The thermal stability of a castor bean seed acid phosphatase

The effect of temperature on the activity and structural stability of an acid phosphatase (EC 3.1.3.2.) purified from castor bean (Ricinus communis L.) seeds have been examined. The enzyme showed high activity at 45 degrees C using p-nitrophenylphosphate (p-NPP) as substrate. The activation energy for the catalyzed reaction was 55.2 kJ mol(-1) and the enzyme maintained 50% of its activity even after 30 min at 55 degrees C. Thermal inactivation studies showed an influence of pH in the loss of enzymatic activity at 60 degrees C. A noticeable protective effect from thermal inactivation was observed when the enzyme was preincubated, at 60 degrees C, with the reaction products inorganic phosphate-P (10 mM) and p-nitrophenol-p-NP(10 mM). Denaturation studies showed a relatively high transition temperature (Tm) value of 75 degrees C and an influence of the combination of Pi (10 mM) and p-NP (10 mM) was observed on the conformational behaviour of the macromolecule.     

Thermal inactivation and product inhibition of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase and their role on hydrolysis of naringin solutions

The kinetics of thermal inactivation of A. terreus alpha-rhamnosidase was studied using the substrate p-nitrophenyl alpha-L-rhamnoside between 50 degrees C and 70 degrees C. Up to 60 degrees C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60 degrees C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60 degrees C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.     

Purification and characterization of two thermostable proteases from the thermophilic fungus Chaetomium thermophilum

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by fractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and PhenylSepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63 kDa protease (PRO63) at pH 5.0. The optimum temperature for the two proteases was 65 degrees C. The PRO33 had a K(m) value of 6.6 mM and a V(max) value of 10.31 micromol/l/min, and PRO63 17.6 mM and 9.08 micromol/l/min, with casein as substrate. They were thermostable at 60 degrees C. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at 70 degrees C for 1 h. The thermal stability of the two enzymes was significantly enhanced by Ca2+. The residual activity of PRO33 and PRO63 at 70 degrees C after 60 min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing Ca2+. These properties make them applicable for many biotechnological purposes.     

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of Funastrum clausum

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70 degrees C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0-10.0 for casein and 6.2-6.8 for PFLNA). Kinetic parameters were determined for N-alpha-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s(-1)) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s(-1)). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.     

Streptomyces flavogriseus cellulase: Evaluation under various hydrolysis conditions

Streptomyces flavogriseus CMCase and Avicelase were very stable at 30 degrees C but not at 40 degrees C or higher. beta-Glucosidase was less stable at all temperatures tested. Stabilities were similar at pH values between 5.5 and 7, the optimal range for enzyme activity. Cellulose solubilizing activity was reduced by 40% at a cellobiose concentration of 150mM but glucose inhibited activity by only 10% at this concentration. beta-Glucosidase was inhibited by 40% at a glucose concentration of 10mM (ten times the substrate concentration). Relatively dilute S. flavogriseus cellulase extensively hydrolysed acid-swollen cellulose at concentrations as high as 10%. More highly crystalline forms of cellulose were more resistant to attack.     

Conformational changes and stabilization induced by phosphate binding to 5′-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.     

Purification and characterization of an extracellular beta-glucosidase produced by Phoma sp. KCTC11825BP isolated from rotten mandarin peel

A beta-glucosidase from Phoma sp. KCTC11825BP isolated from rotten mandarin peel was purified 8.5-fold with a specific activity of 84.5 U/mg protein. The purified enzyme had a molecular mass of 440 kDa with a subunit of 110 kDa. The partial amino acid sequence of the purified beta-glucosidase evidenced high homology with the fungal beta- glucosidases belonging to glycosyl hydrolase family 3. Its optimal activity was detected at pH 4.5 and 60 degrees C, and the enzyme had a half-life of 53 h at 60 degrees C. The Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose were 0.3 mM and 3.2 mM, respectively. The enzyme was competitively inhibited by both glucose (Ki=1.7 mM) and glucono-delta-lactone (Ki=0.1 mM) when pNPG was used as the substrate. Its activity was inhibited by 41% by 10 mM Cu2+ and stimulated by 20% by 10 mM Mg2+.     

Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from the extreme halophilic archaeon Halogeometricum borinquense strain TSS101

A novel haloalkaliphilic, thermostable serine protease was purified from the extreme halophilic archaeon, Halogeometricum borinquense strain TSS101. The protease was isolated from a stationary phase culture, purified 116-fold with 18% yield and characterized biochemically. The molecular mass of the purified enzyme was estimated to be 86 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10.0 in 20% NaCl. The enzyme had high activity over the pH range from 6.0 to 10.0. Enzymatic activity was strongly inhibited by 1 mM phenyl methylsulfonyl fluoride, but activity was increased 59% by 0.1% cetyltrimethylammonium bromide. The enzyme exhibited relatively high thermal stability, retaining 80% of its activity after 1 h at 90 degrees C. Thermostability increased in the presence of Ca2+. The stability of the enzyme was maintained in 10% sucrose and in the absence of NaCl.     

The varied responses of different F1-ATPases to chlorpromazine

The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined. While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature. Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C. Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C. At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature. Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5. Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation. Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5. Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA. The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison. Chlorpromazine protected MF1 and EF1 against cold inactivation. Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine. Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard. Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP.     

A halophilic extracellular protease from a halophilic archaebacterium strain 172 P1

An unidentified halophilic archaebacterium strain 172 P1 produced three extracellular proteases in media containing 15-27% salts. One component, F-II, was purified to homogeneity. It is a serine protease that can be inhibited by phenylmethylsulfonyl fluoride and chymostatin. A high concentration of NaCl was required for its stability; in the presence of 25% NaCl, only 4% of the activity was lost by incubating at 60 degrees C for 30 min, while complete inactivation occurred in the presence of 5% NaCl. F-II is a thermophilic and halophilic protease. High activity was obtained at 75-80 degrees C when F-II was assayed in the presence of 25% NaCl. The optimal concentration of NaCl required was 10-14% when assayed at 70 degrees C with azocasein as substrate, though a halophilic characteristic was not distinct at lower temperatures. Hydrolyses of the synthetic substrates succinyl-alanyl-alanyl-prolyl-phenylalanyl-4-methylcoumaryl-7-amide or succinyl-alanyl-alanyl-alanyl-p-nitroanilide at 26 degrees C were maximal at 25 and 30% NaCl, respectively. F-II was most stable at pH 6-7, and its optimal pH was 10.7. Its molecular weight was estimated as 44,000-46,000 by sodium dodecyl sulfate--polyacrylamide gel electrophoresis and by gel filtration--high-pressure liquid chromatography. The sequence of the 35 N-terminal amino acid residues was determined and compared with that of other serine proteases.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Inactivation of alpha(2)-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active alpha(2)- macroglobulin (80 mug/ml) totally inhibits the proteolytic activity of trypsin (14 mug/ml) by trapping this protease. But after a 20 min incubation of alpha(2)-macroglobulin at 37 degrees C with 2 x 10(6) human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10(-7) M) and cytochalasin B (10(-8) M), 100% of trypsin activity was recovered, indicating a total inactivation of alpha(2)-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates alpha(2)-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10(-7) M also inactivates alpha(2)-macroglobulin, which indicates that an important cause of alpha(2)-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.     

Temperature- and time-dependent inactivation of pyrroline-5-carboxylate synthase: suggestive evidence for an allosteric regulation of the enzyme

When pyrroline-5-carboxylate (PC) synthase activity in the membrane of mitochondria of rat small intestine mucosa was assayed in the presence of 0.5 mM ornithine, the time course of inactivation showed that the activity disappeared entirely by about 8 min at 30 degrees C, whereas there was no decrease in the activity at 15 degrees C. A prior incubation of the enzyme with ornithine at 30 or 37 degrees C in the presence of 50% sorbitol as a thermal stabilizer resulted in a marked loss of the activity, while that at 0 or 15 degrees C did not lose any. This suggests that PC synthase is inactivated by ornithine regardless of the presence of substrates. The inactivation at 30 degrees C proceeded gradually for about 7 h, until an equilibrium was attained. Extensive dialysis allowed the inactivated enzyme to regain about 60% of the original activity. These results suggest that the inactivation is reversible. The concentration of ornithine and the percentage of inactivation at equilibrium was correlated by the Hill equation and displayed a sigmoidicity with n = 1.47 and [S]50 = 0.036 mM. In the presence of sorbitol, the inactivation was prevented by 0.2 mM ATP or ADP. The role of the nucleotides in PC synthase regulation is discussed.     

Purification and characterization of an intracellular chymotrypsin-like serine protease from Thermoplasma volcanium

An intracellular serine protease produced by Thermoplasma (Tp.) volcanium was purified using a combination of ammonium sulfate fractionation, ion exchange, and alpha-casein agarose affinity chromatography. This enzyme exhibited the highest activity and stability at pH 7.0, and at 50 degrees C. The purifed enzyme hydrolyzed synthetic peptides preferentially at the carboxy terminus of phenylalanine or leucine and was almost completely inhibited by PMSF, TPCK, and chymostatin, similarly to a chymotrypsin-like serine protease. Kinetic analysis of the Tp. volcanium protease reaction performed using N-succinyl-L-phenylalanine-p-nitroanilide as substrate revealed a Km value of 2.2 mM and a Vmax value of 0.045 micromol(-1) ml(-1) min(-1). Peptide hydrolyzing activity was enhanced by >2-fold in the presence of Ca2+ and Mg2+ at 2-12 mM concentration. The serine protease is a monomer with a molecular weight of 42 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram activity staining.     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.     

Essential arginyl residues in the H+-translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe

The H+-translocating adenosine-5'-triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3-butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3-butanedione. The extent of inactivation is half maximal at 10 mM 2,3-butanedione for an incubation of 30 min at 30 degrees C at pH 7.0. Under the same conditions, the time-dependence of inactivation is biphasic in a semi-logarithmic plot with half-lives of 10.9 min and 65.9 min. Incubation with 2,3-butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity. Magnesium adenosine 5'-triphosphate (MgATP) and magnesium adenosine 5'-diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3-butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.     

Human fat cell adenylate cyclase. Enzyme characterization and guanine nucleotide effects on epinephrine responsiveness in cell membranes.

Human adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) has been studied in preparations of fat cell membranes ("ghosts"). As reported earlier, under ordinary assay conditions (1.0 mM ATP, 5 mM Mg2+, 30 degrees C, 10 min incubation) the enzyme was activated 6-fold by epinephrine in the presence of the GTP analog, 5'-guanylyl-imidodiphosphate [GMP-P(NH)P] (Cooper, B. et al. (1975) J. Clin. Invest. 56, 1350-1353). Basal activity was highest during the first 2 min of incubation then slowed and was linear for at least the next 18 min. Epinephrine, added alone, was often without effect. but sometimes maintained the initial high rate of basal activity. GMP-P(NH)P alone produced inhibition ("lag") of basal enzyme early in the incubation periods. Augmentation of epinephrine effect by GMP-P(NH)P, which also proceeded after a brief (2 min) lag period, was noted over a wide range of substrate (ATP) concentrations. GTP inhibited basal levels of the enzyme by about 50%. GTP also allowed expression of an epinephrine effect, but only in the sense that the hormone abolished the inhibition by GTP. Occasionally a slight stimulatory effect on epinephrine action was seen with GTP. At high Mg2+ concentration (greater than 10 mM) or elevated temperatures (greater than 30 degrees C) GMP-P(NH)P alone activated the enzyme. Maximal activity of human fat cell adenylate cyclase was seen at 50 mM Mg2+, 1.0 mM ATP, pH 8.2, and 37 degrees C in the presence of 10(-4) M GMP-P(NH)P; under these conditions addition of epinephrine did not further enhance activity. Human fat cell adenylate cyclase of adults was insensitive to ACTH and glucagon even in the presence of GMP-P(NH)P.     

Reassessment of the cold-labile nature of phosphofructokinase from a hibernating ground squirrel.

This study reassesses the proposal that cellular conditions of low temperature and relative acidosis during hibernation contribute to a suppression of phosphofructokinase (PFK) activity which, in turn, contributes to glycolytic rate suppression during torpor. To test the proposal that a dilution effect during in vitro assay of PFK was the main reason for activity loss (tetramer dissociation) at lower pH values, the influence of the macromolecular crowding agent, polyethylene glycol 8000 (PEG), on purified skeletal muscle PFK from Spermophilus lateralis was evaluated at different pH values (6.5, 7.2 and 7.5) and assay temperatures (5, 25 and 37degrees C). A 78 +/- 2.5% loss of PFK activity during 1 h incubation at 5 degrees C and pH 6.5 was virtually eliminated when 10% PEG was present (only 7.0 +/- 1.5% activity lost). The presence of PEG also largely reversed PFK inactivation at pH 6.5 at warmer assay temperatures and reversed inhibitory effects by high urea (50 or 400 mM). Analysis of pH curves at 5 degrees C also indicated that approximately 70% of activity would remain at intracellular pH values in hibernator muscle. The data suggest that under high protein concentrations in intact cells that the conditions of relative acidosis, low temperature or elevated urea during hibernation would not have substantial regulatory effects on PFK.