Proportionate representation of rDNA and Balbiani ring DNA in polytene chromosomes of Chironomus tentans

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ -free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first-order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta-glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01-0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.     

Human DNA topoisomerase IIalpha-dependent DNA cleavage and yeast cell killing by anthracycline analogues

Anthracyclines are among the most clinically useful topoisomerase II poisons. A complete understanding of their molecular mechanism is thus fundamental for a rational design of novel agents. We evaluated four anthracycline analogues with respect to human topoisomerase IIalpha-dependent DNA cleaving activity, efficiency in killing yeast cells, and uptake and retention in yeast and compared the yeast system to tumor cell line models. The yeast JN394top2-4 strain was used because it has a topoisomerase II ts gene mutation: enzyme activity is much less at 30 degrees C than at 25 degrees C and is completely lost at 35 degrees C. Untransformed JN394top2-4 cells were 33-fold more sensitive to idarubicin at 25 degrees C than at 30 degrees C, showing that topoisomerase II is the primary drug target. Overexpression of human topoisomerase IIalpha was toxic to yeast cells when the yeast enzyme was inactivated. Drug-dependent killing of yeast cells expressing low levels of the human alpha isoenzyme at 35 degrees C showed that the analogues spanned a 3-log range of cytotoxic potency in yeast, as they did in tumor cells. However, the compounds were much less active against the yeast strain than mammalian tumor cell lines. Drug uptake was determined and found to be altered in yeast with respect to tumor cells. Although DNA cleavage stimulated by anthracyclines roughly correlated with cytotoxicity, the cleavage level:cytotoxicity ratios were different for the studied drugs. Thus, the results suggest that other drug-dependent molecular factors contribute to drug activity in addition to the cellular content of topoisomerase IIalpha and drug uptake.     

Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase

Plasmin triggers a strong metabolic activation in human platelets, leading to shape change and granule exocytosis. However, its capacity to induce cell aggregation remains discussed and, when observed, this aggregation is preceded by a remarkable lag phase. We have thus investigated the effect of plasmin on the adhesive proteins which can be secreted by isolated platelets and mediate cell-to-cell interactions, but are also substrates for the enzyme. Immunoblot analysis of fibrinogen (Fg), thrombospondin-1 (TSP-1), fibronectin (Fn) and von Willebrand factor (vWf) was performed on extracts of platelets exposed under stirring to increasing concentrations of plasmin for up to 10 min at 37 degrees C. Under conditions leading to formation of large aggregates, Fg, Fn and TSP-1 are extensively degraded concomitantly with their secretion, and readily lost from the surface of aggregated cells. Part of the monomers in the platelet vWf are cleaved during secretion into two main fragments with Mr approximately 180,000 and approximately 145,000. However, multimer distribution analysis shows only a slight decrease in the very high molecular weight multimers, and most of the fragmented as well as intact vWf remains associated with the platelet surface when aggregation is maximal. That indeed vWf largely supports plasmin-induced aggregation is suggested by the observation that platelets from a patient with type 3 von Willebrand's disease, who totally lacks vWf, show little aggregation in response to the enzyme. Finally, plasmin-induced aggregation can be totally inhibited by antagonists of the alpha(IIb)beta3 integrin. The present study thus indicates a major role for secreted vWf in platelet aggregation induced by plasmin, through its likely interaction with the multifunctional receptor alpha(IIb)beta3.     

The influence of thrombopoietin (TpO) on human erythropoiesis. In vitro studies--clinical implications

beta-Glucuronidase of human or bacterial origin may deconjugate bilirubin diglucuronide, causing pigment gallstones. Intrinsic interference by biliary compounds must be minimized for accurate assay of beta-glucuronidase. We report a modified ion-pair extraction of interfering substances by tetrahexylammonium chloride (THAC) in ethyl acetate in the presence of albumin, and a microtitre plate assay for biliary beta-glucuronidase activity in bile with the substrate p-nitrophenol-glucuronide. Adding albumin improved the recovery of beta-glucuronidase activity to 99.8% (CV 1.9%), and 92.2% of the bilirubin in bile samples was extracted in one step. Competitive inhibition was overcome by increasing the substrate concentration. In endoscopically obtained common duct bile from 44 patients, five different beta-glucuronidase activity peaks were identified, at pH 3.9, 4.8, 5.3, 5.8 and 7.2. The pH profiles were classified into one bacterial pattern and five patterns for presumed human beta-glucuronidase. Of the latter patterns, four displayed dual activity peaks. In a second sample, obtained at follow up in four patients, their original pH profile was maintained. In conclusion, using the modified purification and assay system, we found functionally diverse subcategories of human beta-glucuronidase with respect to activity at variable pH. Our results indicate that several pH optima have to be taken into consideration in order to clarify the role of human biliary beta-glucuronidase in the pathogenesis of pigment gallstones. Bacterial beta-glucuronidase activity was associated with duodenal diverticula (p < 0.05) and common duct stones (p < 0.05).     

Encapsulation of various recombinant mammalian cell types in different alginate microcapsules

Microencapsulation of recombinant "universal" cells with immunoprotective membranes is an alternate approach to somatic gene therapy. Therapeutic gene products secreted by these cells can be delivered to different patients without immunosuppression or genetic modification of the host's cells. The encapsulation of different mammalian cell types (epithelial cells, fibroblasts, and myoblasts) is compared among three alginate-based microcapsules: (1) calcium-linked alginate microcapsules with a solubilized core and a poly-L-lysine-alginate-laminated surface; (2) barium-linked alginate beads with a gelled core; and (3) a hybrid formulation of barium-linked alginate beads with a poly-L-lysine-alginate-laminated surface. The mechanical stability of the different microcapsule types, as measured with a cone-and-plate shearing apparatus, was superior in the two barium-linked alginate beads. All cell types maintained high viability (65-90%) in culture after encapsulation. The recombinant gene products secreted by these cells (human growth hormone MW = 22,000, human factor IX MW = 57,000, and murine beta-glucuronidase MW = 300,000) were able to traverse the three microcapsule types at similar rates. Cell numbers within the microcapsules increased twofold to > 20-fold over 4 weeks, depending on the cell type. Epithelial and myoblast cell numbers were not affected by microcapsule formulation; however, fibroblasts proliferated the most in the calcium-linked alginate spheres. These results show that for culturing fibroblasts in a mechanically stable environment the classical calcium-linked microcapsules are adequate. However, where mechanical stability is a more critical requirement, the solid barium-linked gelled beads are more appropriate choices.     

Development of animal models for adeno-associated virus site-specific integration

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.     

Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency

Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders. Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playing a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequence. Using a polyclonal antiserum raised against alkyl-dihydroxyacetonephosphate synthase, levels of this enzyme were examined in fibroblast cell lines from patients affected by peroxisomal disorders. Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rhizomelic chondrodysplasia punctata patients, indicating that the enzyme is not stable in the cytoplasm as a result of defective import into peroxisomes. In a neonatal adrenoleukodystrophy patient with an isolated import deficiency of proteins carrying the peroxisomal targeting signal type 1, the precursor form of alkyl-dihydroxyacetonephosphate synthase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization. A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphate (DHAP) synthase activity had normal levels of this protein. Analysis at the cDNA level revealed a missense mutation leading to a R419H substitution in the enzyme of this patient. Expression of a recombinant protein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, providing further proof that this substitution is responsible for the inactivity of the enzyme and the phenotype. In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be inactivated by the arginine-modifying agent phenylglyoxal. The enzyme is efficiently protected against this inactivation when the substrate palmitoyl-DHAP is present at a saturating concentration. The gene encoding human alkyl-dihydroxyacetonephosphate synthase was mapped on chromosome 2q31.     

A possible mechanism of eighteen patient deaths caused by interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs

A toxicokinetic study was performed using rats to investigate the possible mechanism of 18 acute deaths in Japanese patients with cancer and herpes zoster by interactions of the new oral antiviral drug, sorivudine (SRV), with one of the oral 5-fluorouracil (5-FU) prodrugs within 40 days after approval of the use of SRV. Tegafur, an anticancer 5-FU prodrug suggested to be used by most of the patients who died, and SRV were orally administered to rats simultaneously once daily. All of these rats died within 10 days, whereas rats given SRV or tegafur alone under the same dosage conditions showed no appreciable change over 20 days compared with controls. In the rats given both drugs, bone marrow and intestinal membrane mucosa were greatly damaged at an early stage of the coadministration, and before death, the animals showed marked decreases in white blood cell and platelet counts, diarrhea with bloody flux, and severe anorexia, as was also manifested by the patients who subsequently died. In the rats given both drugs for 6 days, extremely enhanced 5-FU levels were observed from the first day of administration in plasma and in all tissues examined, including bone marrow and intestines. The extreme enhancement of the tissue 5-FU levels was attributable to the facile inactivation by (E)-5-(2-bromovinyl)uracil (BVU) of hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme regulating the systemic 5-FU level in the rat and human. BVU, a major metabolite formed from SRV by gut flora, was found at considerable levels in the liver of rats orally administered SRV alone or SRV and tegafur, and there was a marked decrease in hepatic DPD activity. In the presence of NADPH, DPD purified from rat liver cytosol was rapidly and irreversibly inactivated by [14C]BVU as a suicide inhibitor with concomitant incorporation of the radioactivity into the enzyme protein, although SRV showed no inhibitory effect on DPD under the same conditions. Human liver DPD was recently demonstrated by us to be inactivated with BVU in a manner very similar to rat DPD.     

The trouble with cognitive subtraction

The mechanism(s) by which zinc is transported into cells has not been identified. Since zinc uptake is inhibited by reducing the temperature, zinc uptake may depend on the movement of plasma membrane micoenvironments, such as endocytosis or potocytosis. We investigated the potential role of potocytosis in cellular zinc uptake by incubating normal and acrodermatitis enteropathica fibroblasts with nystatin, a sterol-binding drug previously shown to inhibit potocytosis. Zinc uptake was determined during initial rates of uptake (10 min) following incubation of the fibroblasts in 50 micrograms nystatin/mL or 0.1% dimethylsulfoxide for 10 min at 37 degrees C. The cells were then incubated with 1 to 30 microM 65zinc. Michaelis-Menten kinetics were observed for zinc uptake. Nystatin inhibited zinc uptake in both the normal and AE fibroblasts. Reduced cellular uptake of zinc was associated with its internalization, not its external binding. In normal fibroblasts, nystatin significantly reduced the K(m) 56% and the Vmax 69%. In the AE fibroblasts, nystatin treatment significantly reduced the Vmax 59%, but did not significantly affect the K(m). The AE mutation alone affected the Vmax for cellular zinc uptake. The control AE fibroblasts exhibited a 40% reduction in Vmax compared to control normal fibroblasts. We conclude that nystatin exerts its effect on zinc uptake by reducing the velocity at which zinc traverses the cell membrane, possibly through potocytosis. Furthermore, the AE mutation also affects zinc transport by reducing zinc transport.     

Thermostable farnesyl diphosphate synthase of Bacillus stearothermophilus: molecular cloning, sequence determination, overproduction, and purification

The structural gene for thermostable farnesyl diphosphate synthase from Bacillus stearothermophilus was cloned, sequenced, and overexpressed in Escherichia coli cells. A 1,260-nucleotide sequence of the cloned fragment was determined. This sequence specifies an open reading frame of 891 nucleotides for farnesyl diphosphate synthase. The deduced amino acid sequence shows a 42% similarity with that of E. coli FPP synthase [Fujisaki et al. (1990) J. Biochem. 108, 995-1000]. Comparison with prenyltransferases from a wide range of organisms, from bacteria to human, revealed the presence of seven highly conserved regions. In contrast to thermolabile prenyltransferases, which have four to six cysteine residues, the thermostable farnesyl diphosphate synthase carries only two cysteine residues. This enzyme is also unique in that some of the amino acids that are fully conserved in equivalents from other sources are replaced by functionally different amino acids. Construction of an overproducing strain provided a sufficient supply of this enzyme and it was purified to homogeneity. The purified recombinant enzyme is immunochemically identical with the native B. stearothermophilus enzyme, and it is not inactivated even after treatment at 65 degrees C for 70 min.     

Salt-tolerant and thermostable alkaline protease from Bacillus subtilis NCIM no. 64

The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11. The partially purified enzyme was stable at 60 degrees C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.     

Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic activity

Mutations in the acid beta-glucuronidase gene lead to systemic accumulation of undegraded glycosaminoglycans in lysosomes and ultimately to clinical manifestations of mucopolysaccharidosis VII (Sly disease). Gene transfer by retrovirus vectors into murine mucopolysaccharidosis VII hematopoietic stem cells or fibroblasts ameliorates glycosaminoglycan accumulation in some affected tissues. The efficacy of gene therapy for mucopolysaccharidosis VII depends on the levels of beta-glucuronidase secreted by gene-corrected cells; therefore, enrichment of transduced cells expressing high levels of enzyme prior to transplantation is desirable. We describe the development of a fluorescence-activated cell sorter-based assay for the quantitative analysis of beta-glucuronidase activity in viable cells. Murine mucopolysaccharidosis VII cells transduced with a beta-glucuronidase retroviral vector can be isolated by cell sorting on the basis of beta-glucuronidase activity and cultured for further use. In vitro analysis revealed that sorted cells have elevated levels of beta-glucuronidase activity and secrete higher levels of cross-correcting enzyme than the population from which they were sorted. Transduced fibroblasts stably expressing beta-glucuronidase after subcutaneous passage in the mucopolysaccharidosis VII mouse can be isolated by cell sorting and expanded ex vivo. A relatively high percentage of these cells maintain stable expression after secondary transplantation, yielding significantly higher levels of enzymatic activity than that generated in the primary transplant.     

A novel implantable electromechanical ventricular assist device. First acute animal testing

A method for the simultaneous determination of eight kinds of conjunct bile acids in human bile was developed by HPLC. They were separated on a YWG-C18 (3 microns) column at 30 degrees C, with methanol/water (65/35, V/V, pH3.0) as mobile phase, and detection wavelength at UV 210 nm. The linear ranges were 50-1,000 microns.ml-1, the recoveries were 91.2%-108.6%. The biles of 30 cases with cholelithiasis cholecystolithiasis and 20 cases without gallstone were detected by HPLC. The results showed that the constitution of bile acids was different between patients with cholelithiasis cholecystolithiasis and patients without gallstone.     

Expression of long-patch and short-patch DNA mismatch repair proteins in the embryonic and adult mammalian brain

Expression of the DNA mismatch repair (MMR) pathway was examined in the adult and developing rat brain. Rat homologues of human GTBP and MSH2, which are essential components of the post-replicative DNA MMR system, were identified in nuclear extracts from the adult and developing rat brain. Developmental studies showed that both GTBP and MSH2 levels were higher in nuclei isolated from the embryonic brain (day 16) than adult brain. However, this difference was not as dramatic as the difference in the number of proliferating cells. Levels of thymine DNA glycosylase (TDG), the enzyme which catalyzes the first step in short patch G:T mismatch repair, were also decreased in adult compared to embryonic brain. In the adult brain, MMR proteins were elevated in nuclear extracts enriched for neuronal nuclei. These results suggest that adult brain cells have the capacity to carry out DNA mismatch repair, in spite of a lack of ongoing DNA replication.     

Altered cytotoxicity of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea in human glioma cell lines SK-MG-1 and SKI-1 correlates with differential transport kinetics

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the chloroethylnitrosourea-sensitive Mer- SK-MG-1 and -resistant Mer- SKI-1 human glioma cell lines. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H]SarCNU was found to be temperature dependent in SK-MG-1 and SKI-1, but less so in SKI-1. At 37 degrees C, uptake of 50 microM [3H]SarCNU was linear up to 4 s in both cell lines, with uptake being significantly faster in SK-MG-1 than in SKI-1 under initial rate conditions. There was no significant difference in the rate of influx at 22 degrees C between both cell lines. Equilibrium was approached after 1 min at 22 and 37 degrees C. At 37 degrees C, steady state accumulation of SarCNU at 30 min was reduced significantly (35%) in SKI-1 cells compared with SK-MG-1 cells, although accumulation was similar at 22 degrees C. In SK-MG-1 cells, uptake of [3H]SarCNU at 37 degrees C was found to be saturable, but uptake in SKI-1 cells was not saturable over a 1000-fold range of concentrations. Analysis of efflux in cells preloaded with 50 microM [3H]SarCNU revealed that the rate of efflux was equivalent in both cell lines but that the efflux rate was more rapid at 37 degrees C compared with 22 degrees C. Metabolism of SarCNU at 37 degrees C was not different in either cell line after a 60-min incubation, as determined by thin layer chromatography. SKI-1 cells, compared with SK-MG-1 cells, were 3-fold more resistant to SarCNU at 37 degrees C but only 2-fold more resistant at 22 degrees C, a temperature at which SarCNU accumulation was similar in both cell lines. The 2-fold resistance at 22 degrees C was similar to that of 1,3-bis(2-chloroethyl)-1-nitrosourea at 37 and 22 degrees C. These findings indicate that increased cytotoxicity in SK-MG-1 cells is associated with a greater accumulation of SarCNU via an epinephrine-sensitive carrier that is not detectable in SKI-1 cells. However, part of the chloroethylnitrosourea resistance in SKI-1 cells is not secondary to decreased accumulation.     

Uptake of basic drugs into rat lung granule fraction in vitro

Although basic drugs are distributed widely in various tissues, they are characteristically concentrated in the lung granule fraction. We examined the uptake of seven lipophilic basic drugs into rat lung granule fraction (P2) in vitro and investigated the contributions of drug lipophilicity and lysosomal trapping to the characteristic lung P2 distribution. The uptake of each drug into P2 was examined at various pH values. The drug concentration in P2 was determined by gas chromatography. Biperiden (BP) was rapidly taken up into P2, reaching a maximal concentration within 1 min at pH 7.4 at both 4 degrees C and 37 degrees C. Both BP and chlorpromazine uptake into P2 was biphasic. Though the uptake rates of the seven drugs into P2 increased with rising pH, the rate of increase varied for each drug. There was a good correlation between the octanol-water partition coefficient of the non-ionized form (P(oct)) of each drug and the uptake into P2 in the presence or absence of NH4Cl, which inhibits lysosomal trapping. However, uptake into P2 in the presence of NH4Cl showed a stronger P(oct)-dependency. We conclude that the distribution of basic drugs into lung P2 is dependent on both drug lipophilicity and lysosomal uptake.     

Inhibition of glycohydrolase enzymes by aqueous extracts of Chinese medicinal herbs in a microplate format

A microplate assay, for use with a variety of glycohydrolase enzymes, was developed to aid the screening of Chinese medicinal herb extracts for the presence of potential anti-viral and anti-lymphoma compounds. The microplate assay method described offers greater convenience, speed and reproducibility over existing methods. The enzymes tested were alpha-glucosidase, beta-glucosidase and beta-glucuronidase. The assay can be easily adapted for use with other glycohydrolase enzymes. Of the 12 herb extracts examined four did not inhibit any of the enzymes (< 50% inhibition), one inhibited alpha-glucosidase only (> 50% inhibition), six inhibited beta-glucuronidase only, and one inhibited both alpha-glucosidase and beta-glucuronidase. None of the extracts were capable of inhibiting beta-glucosidase to any significant extent.     

cAMP-dependent protein kinase from brown adipose tissue: temperature effects on kinetic properties and enzyme role in hibernating ground squirrels

Arousal from hibernation requires thermogenesis in brown adipose tissue, a process that is stimulated by beta-adrenergic signals, leading to a rise in intracellular 3',5'-cyclic adenosine monophosphate AMP (cAMP) and activating cAMP-dependent protein kinase A (PKA) to phosphorylate a suite of target proteins and activate lipolysis and uncoupled respiration. To determine whether specific adaptations (perhaps temperature-dependent) facilitate PKA kinetic properties or protein-phosphorylating ability, the catalytic subunit of PKA (PKAc) from interscapular brown adipose of the ground squirrel Spermophilus richardsonii, was purified (final specific activity = 279 nmol phosphate transferred per min per mg protein) and characterized. Physical properties of PKAc included a molecular weight of 41 kDa and an isoelectric point of 7.8 +/- 0.08. A change in assay temperature from a euthermic value (37 degrees C) to one typical of hibernating body temperature (5 degrees C) had numerous significant effects on ground squirrel PKAc including: (a) pH optimum rose from 6.8 at 37 degrees C to 8.7 at 5 degrees C, (b) K(m) values at 37 degrees C for Mg.ATP (49.2 +/- 3.4 microM) and for two phosphate acceptors, Kemptide (50.0 +/- 5.5 microM) and Histone IIA (0.41 +/- 0.05 mg/ml) decreased by 53%, 80% and 51%, respectively, at 5 degrees C, and (c) inhibition by KCl, NaCl and NH4Cl was reduced. However, temperature change had little or no effect on K(m) values of rabbit PKAc, suggesting a specific positive thermal modulation of the hibernator enzyme. Arrhenius plots also differed for the two enzymes; ground squirrel PKAc showed a break in the Arrhenius relationship at 9 degrees C and activation energies that were 29.1 +/- 1.0 kJ/mol for temperatures > 9 degrees C and 2.3-fold higher at 68.1 +/- 2.1 kJ/mol for temperatures < 9 degrees C, whereas the rabbit enzyme showed a breakpoint at 17 degrees C with a 13-fold higher activation energy over the lower temperature range. However, fluorescence analysis of PKAc in the absence of substrates, showed a linear change in fluorescence intensity and wavelength of maximal fluorescence over the entire temperature range; this suggested that the protein conformational change indicated by the break in the Arrhenius plot was substrate-related. Temperature change also affected the Hill coefficient for cAMP dissociation of the ground squirrel PKA holoenzyme which rose from 1.12 +/- 0.18 at 37 degrees C to 2.19 +/- 0.07 at 5 degrees C, making the release of catalytic subunits at low temperature much more responsive to small changes in cAMP levels. Analysis of PKAc function via in vitro incubations of extracts of ground squirrel brown adipose with 32P-ATP + cAMP in the presence versus absence of a PKA inhibitor, also revealed major differences in the patterns of phosphoproteins, both between euthermic and hibernating animals as well as between 37 and 5 degrees C incubation temperatures; this suggests that there are both different targets of PKAc phosphorylation in the hibernating animal and that temperature affects the capacity of PKAc to phosphorylate different targets. Both of these observations, plus the species-specific and temperature-dependent changes in ground squirrel PKAc kinetic properties, suggest differential control of the enzyme in vivo at euthermic versus hibernating body temperatures in a manner that would facilitate a rapid and large activation of the enzyme during arousal from torpor.