderately enhanced by phenobarbital alone in these cells. These results suggest that phenobarbital may affect the conversion of adipoblasts into preadipocytes and thereby increase the proportion of cells susceptible to the differentiating stimulus.
Secretion of triglyceride lipase from rat hepatocytes in culture: modulation by insulin and phenobarbital
Authors: Parkes Joel G. , Chan Peter , Goldberg David M.
Published: December 2009

Hepatic triglyceride lipase (HTGL) was measured in primary rat hepatocytes maintained for 3 days under three different culture conditions: basal medium, basal medium plus insulin, and basal medium plus insulin and phenobarbital. The activity of HTGL secreted by these cells was measured by treating intact cells with heparin; intracellular enzyme was subsequently measured in cell homogenates. Insulin stimulated intracellular triglyceride lipase activity by 48% and extracellular lipase by 30%. Phenobarbital, an enzyme-inducing drug, caused a further 15% increase in extracellular hepatic triglyceride lipase; whereas, the intracellular activity was reduced. The presence of insulin greatly stimulated the rate of enzyme secretion, and this rate was not notably affected by the presence of phenobarbital. After 3 days in culture, the short term (2–8 h) synthesis and secretion of enzyme from cultures treated with insulin or insulin plus phenobarbital were equally inhibited by cycloheximide. Monensin also inhibited enzyme secretion in both cultures and caused a similar increase in intracellular lipase activities. Insulin did not significantly affect the proportion of intracellular enzyme (17.7% basal vs. 15.8% insulin). On the other hand phenobarbital produced a 20–30% reduction in the proportion of intracellular enzyme (12.5 vs. 17.7% basal or 15.8% insulin). These findings suggest a drug-induced redistribution of triglyceride lipase.
Selective isolation of free or blocked amino-terminal peptides from protein digests by high performance liquid chromatography: application to the crystal protein of Bacillus thuringiensis
Authors: Kaplan H. , Oda G. , Bietlot H. , Carey P. , Pozsgay M. , Fast P.
Published: December 2009

A method for the selective isolation of amino-terminal peptides from enzymatic digests of proteins using high performance liquid chromatography (HPLC) is described. The rationale behind this method is based on the condition that after citraconylation of the free amino groups of the protein, followed by enzymatic digestion and reaction with fluorodinitrobenzene only peptides derived from the amino terminus will not contain a dinitrophenyl (Dnp) group. After removal of citraconyl groups at acid pH, the peptide mixture was subjected to HPLC using an Altex Ultrasphere octadecylsilane column. The strong adsorption of the Dnp group retards the elution of all peptides, except the amino-terminal peptides which do not contain a Dnp group. A shallow gradient employing increasing acetonitrile to 28% over 30 min was used to elute the peptides. For all test proteins, only peptides derived from the amino terminus eluted in the shallow portion of the gradient. As the procedure involves the reversible blocking of any free amino terminus, peptides are isolated with their amino-termini free. However, proteins which are naturally blocked at their amino terminus will yield amino-terminal peptides with the blocking groups intact. The demonstrated sensitivity of the method is in the 1- to 5-nmol range of protein. Application of this procedure to the crystal protein from Bacillus thuringiensis var. kurstaki, which was solubilized at pH 10.5 in 2% β-mercaptoethanol, gave multiple amino-terminal peptides. However, prior treatment of the crystals with 8 M urea at pH 3, followed by solubilization at pH 10.5, gave only one major amino-terminal peptide. These results show that there is probably only one major protein present in the crystals and that the proteolysis results from processes occurring upon solubilization and not by the action of the proteases within the intact crystal.
Production of antibodies against sorghum leaf phosphoenolpyruvate carboxylase monomer and their use in monitoring phosphoenolpyruvate carboxylase levels in sorghum tissues
Authors: Penrose Donna M. , Glick Bernard R.
Published: December 2009

The enzyme phosphoenolpyruvate carboxylase (PEP-C) was purified from leaves of the C4 plant Sorghum bicolor. The purified enzyme had a specific activity of 30 U/mg protein and an apparent molecular mass of 415 000 daltons. Rabbit antibodies produced against purified PEP-C monomer were used to develop an enzyme-linked immunosorbent assay for PEP-C. Titration of rabbit antiserum against both crude and purified PEP-C indicated a 10-fold difference in response, suggesting a 10-fold purification of the enzyme, consistent with the 10-fold purification indicated by densitometric traces of sodium dodecyl sulphate – polyacrylamide gels of crude and purified PEP-C and the 12-fold purification indicated by enzyme assays. Rabbit antibodies were used to monitor PEP-C protein in sorghum tissues during plant development. A rapid increase in PEP-C protein, paralled by an increase in PEP-C activity, was observed in young leaves during the greening stage following germination. PEP-C protein (and activity) were present at moderate and relatively constant levels in shoots. PEP-C protein peaked sharply in roots and attained a moderately high level while PEP-C activity was relatively constant.
The relative degradation of [14C]eicosapentaenoyl and [3H]arachidonoyl species of phosphatidylinositol and phosphatidylcholine in thrombin-stimulated human platelets
Authors: Weaver Bonnie J. , Holub Bruce J.
Published: December 2009

The platelet-rich plasma from volunteers who had consumed a supplement containing eicosapentaenoate (EPA) was incubated with [3H]arachidonic acid (AA) and [14C]EPA so as to provide for the labelling of these fatty acids in the individual platelet phospholipids. Washed dual-labelled platelet suspensions were prepared and incubated with and without thrombin in the presence of BW755C and in the presence and absence of trifluoperazine (TFP) or indomethacin. The platelet lipids were extracted and the individual phospholipids, as well as diacylglycerol and phosphatidic acid, were separated by thin-layer chromatography and the radioactivity in each fraction was determined. The [H]AA/[14C]EPA dpm ratio for the loss of radioactivity from phosphatidylcholine (PC) upon thrombin stimulation, as well as that in the residual PC remaining after stimulation, was similar to that in PC in the resting platelets. This suggests no marked selectivity in the degradation of EPA- versus AA-containing species of PC during platelet activation. The [3H]/[14C] ratios for the increased radioactivity appearing in diacylglycerol (DG) and phosphatidic acid (PA) upon thrombin stimulation were not significantly different from the corresponding ratio in phosphatidylinositol (PI) from resting platelets, suggesting little or no preference for 1-acyl-2-eicosapentaenoyl PI over 1-acyl-2-arachidonoyl PI in the pathway from PI to DG to PA. These results suggest that the relative formation of the 2- and 3-series prostaglandins, including thromboxane (Tx) A2 and A3, in stimulated platelets is not regulated by a preferential loss of one of the corresponding eicosanoid precursors over the other from membrane PC and PI.
Posttranslational modifications of proopiomelanocortin in rat intermediate lobe cells
Authors: Bourbonnais Yves , Fortin Suzanne , Crine Philippe
Published: December 2009

Proopiomelanocortin (POMC), the common precursor to β-endorphin and α-melanocyte-stimulating hormone synthesized in rat intermediate lobe cells, exhibits both charge and size heterogeneity on two-dimensional gels. Pulse-labeling and pulse–chase studies revealed that this heterogeneity is due to co- and post-translational modifications of a single common polypeptide. Short 5-min-pulse incubation with [3H]phenylalanine allowed the preferential labeling of two major forms characterized by an identical isoelectric point (8.2), but slightly different apparent molecular weights (MW = 34 000 and 36 000). These peptides could be labeled with [3H]mannose and the analysis of their tryptic fragments by high-pressure liquid chromatography revealed that they correspond to polypeptides bearing one or two N-linked carbohydrate side chains. Accumulation of more acidic forms was observed during subsequent chase incubations in the absence of phenylalanine. These acidic forms were shown to incorporate sulfate and (or) phosphate groups. Sulfation and phosphorylation occurred on POMC within 5 min after its synthesis and were concomitant with the processing of the N-linked carbohydrates from the high mannose to the complex structure. Finally, partial digestion of the phosphorylated and nonphosphorylated analogs of POMC with either Staphylococcus aureus (V8 strain) protease or chymotrypsin suggests that the presence of a phosphate group may alter POMC sensitivity to exogenously added proteases.
The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles
Authors: Still J. S. , Manery J. F. , Dryden E. E.
Published: December 2009

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 μmol inorganic phosphate/g muscle in 20 min at 20 °C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 °C for 18 h caused ectoenzyme inactivation which plateaued at 35–40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 μg ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by α-methyl D-mannopyranoside. As the temperature increased from 10 to 40 °C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 °C, with a "break point" and a clear change in slope at 35 °C. When treated with ConA the activity increased linearly from 10 to 40 °C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 °C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.
Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Pseudomonas putida
Authors: Alonso Josefa M. , Garrido-Pertierra Amando
Published: December 2009

5-Carboxymethyl-2-hydroxymuconic semialdehyde (CHMSA) dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway was purified from Pseudomonas putida by gel filtration, anion-exchange, and affinity chromatographies. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis analysis suggested an approximate tetrameric molecular weight of 200 000. The purified enzyme showed a pH optimum at 7.8. The temperature–activity relationship for the enzyme from 27 to 45 °C showed broken Arrhenius plots with an inflexion at 36–37 °C. Under standard assay conditions, the enzyme acted preferentially with NAD. It could also catalyze the reduction with NADP (which had a higher Km), at 18% of the rate observed for NAD. The following kinetic parameters were found: Km(NAD) = 20.0 ± 3.6 μM, Km(CHMSA) = 8.5 ± 1.8 μM, and Kd(enzyme–NAD complex) = 7.8 ± 2.0 μM. The product NADH acted as a competitive inhibitor against NAD.
Structure of the O-chain of the lipopolysaccharide of Haemophilus pleuropneumoniae serotype 1
Authors: Altman Eleonora , Brisson Jean-Robert , Perry Malcolm B.
Published: December 2009

By phenol-water extraction an aqueous-phase soluble cellular lipopolysaccharide was isolated from Haemophilus pleuropneumoniae serotype 1. It was shown by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, hydrolysis, methylation, and both one- and two-dimensional 1H and 13C nuclear magnetic resonance studies to be an S-type lipopolysaccharide, which could be cleaved to yield a lipid A and an O-chain polysaccharide identified as a high molecular weight branched polymer of a tetrasaccharide repeating unit having the structure:[Formula: see text]
Multiple forms of ribonuclease II in mouse liver: relative sizes, subcellular distributions, and pH–activity profiles
Authors: Kimball Scot R. , Meyer William L.
Published: December 2009

Multiple forms of ribonuclease II (EC 3.1.27.5) have been resolved from extracts of crude fractions of mouse liver by ion-exchange chromatography on phosphocellulose and gel permeation chromatography. The forms are designated 6S, 6L, 5S, 5L, 4S, 4L, 3S, 3L, 2, and 1 in increasing order of apparent cationic character. The forms fall into two series of apparent molecular weight. The small series increases from molecular weight equal to 9000 for form 1 to 14 000 for form 6S. The large series increases from molecular weight equal to 22 000 for form 2 to 44 000 for form 6L. All forms have pH–activity profiles with maxima near pH 7. Activity falls to no less than 30% of this maximum at pHs 5 and 8.5. Relative to the other forms, form 1 has a higher ratio of activity in the alkaline compared with acid pH range. Form 1 is found in the cytosolic, "light" particle, and "heavy" particle fractions. The other forms are largely restricted to the heavy particle fraction. In this fraction the proportion of total activity attributable to each form generally decreases in order from form 1 down to form 6. The results are accommodated by models in which one or more gene products give rise to multiple forms of ribonuclease II by processes involving dimerization and glycosylation.
Interaction of hepatic asialoglycoprotein receptor with dimyristoyl phosphatidylcholine vesicles
Authors: Krebs Anita , Fannon Allison , Racey Thomas J. , Rochon Paul , Depew William T. , Singer Michael A.
Published: December 2009

The hepatocyte membrane asialoglycoprotein receptor (ASGP-R) was extracted from rabbit liver, purified, and then incubated with preformed vesicles of dimyristoyl phosphatidylcholine. The association of protein with lipid was dependent on vesicle size and the best results were achieved with small vesicles of about 20 nm diameter. The ligand binding capacity of ASGP-R–vesicle complexes was also measured and found to be approximately sevenfold greater than free receptor in aqueous buffer and twofold greater than receptor solubilized in Triton X-100. Most likely, the reconstitution procedure used in these experiments does not result in transmembrane insertion of the receptor. ASGP-R probably resides on the surface of the vesicle, held there primarily by weak hydrophobic forces.
ADP-ribosylation of histones and acid-soluble, high mobility group like chromosomal proteins from Physarum polycephalum
Authors: Poirier G. G. , Coté Serge , Pallotta Dominic
Published: December 2009

Nuclei from the cellular slime mould Physarum polycephalum were incubated with [32P]NAD. The chromosomal basic proteins were acid extracted and separated by two-dimensional polyacrylamide gel electrophoresis. After autoradiography the poly(ADP-ribosylated) proteins were identified. Histone H1 was the major acceptor. Histones H2B and H2A were significantly modified, although to a lesser extent than H1. In addition, the acid-soluble, high mobility group-like proteins AS-2 and AS-3 and the protein A-24 showed some modification. Histones H3 and H4 were not modified. The pattern of ADP-ribosylation did not change with NAD concentrations between 1 and 100 μM NAD.
Actions of dietary orotic acid on liver synthesis of phosphatidylcholine and phosphatidylethanolamine in rats
Authors: Haines D. S. Milton , Tokmakjian Sonya D.
Published: December 2009

The in vivo rates of the reactions of the cytidine pathways of liver phosphatidylcholine and phosphatidylethanolamine synthesis were measured in rats after 1 day of feeding on a semisynthetic diet containing 1% orotic acid. The calculations were made from the specific and total radioactivity versus time curves of the precursors and products following intraportal injection of [1,2-14C]choline, [2-14C]ethanolamine, and [2-3H]glycerol. The liver CTP level was increased twofold and the rates of CDP-choline and phosphatidylcholine synthesis were stimulated 4.5-fold in the rats fed orotic acid. The rate of CDP-ethanolamine synthesis was increased but could not be accurately quantified because of its extreme rapidity. No change occurred in the rate of the ethanolaminephosphotransferase reaction and the overall rate of phosphatidylethanolamine synthesis was unchanged by orotic acid feeding. The catalytic activities of the enzymes of the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis were not affected by feeding orotic acid for 1 day. Similar findings were obtained 3 h following intragastric administration of 100 mg of orotic acid. The results suggest the possibility that changes in the levels of liver CTP may play a role in regulation of the cytidine pathway of liver phosphatidylcholine synthesis but not of phosphatidylethanolamine synthesis, because the latter pathway appears to be tightly controlled at the ethanolaminephosphotransferase step.
Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles
Authors: Dupuis Gilles , Letellier Marc
Published: December 2009

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65–70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towads the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 ± 0.05 μg of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120 000 × g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 μg of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.
Effects of prolonged ethanol feeding on methionine metabolism in rat liver
Authors: Barak Anthony J. , Beckenhauer Harriet C. , Tuma Dean J. , Badakhsh Shahrokh
Published: December 2009

Pairs of rats were fed control and alcohol liquid diets for periods of 1, 2, 3, and 4 months. The animals were then killed, and their livers analyzed for betaine, S-adenosylmethionine (SAM), methionine synthetase activity, and betaine–homocysteine methyltransferase (BHMT) activity. The results of this time-course study showed that chronic ethanol feeding inhibited the activity of the methionine synthetase throughout the study, but increased the activity of BHMT and lowered betaine levels. These data suggest that the rat, because of its ability to produce betaine from choline, has the capacity to compensate for the ethanol-induced impairment of methionine synthetase and maintain vital tissue levels of SAM over prolonged periods of time via an adaptive increase in BHMT activity.
On the existence of an amphotericin B – sterol complex in lipid vesicles and in propanol–water systems
Authors: Gruda Ilona , Bolard Jacques
Published: December 2009

The amphotericin B (AmB) – ergosterol complex, formed by interaction of the antibiotic with ergosterol-containing phospholipid vesicles, is associated with the lipid bilayer. It has been shown by circular dichroism studies that the AmB–ergosterol complex formed in water–propanol binary mixtures has a similar structure to that observed in phospholipid vesicles. A positive cooperativity is found for the interaction of AmB with ergosterol. The similar AmB–cholesterol complex is much less stable and rearranges rapidly to a different conformation.
Immunochemical detection and quantitation of brown adipose tissue uncoupling protein
Authors: Henningfield Mary F. , Swick Robert W.
Published: December 2009

A polyclonal antisera against rat brown adipose tissue mitochondrial uncoupling protein was used to examine mitochondrial samples from liver and white and brown adipose tissue from several mammalian species. A sodium dodecyl sulfate – polyacrylamide gel electrophoretic separation of proteins combined with an immunochemical method allowed for visualization of antigen–antibody complexes on nitrocellulose blots. Hamster, cavy, monkey, and mouse brown adipose tissue mitochondrial samples cross-reacted with the antisera. Mitochondria prepared from white fat obtained from young swine and sheep contained two closely migrating, antigenically active proteins. Hepatic mitochondria samples did not contain antigenically active protein. Reflectance densitometry was used for quantitation of the uncoupling protein in various mitochondrial samples. In rats fed diets low in protein, there appears to be a dissociation between the concentration of uncoupling protein and the number of nucleotide binding sites as given by the [3H]GDP binding assay. These results are indicative of a physiological activation of the uncoupling protein.
Effect of phalloidin and viroisin on Acanthamoeba castellanii after permeabilization of the cell
Authors: Joos Luc , Gicquaud Claude
Published: December 2009

We have developed a new technique for the permeabilization of the membrane of Acanthamoeba castellanii. This technique involves the use of digitonin which alters neither the morphology nor the motility of the cell, but favours the penetration of phalloidin and viroisin. Treatment of permeabilized cells with phalloidin or viroisin induces, in the cortex of the cell, an intensive proliferation of filaments which have been identified as actin. This cortical filamentous layer detaches from the membrane and slowly contracts, acting as a fine mesh sieve which concentrates the organelles in the middle of the cell, causing therefore the formation of a central granuloplasm and a cortical hyaloplasm. During this process, cell motility is irreversibly lost. The results indicate that extensive proliferation and reorganization of actin filaments cannot support cell motility and they are discussed in terms of a general understanding of amoeboid movement.
Cartilage fucoproteins with sites for cross-linking by transglutaminase
Authors: Bowness J. Michael
Published: December 2009

Slices of various types of cartilage were incubated with either L-[6-3H]fucose or [1,4-3H(N)]putrescine. Homogenization of the slices and fractionation of the homogenates showed for both labels that an insoluble collagenase-resistant fraction had the highest specific activity (dpm/mg dry weight). Examination of an exhaustive proteolytic digest of this insoluble fraction by ion-exchange high performance liquid chromatography showed the presence of γ-glutamyl[3H]putrescine. Chromatography of solubilized [3H]fucoprotein fractions showed the presence of several low molecular weight peaks, as well as high molecular weight material. Incubation of [3H]fucoprotein extracts with transglutaminase increased the high molecular weight peaks and decreased the low molecular weight ones. Incubation of the cartilage slices with L-[3H]fucose plus 0.5 mM dansylcadaverine, an inhibitor of transglutaminase, caused a decrease in the insoluble and high molecular weight fraction relative to the low molecular weight peaks. It is hypothesized that this is due to inhibition of cross-link formation between fucoprotein components of the cartilage which are transglutaminase substrates. One major low molecular weight peak, which labels with both fucose and putrescine, corresponds in size with the 15 000 subunit of collagen III aminopropeptide, which is known to be a substrate for transglutaminase.
Amino acid sequence differences in the alpha chains of pea seed isolectins: C-terminal processing
Authors: Rini James Michael , Hofmann Theo , Carver Jeremy P.
Published: December 2009

The complete amino acid sequence of the alpha chains of both isolectins found in pea seeds has been determined using automated Edman degradation. We show that the alpha chains of these two proteins differ only at their C-termini: isolectin B is two amino acids longer than isolectin A. Furthermore, the alpha chains of both isolectins are shorter than would be predicted from the nucleotide sequence of a cDNA clone for pea lectin. We suggest, therefore, that these proteins arise from differential C-terminal processing. Amino acid composition data and C-terminal analysis show that the beta chains have also been processed at their C-termini, but in this case identical chains for both isolectins are produced.
Preparation and analysis of mass amounts of germin: demonstration that the protein which signals the onset of growth in germinating wheat is a glycoprotein
Authors: Lane Byron , Grzelczak Zbyszko , Kennedy Theresa , Hew Choy , Joshi Shashikant
Published: December 2009

(1) A rapid method (2–3 weeks) is described for preparing milligram amounts of germin, in high yield, from kilogram quantities of germinated wheat embryos. An ammonium-sulphate fraction of proteins in the low-speed supernatant from an embryo homogenate is suspended in buffer for pepsin digestion, and the resulting digest is filtered through Ultrogel AcA34 to obtain germin, virtually free of contamination (<1%) by other proteins.(2) An acid hydrolysate of germin contains a full complement of the 20 different amino acids. The proportions of half-cystine, tyrosine, and tryptophan are conspicuously low (<1.5%), the proportions of aspartic acid and glycine (each ca. 11%) as well as proline (ca. 8%) are relatively high, and the cumulative proportion of hydrophobic amino acids (valine, leucine, isoleucine, phenylalanine) is also high (ca. 25%). The N-terminal sequence of germin, as determined by Edman degradation, is enriched with respect to aspartic acid, which occupies 5 of the first 15 positions.(3) There are two forms of germin, one (G) being the dominant species in the soluble fraction of homogenates of germinated embryos and the other (G′) being the dominant species in a fraction obtained by rinsing growing embryos. Consistent differences between the amino-acid compositions of G and G′ were not observed. Similarly, wherever identification was possible, the N-terminal amino-acid sequences (residues 1–22) of G and G′ were not different.(4) The polymeric and monomeric forms of germin (G or G′) give a positive Schiff reaction, characteristic of glycoproteins, when electrophoretically separated in sodium dodecyl sulphate (SDS) – poly aerylamide. Treatment with trifluoromethanesulfonic acid affirmed the glycoprotein character of germin, as did labeling, in vivo, from exogeneously supplied tritiated sugars.(5) The G and G′ forms of germin were among the most heavily labeled proteins seen in SDS–polyacrylamide separations if wheat-embryo proteins were labeled, in vivo, from exogenous supplies of tritiated mannose, glucosamine, or fucose. Differences between the patterns of glucosamine labeling and fucose labeling of G and G′ suggest the possibility that the two forms differ with respect to their carbohydrate components.
Quaternary structure of intestinal maltase–glucoamylase in pancreatectomized rats
Authors: Lee L. , Forstner G.
Published: December 2009

Detergent-solubilized intestinal maltase–glucoamylase was isolated 1 week postpancreatectomy (dMpanc) and purified in the presence of detergent and protease inhibitors. Upon sodium dodecyl sulfate – polyacrylamide gel electrophoresis under nondissociating conditions, the major band had a molecular weight of 280 000, slightly smaller than similar bands from detergent (dM) and papain (pM) solubilized maltase from nonpancreatectomized rats. Upon octyl-Sepharose CL-4B chromatography, 57% of the enzyme was eluted by aqueous buffer, unlike pM which was almost completely eluted or dM, 95% of which bound to the column. All fractions of dMpanc from octyl-Sepharose 4B were reduced, by boiling ± β-mercaptoethanol, to monomelic subunits, indicating that processing by pancreatic enzymes at the level of the brush border is not a requirement for the appearance of subunits in the rat. As well, under these dissociating conditions, the 145 000 subunit previously identified with the apolar terminus was present in all fractions of dMpanc, including the aqueous fraction, whereas pM contained only the 130 000 subunit. The presence of dMpanc in the aqueous fraction cannot be explained, therefore, by proteolytic cleavage of an apolar anchor segment from the 145 000 subunit. Pancreatic enzymes may affect the enzyme in a minor fashion, however, since aqueous solubility was enhanced and the apparent molecular weight was reduced by pancreatectomy, suggesting a more compact conformation with shielding of apolar segments.
The role of the female Syrian hamster protein on the interaction between serum lipoproteins and heparin
Authors: Saxena Uday , Nagpurkar Arun , Coe John E. , Mookerjea Sailen
Published: December 2009

We have previously shown the effect of phosphorylcholine-binding proteins from rat (PCBP) and rabbit (CRP) on the precipitation of serum lipoproteins by heparin in presence of Ca2+. The present paper describes the effect of a phosphorylcholine-binding protein from the female Syrian hamster (FP) on the lipoprotein precipitation reaction. The precipitation of lipoproteins by heparin was lower in assays using female hamster serum in which FP is a prominent protein, compared with assays with male serum in which FP is present in very low concentration. Depletion of FP from female serum resulted in increased lipoprotein precipitation. The addition of purified FP to assays using human very low density lipoprotein (VLDL) inhibited the precipitation reaction. The precipitation of lipoproteins was also examined using serum from male hamsters treated with diethylstilbestrol and female hamsters treated with testosterone, treatments known to modulate the levels of FP. Results indicate an inverse relationship between serum FP levels from normal and hormone-treated hamsters and the precipitation of lipoproteins from their serum. The partially desialylated FP when added to precipitation assays using human VLDL resulted in reduced inhibition of VLDL precipitation.
Studies of the process of renaturation and assembly of Escherichia coli succinyl-CoA synthetase from its α and β subunits
Authors: Wolodko William T. , Bridger William A.
Published: December 2009

Succinyl-CoA synthetase catalyzes the substrate-level phosphorylation step of the tricarboxylic acid cycle. The enzyme, as isolated from Escherichia coli, has an α2β2 subunit structure. It is known that substrate-binding sites are distributed between both subunit types and that the active enzyme is the nondissociating tetramer. This paper describes a study of the process of assembly of the enzyme from its denatured constituent subunits. Starting with equimolar mixtures of the subunits that are prepared in denaturing conditions (6 M urea, 5% acetic acid), rapid renaturation to produce virtually a fully active enzyme occurs after neutralization and dilution under suitable conditions. This process occurs most efficiently in the presence of either ATP or Pi, indicating that occupation of the phosphoryl-binding site on the refolding α subunit facilitates productive intrasubunit interactions. We have determined conditions of protein concentration, pH, temperature, final urea concentration, and buffer composition that optimize both the rate and extent of production of active enzyme. The final refolded product is indistinguishable from the native species with respect to its specific catalytic activity, size, and other physical properties. To probe further the mechanism and route of renaturation, we have shown that the rate of appearance of activity has first-order dependence on each of the two subunits. The step that determines the rate of assembly is thus bimolecular, such as the association of structural monomers to form a dimeric transient species. The highly specific mutual interactions between the refolding transient species of subunits must be essential for the correct assembly of this enzyme from the two gene products in vivo.
Size and composition of lymph chylomicrons following feeding corn oil or its fatty acid methyl esters
Authors: Yang Lu-Ying , Kuksis Arnis
Published: December 2009

Male rats with thoracic duct cannulae were intubated with com oil or fatty acid methyl esters and the lymph was collected over the next 2–72 h. The apoprotein (apo) composition of the chylomicrons, isolated by conventional ultracentrifugation, was determined by sodium dodecyl sulfate – polyacrylamide – glycerol gel electrophoresis and isoelectric focusing. The lipid content and composition was assessed by gas–liquid chromatography. The particle size was obtained by calculation and confirmed by electron microscopy. The study demonstrates that both the monoacylglycerol (corn oil feeding) and the phosphatidic acid (methyl ester feeding) pathways of triacylglycerol biosynthesis yield chylomicrons with closely similar apoprotein profiles representing apo B-48, apo A-IV, apo E, apo A-I, and the apo C components. A protein band corresponding to apo B-100 was occasionally observed as a minor component of the chylomicrons from both groups of animals. The chylomicrons from com oil feeding had about two times larger diameters than those from methyl ester feeding. There were no significant differences in the composition of the apoproteins, although the smaller particles had two times higher apoprotein/triacylglycerol ratios. It was calculated that the amount of apo B per lipid particle for the ester fed rats ranged from one to eight molecules and was closely correlated with the particle size. The corn oil fed rats yielded about three molecules apo B per lipid particle regardless of the particle size. It is concluded that the pathway of intestinal triacylglycerol biosynthesis has a significant effect on the apoprotein mass and to a lesser extent on the apoprotein and lipid composition of the chylomicrons. The phosphatidic acid pathway produces smaller particles and transfers to the bloodstream twice as much apoprotein per gram of fat than the monoacylglycerol pathway, which yields the larger particles. Possible variations in the site and rate of biosynthesis of the triacylglycerols could not be entirely excluded as contributing factors.
A neutral sphingomyelinase in spermatozoal plasma membranes
Authors: Hinkovska Vania Tz. , Petkova Diana H. , Koumanov Kamen S.
Published: December 2009

A highly active neutral sphingomyelinase was observed for the first time in ram spermatozoal plasma membranes. The optimal conditions for the enzyme activity are pH 7.4, 40 mM MgCl2, 40 min of incubation, and 267 nmol sphingomyelin. Ca2+ and cholesterol were found to inhibit sphingomyelinase activity.
Effects of ribosome dissociation on the structure of the ribosome-associated 5.8S RNA
Authors: Lo Amy C. , Liu Wangyi , Culham Doreen E. , Nazar Ross N.
Published: December 2009

Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA – protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.
Relief of temperature sensitivity in a concanavalin A resistant Chinese hamster ovary cell line auxotrophic for cholesterol
Authors: Hurta Robert A. R. , Burton David N.
Published: December 2009

The concanavalin A resistant, glycosylation-deficient, Chinese hamster ovary cell variant CR-7 is auxotrophic for cholesterol owing to an inability to adequately convert lanosterol to cholesterol. It is also temperature sensitive for growth, being unable to proliferate at 39 °C. Temperature sensitivity was relieved by addition of mevalonolactone, dolichol, or dolichyl-P to the growth medium, provided that cholesterol was also present in amounts sufficient to overcome cholesterol auxotrophy at 34 °C. Other metabolites of mevalonolactone (squalene, ubiquinone, lanosterol, and isopentenyladenine) were inactive in this regard. Measurement of dolichol levels in CR-7 and wild-type cells at 34 °C and after exposure to 39 °C showed that dolichol increased at 39 °C to an approximately equal extent in both cell types. Dolichol, dolichyl-P, ubiquinone, and isopentenyladenine had no effect on the sensitivity of either wild-type or CR-7 cells to the cytotoxic effects of concanavalin A. Mevalonolactone or lanosterol markedly increased the resistance of CR-7 to the lectin, but had no effect on wild-type cells. This raises the possibility that the presence of unusually large amounts of lanosterol, coupled with low amounts of cholesterol, in the membranes of CR-7 may be related to its concanavalin A resistance and other characteristic phenotypic abnormalities.
The effect of the hypoferremic response on iron acquisition by and growth of murine lymphoma cells
Authors: Caldwell Margaret , Archibald Frederick
Published: December 2009

The hypoferremic response in C57 black mice during EL4 lymphoma cell growth was followed to determine if a lowered transferrin iron saturation inhibited iron acquisition and growth by these tumor cells. Although the lymphoma induced a strong hypoferremic response, it occurred late in the disease when the tumor burden was high and did not appear to effectively limit tumor cell growth, as all mice subsequently died. The induction of a hypoferremic response early in the disease with subcutaneous injections of turpentine also did not inhibit tumor cell growth. Parallel in vitro cell growth experiments revealed iron to be readily available to the lymphoma cells, regardless of whether the transferrin pool was 100, 50, or 10% saturated with iron. The dynamics of 59Fe uptake by the lymphoma cells from 50% saturated ferritransferrin were followed and saturation of iron uptake was found to occur at 150 nM iron and transferrin, far below physiological levels. Lowering transferrin saturation from 50 to 10% did not result in a proportional decrease in 59Fe uptake. Thus the ability of the EL4 tumor cells to obtain iron appeared to exceed the ability of a vigorous murine hypoferremic response to sequester it, suggesting that the hypoferremic response does not effectively limit EL4 ascites tumor cell growth.
Submitochondrial localization of kidney 25-hydroxycholecalciferol 1α-hydroxylase in vitamin D repleted weanling guinea pigs
Authors: Yan Xue , Charette Guy , Delvin Edgard E.
Published: December 2009

We have studied the submitochondrial localization of guinea-pig kidney 25-hydroxycholecalciferol 1α-hydroxylase. Treatment of the mitochondrial-enriched fraction with recrystallized digitonin produced mitoplasts bordered by a single membrane and with intact matrix. They contained nearly 90% of the 25-hydroxycholecalciferol 1α-hydroxylase activity and nearly 100% of the cytochrome-c: oxygen oxidoreductase. Amine: oxygen oxidoreductase activity remained mainly in the outer membrane fraction. These data show that 25-hydroxycholecalciferol 1α-hydroxylase has a distribution similar to that of steroid hydroxylases.
Enamel protein and collagen production by cells subcultured from porcine tooth bud explants
Authors: Limeback Hardy
Published: December 2009

Fibroblast (F) and epithelial (E) cells were obtained as primary outgrowths from explants of fetal porcine maxillary molars and subcultured up to four passages in monolayers enriched with either cell type. Histology of a tooth bud after 1 day in culture showed intact odontogenic E cell layers which were the probable source of the E cell outgrowths. After 2 months in culture, the fourth passage E cells demonstrated morphological differentiation by an alteration in cell packing and the formation of domes and nodules, when E and F cells were cocultured. Occasionally the nodules grew to considerable size, indicating the potential of these cells to aggregate and reorganize into odontogenic tissues even on culture dishes. The cells were characterized in monolayer culture by immunocytochemical staining. Laminin and type IV collagen staining was distributed diffusely throughout the culture, whereas type I collagen and osteonectin staining was predominantly localized in the F cells. Radio-labelled proteins from both E and F cell media produced similar collagen patterns (95% type I, 4% type V, 1% other), except that the F cells appeared to produce active collagenase. In addition, the E cells produced two radiolabelled proteins (relative masses of 50 000 and 53 000) that reacted with an affinity-purified antibody directed against porcine amelogenin. These experiments show that cells subcultured from tooth buds and grown in monolayer cultures can be used to study tooth organogenesis in vitro, as well as enamel protein biosynthesis.