Isoelectric focusing studies of human pancreatic secretion

Pure bile, pancreatic and duodenal human juices have been analyzed by isoelectric focusing, either at rest or upon stimulation with caerulein. In rats, stimulation has also been performed with secretin. Twenty bands have been resolved and quantified in the pancreatic secretion. By developing zymograms, a number of isozymes have been identified: 6 isoamylases [pI's 7.2, 7.1 and 6.6 (major) and pI's 7.4, 6.7 and 5.8 (minor)], 3 lipases [pI's 7.0 and 6.8 (major) and 6.4 (minor)], two major alkaline proteases (pI's 9.8 and 8.4) and one major acidic protease (pI 4.3) and one band of RNAase activity (pI 8.6). The stimulation kinetics follow a mechanism according to Palade, indicating uniform response to secretogogues, parallel intracellular transport and parallel discharge of pancreatic exocrine proteins.     

A Model of the Quaternary Structure of Enolases, Based on Structural and Evolutionary Analysis of the Octameric Enolase from Bacillus subtilis

Purified enolase from Bacillus subtilis has a native mass of approximately 370 kDa. Since B. subtilis enolase was found to have a subunit mass of 46.58 kDa, the quaternary structure of B. subtilis is octameric. The pl for B. subtilis enolase is 6.1, the pH optimum (pH_o) for activity is 8.1–8.2, and the K _m for 2-PGA is approximately 0.67 mM. Using the dimeric C structure of yeast dimeric enolase as a guide, these dimers were arranged as a tetramer of dimers to simulate the electron microscopy image processing obtained for the octameric enolase purified from Thermotoga maritima. This arrangement allowed identification of helix J of one dimer (residues 86–96) and the loop between helix L and strand 1 (HL–S1 loop) of another dimer as possible subunit interaction regions. Alignment of available enolase amino acid sequences revealed that in 16 there are two tandem glycines at the C-terminal end of helix L and the HL–S1 loop is truncated by 4–6 residues relative to the yeast polypeptide, two structural features absent in enolases known to be dimers. From these arrangements and alignments it is proposed that the GG tandem at the C-terminal end of helix L and truncation of the HL–S1 loop may play a critical role in octamer formation of enolases. Interestingly, the sequence features associated with dimeric quaternary structure are found in three phylogenetically disparate groups, suggesting that the ancestral enolase was an octamer and that the dimeric structure has arisen independently multiple times through evolutionary history.     

Chromatographic performance of a thin microporous bed of nitrocellulose

Chromatography along thin (125 μm) porous beds of nitrocellulose, layered on top of an polyester backing, shows good separation efficiency with plate heights of 10–20 μm. Flow is controlled by capillary forces and shows low rate variations between the individual disposable devices. Positively charged groups were introduced into the nitrocellulose and efficient separation of transferrin isoforms, differing by only 0.1 pI units, was found after a short migration distance (1 cm). The upper surface is not covered, which allows sample and reagents to be added, and the clear backing permits detection. The chromatography can easily be combined on-line with sensitive immunoassay detection down to the pM (10⁻¹² M) range. This microscaled combination device should have a wide range of applications in analytical biochemistry.     

A Quantitative Approach to Membrane Binding of Human Ubiquitous Mitochondrial Creatine Kinase Using Surface Plasmon Resonance

We have evaluated surface plasmon resonance with avidin-biotin immobilized liposomes tocharacterize membrane binding of ubiquitous mitochondrial creatine kinase (uMtCK). Whilethe sarcomeric sMtCK isoform is well known to bind to negatively charged phospholipids,especially cardiolipin, this report provides the first experimental evidence on the membraneinteraction of an uMtCK isoform. Qualitative measurements showed that liposomes containing16% (w/w) cardiolipin bind octameric as well as dimeric human uMtCK and also cytochromec, but not bovine serum albumin. Quantitative parameters could be derived only for themembrane interaction of octameric human uMtCK using an improved analytical approach.Association and dissociation kinetics of octameric uMtCK fit well to a model for heterogeneousinteraction suggesting two independent binding sites. Rate constants of the two sites differedby one order of magnitude, while their affinity constants were both about 80–100 nM. Thedata obtained demonstrate that surface plasmon resonance with immobilized liposomes is asuitable approach to characterize the binding of peripheral proteins to a lipid bilayer and thatthis method yields consistent quantitative binding parameters.     

Hexameric,Trimeric, Dimeric,and Monomeric Forms of Inorganic Pyrophosphatase from Escherichia coli

The conditions were found for obtaining trimeric, dimeric, and monomeric forms of the Escherichia coli inorganic pyrophosphatase from its native hexameric form. Interconversions of the oligomers were studied, and rate constants for their dissociation and association were determined. All forms were found to be catalytically active, with the activity decreasing in the following order: hexamer–trimer–dimer–monomer. The activity of trimeric and dimeric forms was high enough to study and to compare their catalytic properties. The monomeric form of the enzyme was unstable.     

Arginine and Lysine Aminopeptidase Activities in Chromaffin Granules of Bovine Adrenal Medulla: Relevance to Prohormone Processing

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH₂ and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg-methylcoumarinamide (Arg-MCA) and Lys-MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co-localized with [Met]-enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β-mercaptoethanol (β-ME) and inhibited by p-hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β-ME/NaCl-stimulated Arg-MCA cleaving activity and β-ME-stimulated Lys-MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. K_m(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg-MCA and Lys-MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co²⁺ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni²⁺ and Zn²⁺ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.     

Soluble and bound forms of intracellular acid carboxypeptidase inAspergillus saitoi

The enzymological, physical, and immunological properties of soluble and bound forms of intracellular acid carboxypeptidase isolated from fresh mycelia ofAspergillus saitoi are reported. In the broken mycelia, about 60% of the total activity was found in the 2,000×g precipitate, with most of the remainder in the 100,000×g supernantant. The highly purified enzymes, I_a and I_b, from the 100,000×g supernatant were found to be homogeneous by such criteria as disc gel electrophoresis at pH 9.4 The bound enzyme, II, was solubilized from the 2,000×g precipitate by self-digestion at pH 6.4 and was highly purified by chromotography. The two forms of intracellular enzymes, the soluble enzymes (I_a and I_b) from the 100,00×g supernatant and the solubilized enzyme (II) from the 2,000×g precipitate, were closely related to, but not completely identical with, the extracellular acid carboxypeptidase.     

Protonation states of active‐site lysines of penicillin‐binding protein 6 from Escherichia coli and the mechanistic implications

The protonation states of the two active‐site lysines (Lys69 and Lys235) of PBP 6 of Escherichia coli were explored to understand the active site chemistry of this enzyme. Each lysine was individually mutated to cysteine, and the resultant two mutant proteins were purified to homogeneity. Each protein was denatured, and its cysteine was chemically modified to produce an S‐aminoethylated cysteine (γ‐thialysine) residue. Following renaturation, the evaluation of the kinetics of the dd ‐carboxypeptidase activity of PBP 6 as a function of pH was found consistent with one lysine in its free‐base (Lys69) and the other in the protonated state (Lys235) for optimal catalysis. The experimental estimates for their pK_a values were compared with the pK_a values calculated computationally, using molecular‐dynamics simulations and a thermodynamic cycle. Study of the γ‐thialysine69 showed that lysine at position 69 influenced the basic limb of catalysis, consistent with the fact that the two lysine side chains are in proximity to each other in the active site. Based on these observations, a reaction sequence for PBP 6 is proposed, wherein protonated Lys235 serves as the electrostatic substrate anchor and Lys69 as the conduit for protons in the course of the acylation and deacylation half‐reactions. Proteins 2014; 82:1348–1358. © 2013 Wiley Periodicals, Inc.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Genetics and ontogeny of α-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster

-Glycerophosphate dehydrogenase (GPDH) occurs in Drosophila melanogaster in three isozymic forms. These are separable by starch gel electrophoresis and have been tentatively numbered 1, 2, and 3. GPDH-1 is most concentrated in the adult thorax and GPDH-3 in the abdomen; 1 and 3 are in approximately equal amounts in the head. GPDH-2 is relatively weak in all preparations. In larvae, only GPDH-3 is present. Purified GPDH-1 has optimal activity at pH 6.7–7.0. GPDH-3 at pH 7.5, and GPDH-2 is intermediate. Changes in total GPDH activity parallel larval growth, pupal histolysis, and differentiation of adult tissues. In the latter period the ratio of activity at pH 6.7 to pH 7.6 increases, reflecting the shift from GPDH-3 to GPDH-1. Two types of homozygous GPDH patterns which differ in the electrophoretic mobilities of all three isozymes have been found in inbred strains. In heterozygous adults six bands, the parental forms of GPDH-1 and GPDH-3 and hybrid forms of each, can be resolved. Analysis of F₂ and backcross progeny suggests that a single genetic locus affects all three isozymes. Heterozygous embryos have only the maternal form of GPDH-3 until just before they hatch as first instar larvae. At this stage they have maternal and paternal GPDH-3 plus an intermediate band.This project was supported in part by National Institutes of Health research grant GM-15597.     

Glutamine synthetase oligomers and isoforms in sugarbeet (Beta vulgaris L.)

The -amino-N compounds that accumulate in the thickening storage root of sugarbeet (Beta vulgaris L.) were synthesized in the leaves (NO ₃ ^– nutrition) and also in the lateral roots (NH ₄ ⁺ nutrition). Ammonium stimulated glutamine synthetase (GS, EC 6.3.1.2) activity, especially in the lateral roots. With non-denaturing polyacrylamide-gel isoelectric focussing, simultaneously active charge-isomers of GS were separated in both leaves and roots. The leaf isoforms were active in an octameric and also in a tetrameric form. In the root only octameric isoforms were found. The tetramer was more active than the octamer in the leaf blade and vice versa in the leaf stem. Only the tetramer needed -mercaptoethanol for activity stabilization in vitro. A reactivation, however, of an inactive tetramer by the addition of thiol/thioredoxin was not possible. The same isoforms of GS were separated in different organs of sugarbeet but with different patterns of relative activity. The activity pattern depended also on the N-source of the plant. With increasing age of the plant the number of active GS isoforms declined in both leaves and roots although the in-vitro activity remained unchanged (NO ₃ ^– -fed plants) or even increased (NH ₄ ⁺ -fed plants).Abbreviations GS glutamine synthetase (E.C. 6.3.1.2.) - IEF isoelectric focussing - PAGE polyacrylamide gel electrophoresis This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Structural and functional implications of the amino acid sequences of dimeric, cytoplasmic and octameric mitochondrial creatine kinases from a protostome invertebrate.

The cDNA and deduced amino-acid sequences for dimeric and octameric isoforms of creatine kinase (CK) from a protostome, the polychaete Chaetopterus variopedatus, were elucidated and then analysed in the context of available vertebrate CK sequences and the recently determined crystal structure of chicken sarcomeric mitochondrial CK (MiCK). As protostomes last shared a common ancestor with vertebrates roughly 700 million years ago, observed conserved residues may serve to confirm or reject contemporary hypotheses about the roles of particular amino acids in functional/structural processes such as dimer/octamer formation and membrane binding. The isolated cDNA from the dimeric CK consisted of 1463 nucleotides with an open reading frame of 1116 nucleotides encoding a 372-amino-acid protein having a calculated molecular mass of 41.85 kDa. The percentage identity of C. variopedatus dimeric CK to vertebrate CK is as high as 69%. The octameric MiCK cDNA is composed of 1703 nucleotides with an open reading frame of 1227 nucleotides. The first 102 nucleotides of the open reading frame encode a 34-amino-acid leader peptide whereas the mature protein is composed of 375 amino acids with a calculated molecular mass of 42.17 kDa. The percentage identity of C. variopedatus MiCK to vertebrate CK is as high as 71%. This similarity is also evident in residues purported to be important in the structure and function of dimeric and octameric CK: (a) presence of seven basic amino acids in the C-terminal end thought to be important in binding of MiCK to membranes; (b) presence of a lysine residue (Lys110 in chicken MiCK) also thought to be involved in membrane binding; and (c) presence of a conserved tryptophan thought to be important in dimer stabilization which is present in all dimeric and octameric guanidino kinases. However, C. variopedatus MiCK lacks the N-terminal heptapeptide present in chicken MiCK, which is thought to mediate octamer stabilization. In contrast with vertebrate MiCK, polychaete octamers are very stable indicating that dimer binding into octamers may be mediated by additional and/or other residues. Phylogenetic analyses showed that both octamer and dimer evolved very early in the CK lineage, well before the divergence of deuterostomes and protostomes. These results indicate that the octamer is a primitive feature of CK rather than being a derived and advanced character.     

The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a ∼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of ∼24 kDa with pI values of ∼6.7, 6.9, and 7.0 and two spots of ∼22 and ∼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.     

Inhibition by cupric ions of the hydration of CO2 catalyzed by carbonic anhydrase II

The inhibition by cupric ions of the hydration of CO₂ catalyzed by carbonic anhydrase II is interesting because of the results of Tuet al. obtained at chemical equilibrium, indicating that Cu²⁺ inhibits specifically a proton transfer in the catalytic pathway. We have measured this inhibition at steady state, using stopped-flow methods. The inhibition by Cu²⁺ of the hydration of CO₂ catalyzed by carbonic anhydrase II had aK _I near 1×10^–6 M atpH 7.0 and gave inhibition that is noncompetitive atpH 6.0 and mixed, but close to uncompetitive, atpH 6.8. ThepH dependence of this binding is consistent with a binding site for Cu²⁺ on the enzyme with apK _a near 7. The binding interaction between Cu²⁺ and the fluorescent inhibitor 5-dimethylaminonaphthalene-l-sulfonamide on carbonic anhydrase II was noncompetitive, indicating that the binding site for Cu²⁺ is distinct from the coordination sphere of zinc in which the actual interconversion of CO₂ and HCO ₃ ^– and the binding of sulfonamides takes place.     

Phosphatase and ATPase activities in isonuclear lines of cytoplasmic male-sterile and male-fertile petunia

Summary Soluble and membrane-bound fractions of plant leaves, cell suspension cultures and seedlings of petunia were examined for phosphohydrolase activity on p-nitrophenyl phosphate (pNPPase) and adenosine triphosphate (ATPase). One cytoplasmic male-sterile (CMS) and one fertile (F) line was examined for each tissue. Both pNPPase and ATPase exhibited a broad optimal activity between pH 5.5–7.0 for the membrane-bound fraction and between 4.5–7.0 for the soluble fractions. The activity of both were inhibited by divalent ions including Mg²⁺. At pH 7.2, the activities on various triphosphonucleotides were similar and they were hydrolyzed by a rate of 20–50% of that of ATP. Significant differences between CMS and F extracts were: (a) higher activities in CMS membranes; (b) lower Ea (energy of activation) values for activities in CMS membrane functions; (c) seedling and cell-culture CMS extracts exhibited a higher sensitivity to high temperature denaturation; (d) the hydrolase activity on monoand triphospho-cytosine compounds was significantly higher in CMS than in F membranes.Contribution from the Agricultural Research Organization, The Volcani Center, Bet-Dagan, Israel No. 355-E, 1992 series     

Purification of homogeneous forms of fungal peroxygenase

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.     

Acid glycosidases from hen oviduct and egg albumen

Activities of seven acid glycosidases: β-N-acetylhexosaminidase (β-HEX), α- and β-galactosidase (α- and β-GAL), α- and β-mannosidase (α- and β-MAN), α-glucosidase and α-fucosidase in magnum region of hen (Gallus gallus domesticus) oviduct, and four acid glycosidases: β-HEX, β-GAL, α- and β-MAN in egg albumen, were investigated. β-HEX from magnum and egg albumen hydrolysed 4-methylumbelliferyl-β-N-acetylhexosamine-6-sulphate (4-MeUmbGlcNAc-6-SO₄) like mammalian β-HEX form A. Multiple forms of magnum and egg albumen β-HEX, β-GAL, α- and β-MAN were separated by strong anion exchange chromatography and chromatofocusing method. Chromatofocusing of the magnum resulted in the appearance of multiple forms for β-HEX with pI of 6.18, 5.43, 5.55, 5.34, 5.27 and 5.16, for β-GAL with pI of 4.98, 4.84, 4.77, 4.64 and 4.68–4.63, for α-MAN with pI of ≥ 7.4, 6.75, 6.62 and 6.26, and for β-MAN two forms with pI of 6.37 and 5.77. Chromatofocusing of egg albumen yields multiple forms for β-HEX with pI of 6.24, 6.08, 5.55 and 5.35, for β-GAL two forms with pI of 5.10 and 4.86–4.80 for α-MAN multiple forms with pI of ≥ 7.4, 6.80, 6.60 and 6.30, and for β-MAN forms with pI of 6.30 and 5.77. In conclusion, this study was the first to show β-HEX activity against 4-MeUmbGlcNAc-6-SO₄ in the magnum and albumen of bird eggs, corresponding to β-HEX A activity in mammals. Main multiple forms of β-HEX, β-GAL, α- and β-MAN occurring in the magnum were revealed in the egg albumen. Comparison with a cock of the same breed showed that hen egg magnum and albumen has the same multiple forms of the enzymes that are found in the epididymides and seminal plasma of the cock.     

The 40s Ω-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1–38 heme-containing N-fragment with the residues 57–104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega ()-loop region. The fragment complex shows compact structure, native-like -helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine (located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1–38/57–104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (pK_a1=6.72) and a high pK_a-associated state transition (pK_a2=8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition (pK_a=8.9). The data indicate that the 40s -loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein.     

Evidence for a posttranslational covalent modification of liver glyceraldehyde-3-phosphate dehydrogenase in hibernating jerboa (Jaculus orientalis)

The specific activity of d-glyceraldehyde-3-phosphate (G3P) dehydrogenase (phosphorylating) (GPDH, EC 1.2.1.12) found in liver of induced hibernating jerboa (Jaculus orientalis) was 2–3-fold lower than in the euthermic animal. However, the comparative analysis of the soluble protein fraction of these tissues by SDS-PAGE and Western blotting showed no significant changes in the intensity of the 36 kDa protein band of the GPDH subunit. After using the same purification procedure, the GPDH from liver of hibernating jerboa exhibited lower values for both apparent optimal temperature and specific activity than the enzyme from the euthermic animal. Similar non-linear Arrhenius plots were obtained, but the E_a values calculated for the GPDH from hibernating tissue were higher. Although in both purified enzyme preparations four isoelectric GPDH isoforms were resolved by chromatofocusing, those of hibernating liver exhibited more acidic pI values (pI 7.3–6.1) than the hepatic isoforms of euthermic animals (pI 8.7–8.1). However, all liver GPDH isoforms exhibited similar native and subunit molecular masses and cross-reacted with an antibody raised against muscle GPDH. The comparison of the kinetic parameters of both purified preparations and the main isoforms isolated from euthermic and hibernating tissues showed the decreased catalytic efficiency of hibernating enzyme being exclusively due to a lower V_max for both substrates G3P and NAD⁺. Phosphodiesterase treatment of cell-free extracts increased GPDH activity in the case of hibernating liver only. The pI of the main isoform purified from this tissue, about 6.9, changed after this treatment to an alkaline value (pI 8.44) similar to those of the euthermic GPDH isoforms. Differential ultraviolet absorption spectra of these isoforms indicated that a substance absorbing at 260 nm, that was released by the phosphodiesterase digestion, was present in the enzyme of hibernating tissue. Incubation of purified GPDH with the NO-releasing agent sodium nitroprussite produced under conditions that promote mono-ADP-ribosylation a dramatic decrease of activity (up to 60%) of both euthermic and phosphodiesterase-treated hibernating preparations but only a marginal inhibition of the hibernating enzyme. These data suggest that liver GPDH of hibernating jerboa exhibits a posttranslational covalent modification, being probably a mono-ADP-ribosylation. The resulting inhibition of enzyme activity could contribute to the wide depression of the glycolytic metabolic flow associated with mammalian hibernation.     

Kurloff cell lysosomal arylsulphatases: Presence of both cationic and highly anionic isoforms of the sole B class

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or LeeVaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4–23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, ‘classical’ cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their K_m (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO₃ were also characteristic of this class of Asase. Finally, chondroitin4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.