Kinetics and calcium-specificity of polyamine uptake in carrot protoplasts

Summary The kinetics of putrescine and spermidine uptake and the influence of calcium on the kinetic parameters of the transport process were investigated in protoplasts isolated from carrot phloem parenchyma. Spermidine uptake dependence on external concentration was biphasic, both in the absence and in the presence of 1 mM CaCl₂. In the first case, saturation was reached at 0.1 to 0.25 mM and the K_m value was 43µM. When calcium was added, the K_m and V_max increased. A similar pattern was found with regard to putrescine uptake. Moreover, in order to clarify the mode of action of calcium on polyamine uptake, lanthanides (lanthanum and gadolinium) were utilised as Ca⁺²-channel antagonists. When protoplasts were preincubated with these lanthanides, the stimulatory effect exerted by Ca⁺² on polyamine uptake was almost totally abolished. On the other hand, if lanthanum was supplied instead of calcium, it gave rise to a small enhancement of polyamine transport. These results induce us to suggest that calcium acts on polyamine uptake both by binding to external sites on the plasmalemma and by penetrating into the cell.     

Tb(III)-ion-binding-induced conformational changes in platelet factor XIII.

Ca(II) ions are crucial during proteolytic conversion of Factor XIII zymogen into the active enzyme Factor XIIIa. Factor XIII proteolyzed by thrombin or trypsin in the presence of 5 mM-EDTA resulted in rapid inactivation of transglutaminase activity. Factor XIIIa formed by thrombin or trypsin in the presence of 40 microM-Tb(III) ions, however, was indistinguishable from Factor XIIIa formed in the presence of 2-5 mM-Ca(II) ions with respect to molecular mass and transglutaminase activity. Thrombin treatment of Factor XIII in the presence of 1-5 microM-Tb(III) ions resulted in three fragments (76 kDa, 51 kDa and 19 kDa) with simultaneous loss of transglutaminase activity. Tb(III) ions at concentrations greater than 40 microM made platelet Factor XIII resistant to proteolysis by either thrombin or trypsin. Other lanthanide(III) ions [Ln(III) ions] tested [Ce(III), La(III) and Gd(III) ions] functioned similarly to Tb(III) ions during proteolytic activation of Factor XIII. Ln(III) ions (10-100 microM) were unable to replace the Ca(II) ions required for transglutaminase activity of Factor XIIIa. Tb(III) ions also inhibited in a non-competitive manner the transglutaminase activity of Factor XIIIa (Ki 71 microM) even when measured in the presence of 200-fold molar excess of Ca(II) ions. Factor XIII selectively bound to a Tb(III)-chelate affinity column, and could not be eluted by 100 mM-CaCl2. Binding of Tb(III) ions to Factor XIII was demonstrated by fluorescence emission due to Forster energy transfer. A 10(4)-fold molar excess of CaCl2, but not NaCl, partially quenched Tb(III) fluorescence. Low concentrations (5-20 microM) of Tb(III) ions also inhibited the binding of Factor XIII to des-A-fibrinogen by about 43%, whereas higher concentrations (40-100 microM) promoted binding. Conformational changes in Factor XIII consequent to the binding of Tb(III) ions could be responsible for the observed effects on protein structure and function.     

Calcium Binding Ability of Recombinant Buffalo Regucalcin: A Study Using Circular Dichroism Spectroscopy

Regucalcin is a calcium regulating multifunctional protein reported to have many important functions like calcium homeostasis, anti-oxidative, anti-apoptotic and anti-cancerous functions. Although it is demonstrated as a calcium regulating protein, the calcium binding ability of regucalcin is still a controversy. The main reason for the controversy is that it lacks a typical EF hand motif which is common to most of the calcium binding proteins. Even though many studies reported regucalcin as a calcium binding protein, there are some studies reporting regucalcin as non-calcium binding also. In the present study, we investigated the calcium binding ability of recombinant buffalo regucalcin by assessing the secondary structural changes of the protein using circular dichroism spectroscopy after adding Ca²⁺ to the protein solution. Two types of calcium binding studies were done, one with different concentration of calcium chloride (0.5 mM CaCl₂, 1 mM CaCl₂, 2 mM CaCl₂) and other at different time interval (no incubation and 10 min incubation) after addition of calcium chloride. Significant structural changes were observed in both studies which prove the calcium binding ability of recombinant regucalcin. A constant increase in the α-helix (1.1% with 0.5 mM CaCl₂, 1.4% with 1 mM CaCl₂, 3.5% with 2 mM CaCl₂) and a decrease in β-sheets (78.5% with 0.5 mM CaCl₂, 77.4% with 1 mM CaCl₂, 75.7% with 2 mM CaCl₂) were observed with the increase in calcium chloride concentration. There was a rapid increase in α-helix and decrease in β-sheets immediately after addition of calcium chloride, which subsides after 10 min incubation.     

Calcium-dependent affinity purification of transglutaminase from rat liver cytosol

Tissue transglutaminase (E.C.2.3.2.13, R-glutaminyl-peptide: amine glutaminyl transferase), was purified from extracts of rat liver by calcium dependent affinity chromatography on casein-Sepharose. In the presence of 5 mM calcium the enzyme binds to casein Sepharose and is subsequently eluted with 5 mM EGTA. The enzyme has a molecular weight of 83,000 and its activity is dependent on calcium and reduced sulfhydryl residues. A widely distributed calcium-dependent protease (E.C. 3.4.22.17) copurified with transglutaminase by gel filtration and ion exchange chromatography. The separation of these activities prior to chromatography on casein-Sepharose is essential for the isolation of a stable transglutaminase by calcium-dependent affinity chromatography. Affinity chromatography using casein-Sepharose or other immobilized substrates may allow the calcium-dependent purification of a variety of transglutaminases.     

Purification and partial characterization of thermostable serine alkaline protease from a newly isolated<Emphasis Type="Italic">Bacillus subtilis</Emphasis> PE-11

The purpose of the research was to study the purification and partial characterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. The enzyme was purified in a 2-step procedure involving ammonium sulfate precipitation and Sephadex G-200 gel permeation chromatography. The enzyme was shown to have a relative low molecular weight of 15 kd by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and was purified 21-fold with a yield of 7.5%. It was most active at 60°C, pH 10, with casein as substrate. It was stable between pH 8 and 10. This enzyme was almost 100% stable at 60°C even after 350 minutes of incubation. It was strongly activated by metal ions such as Ca²⁺, Mg⁺², and Mn⁺². Enzyme activity was inhibited strongly by phenylmethyl sulphonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP) but was not inhibited by ethylene diamine tetra acetic acid (EDTA), while a slight inhibition was observed with iodoacetate,p-chloromercuric benzoate (pCMB), and β-mercaptoethanol (β-ME). The compatibility of the enzyme was studied with commercial and local detergents in the presence of 10mM CaCl₂ and 1M glycine. The addition of 10mM CaCl₂ and 1M glycine, individually and in combination, was found to be very effective in improving the enzyme stability where it retained 52% activity even after 3 hours. This enzyme improved the cleansing power of various detergents. It removed blood stains completely when used with detergents in the presence of 10mM CaCl₂ and 1M glycine.     

Interaction of Calcium Ion with Bovine Caseins

In order to clarify the interaction of calcium ion with casein, the volume change associated with the interaction was measured by dilatometric procedures. When CaCl₂ was added to the casein solutions at neutral pH, a volume increase occurred and reached a constant saturated value of about 700 ml per 10⁶ g protein with increasing CaCl₂ concentrations for whole-, α_s- and β-casein solutions, but there was no volume change for κ-casein solution. On the other hand, the binding of calcium ion to the casein fractions was determined by a gel filtration procedure at pH 6.0 to 9.0. The number of Ca²⁺ ions bound to the caseins increased with the CaCl₂ concentration and pH value, and the relative order of binding capacities for the caseins was: α_s-casein > whole-casein > β-casein > κ-casein.

It was found that the volume changes obtained by the dilatometry were smaller than the calculated volume increases based on the assumption that these are caused by the binding of Ca²⁺ ion to the caseins. Therefore it is necessary to introduce another factor which reduces the volume increase due to the Ca²⁺ ion binding in order to reasonably explain the measured volume changes. At present it is presumed that there occurs the unfolding of peptide chain of casein molecule on Ca²⁺ ion binding, which has been known to decrease the volume of the protein solution.     

The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII. Characterization of the binding of liver transglutaminase to fibrin

The present study represents detailed investigations into the nature of interactions between an intracellular "tissue" transglutaminase and a plasma protein, fibrinogen. We demonstrate a specific, saturable, and reversible binding of transglutaminase to fibrin(ogen). The binding was time- and temperature-dependent, was independent of divalent metal ions, did not require the release of either fibrinopeptide A or B, and was partially inhibited by the presence of sodium chloride or plasma proteins, properties similar to Factor XIII binding to fibrin(ogen). Both Factor XIII and liver transglutaminase also shared similar binding sites on fibrinogen, the A alpha- and the B beta-chains. The binding characteristics of liver transglutaminase were thus similar to Factor XIII binding to fibrin, but there were also important differences. Scatchard analyses of the binding data indicated that the affinity of liver transglutaminase (Kd = 4.17 x 10(-7) M) was at least 40-fold weaker compared with the affinity of Factor XIII to fibrinogen. Consequently, a 20-fold molar excess of Factor XIII a-chains specifically and completely inhibited the binding of liver transglutaminase to des-A-fibrinogen. The association between liver transglutaminase and fibrin(ogen) was also critically controlled by the conformational states of the two proteins. Substances capable of altering the conformation of either transglutaminase (such as guanosine 5'-triphosphate) or of fibrinogen (such as the tetrapeptide Gly-Pro-Arg-Pro and Fragment D) disrupted binding. Excess CaCl2 was able to counteract the effects of guanosine 5'-triphosphate on transglutaminase binding to fibrin. In contrast, Factor XIII binding to fibrin was unaffected by either guanosine 5'-triphosphate, CaCl2, or Gly-Pro-Arg-Pro, suggesting a more stable association between the two proteins. The physiologic implications of transglutaminase-fibrin(ogen) interactions are discussed.     

Isolation, purification and characterization of bovine epidermal transglutaminase.

A crosslinking enzyme, epidermal transglutaminase, was isolated from soluble proteins of glabrous cow snout epidermis. This enzyme stabilized fibrin clots rendering them insoluble in 2% acetic acid. It also catalyzed the incorporation of the fluorescent amine, dansyl cadaverine, into casein. Epidermal transglutaminase was purified by chromatography upon DEAE-Sephadex A-50, zone electrophoresis in Pevikon, and Sephadex G-200 gel permeation chromatography. The highly purified substance, which had a specific activity of 3267 amine-incorporating units/mg per h and a molecular weight of 55000, behaved as a single molecular species in the analytical ultracentrifuge. It had a sedimentation coefficient of 4.4 S and migrated as a gamma-globulin at pH 8.6; it displayed anomalous migration in polyacrylamide gels containing sodium dodecyl sulfate. The enzyme was dependent upon free calcium ions and a reduced sulfhydryl group for activity. The apparent Km for dansyl cadaverine was 1.2 - 10(-4) at pH 7.5. Monospecific antiserum to bovine epidermal transglutaminase precipitated with the enzyme in agar. The antiserum prevented fibrin crosslinking but enhanced incorporation of dansyl cadaverine into casein by the enzyme. The epidermal enzyme differed biochemically and immunochemically from bovine plasma transglutaminase (Factor XIII).     

Regulation of respiration and energy transduction in cytochrome c oxidase isozymes by allosteric effectors

The binding of TNP-ATP (2 or 3-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant K_d = 0.2 µM) and one with lower affinity (K_d = 0.9 µM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (K_d = 3.6 µM). The binding of [³⁵S]ATPaS was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (K_d = 7.5 µM) and six at the liver enzyme (K_d = 12 µM). The Paracoccus enzyme had only one binding site (K_d = 16 µM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H⁺/e^- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H⁺/e^- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H⁺/e^- stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature. (Mol Cell Biochem 174: 131–135, 1997)     

Binding and regulatory properties of phosphofructokinase from swine kidney

Summary The influence of fructose 2,6-bisphosphate on the activation of purified swine kidney phosphofructokinase as a function of the concentration of fructose 6P, ATP and citrate was investigated. The purified enzyme was nearly completely inhibited in the presence of 2 mM ATP. The addition of 20 nM fructose 2,6-P₂ reversed the inhibition and restored more than 80% of the activity. In the absence of fructose 2,6-P₂ the reaction showed a sigmoidal dependence on fructose 6-phosphate. The addition of 10 nM fructose 2,6-bisphosphate decreased the K_0.5 for fructose 6-phosphate from 3 mM to 0.4 mM in the presence of 1.5 mM ATP. These results clearly show that fructose 2,6-bisphosphate increases the affinity of the enzyme for fructose 6-phosphate and decreases the inhibitory effect of ATP. The extent of inhibition by citrate was also significantly decreased in the presence of fructose 2,6-phosphate.The influence of various effectors of phosphofructokinase on the binding of ATP and fructose 6-P to the enzyme was examined in gel filtration studies. It was found that kidney phosphofructokinase binds 5.6 moles of fructose 6-P per mole of enzyme, which corresponds to about one site per subunit of tetrameric enzyme. The K_D for fructose 6-P was 13 µM and in the presence of 0.5 mM ATP it increased to 27 µM. The addition of 0.3 mM citrate also increased the K_D for fructose 6-P to about 40 µM. AMP, 10 µM, decreased the K_D to 5 µM and the addition of fructose 2,6-phosphate decreased the K_D for fructose 6-P to 0.9 µM. The addition of these compounds did not effect the maximal amount of fructose 6-P bound to the enzyme, which indicated that the binding site for these compounds might be near, but was not identical to the fructose 6-P binding site. The enzyme bound a maximum of about 12.5 moles of ATP per mole, which corresponds to 3 moles per subunit. The K_D of the site with the highest affinity for ATP was 4 µM, and it increased to 15 µM in the presence of fructose 2,6-bisphosphate. The addition of 50 µM fructose 1,6-bisphosphate increased the K_D for ATP to 5.9 µM. AMP increased the K_D to 5.9 µM whereas 0.3 mM citrate decreased the K_D for ATP to about 2 µM. The K_D for AMP, was 2.0 µM; the K_D for cyclic AMP was 1.0 µM; the K_D for ADP was 0.9 µM; the K_D for fructose 1,6-bisphosphate was 0.5 µM; the K_D for citrate was 0.4 µM and the K_D for fructose 2,6-bisphosphate was about 0.1 µM. A maximum of about 4 moles of AMP, ADP and cyclic AMP and fructose 2,6-bisphosphate were bound per mole of enzyme. Taken collectively, these and previous studies (9) indicate that fructose 2,6-phosphate is a very effective activator of swine kidney phosphofructokinase. This effector binds to the enzyme with a very high affinity, and significantly decreases the binding of ATP at the inhibitory site on the enzyme.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent protein kinase activity in rat renal cortex cytosol

The effect of regucalcin on Ca²⁺/calmodulin-dependent protein kinase activity in the cytosol of rat renal cortex was investigated. Regucalcin is a calcium-binding protein which exists in rat liver and renal cortex. Protein kinase activity in renal cortex cytosol was markedly increased by the addition of CaCl₂ (0.5 mM) plus calmodulin (10 µg/ml) in the enzyme reaction mixture. This increase was completely prevented by the addition of trifluoperazine (25 µM), an antagonist of calmodulin. The cytosolic Ca²⁺/calmodulin- dependent protein kinase activity was clearly inhibited by the addition of regucalcin; an appreciable effect of regucalcin was seen at 0.01 µM. The cytosolic Ca²⁺/calmodulin-dependent protein kinase activity was fairly increased by increasing concentrations of added Ca²⁺ (100-1000 µM). This increase was markedly blocked by the presence of regucalcin (0.1 µM). The inhibitory effect of regucalcin on the protein kinase activity was also seen with varying concentrations of calmodulin (2-20 µg/ml). These results demonstrate that regucalcin can regulate Ca²⁺/calmodulin-dependent protein kinase activity in renal cortex cells.     

Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence

Metals are thought to play a role in the structure of many viruses. The crystal structure of the T=3 icosahedral cowpea chlorotic mottle virus (CCMV) suggests the presence of 180 unique metal-binding sites in the assembled protein cage. Each of these sites is thought to involve the coordination of the metal by five amino acids contributed from two adjacent coat protein subunits. We have used fluorescence resonance energy transfer (FRET), from tryptophan residues proximal to the putative metal-binding sites, to probe Tb(III) binding to the virus. Binding of Tb(III) was investigated on the wild-type virus and a mutant where the RNA binding ability of the virus was removed. Tb(III) binding was observed both in the wild-type virus (K_d=19 M) and the mutant (K_d=17 M), as monitored by the increase in Tb(III) fluorescence (545 nm) and concomitant decrease in tryptophan fluorescence (342 nm). Competitive binding experiments showed Ca(II) to have about 100-fold less affinity for the binding sites (K_d=1.97 mM). This is the first direct evidence of metal binding to the putative metal-binding sites, originally suggested from the crystal structure of CCMV.     

Embryopathic and embryocidal effects of purified fumonisin B1 orFusarium proliferatum culture material extract on chicken embryos

One hundred eight fertile eggs (Columbia × New Hampshire) were assigned to 10 groups of 10 eggs each (2 control groups had 14 eggs each). Five groups of eggs were inoculated on day 1 of incubation, while the other 5 groups were inoculated on day 10. The inoculum of the 4 treatment groups on both day 1 and 10 consisted of 1,10, or 100 µM purified fumonisin B₁ (FB₁) or a culture material extract (CME) ofFusarium proliferatum, having known amounts of FB₁, FB₂ and moniliformin (FB₁ 20 µM; FB₂ 4 µM and moniliformin 7 µM). Inoculum consisted of the respective toxin(s) dissolved in 100 µl double distilled, autoclaved water (diluent). Control eggs were inoculated with diluent only. Mortality was both dose- and time-responsive in all treatments. Eggs inoculated on day 1 with 1 µM FB₁ had 50% mortality; 10 µM FB₁ had 70% mortality; 100 µM FB₁ had 100% mortality; and CME had 100% mortality. Eggs inoculated on day 10 with 1,10 or 100 µM FB₁ or CME had 30, 60, 90 and 80% mortality, respectively. Normal chicks were hatched from all control eggs. The median death times (MDT₅₀) were inversely dose-responsive in all treatments, ranging from 3.0 to 7.4 days in embryos exposed on day 1 and from 3.2 to 9.0 days in those exposed on day 10. Early embryonic changes in exposed embryos included hydrocephalus, enlarged beaks and elongated necks. Pathologic changes were noted in liver, kidneys, heart, lungs, musculoskeletal system, intestines, testes and brain toxin-exposed embryos.     

Conversion of myelin-associated glycoprotein (MAG) to a smaller derivative by calcium activated neutral protease (CANP)-like enzyme in myelin and inhibition by E-64 analogue

Using the immunoblot technique, we found that an incubation of purified human myelin in 10 mM Tris-HCl buffer at pH 7.5 resulted in the conversion of the myelinassociated glycoprotein (MAG) to a smaller derivative (dMAG). Exogenously added 5 mM CaCl₂ accelerated the conversion of MAG. In buffer containing more than 100 M of EGTA, the conversion was inhibited. In addition, the existence of endogenous calcium in purified myelin was confirmed using atomic absorption spectroscopy. The conversion was also inhibited partially by one of the thiol protease inhibitors, E-64 analogue (E-64-a). These observations suggest that the conversion of MAG is mediated by calcium-activated neutral protease (CANP)-like enzyme.     

Purification of a 107 kilodalton (kDa) casein kinase G substrate from thyroid cytosol

Summary A 107 kDa (pp107) casein kinase G (ck-G) substrate has been purified from mouse and beef thyroid cytosol; ck-G was purified from beef thyroid cytosol. Ck-G and pp107 were found to co-elute on DEAE cellulose chromatography at approximately 300 mM NaCl. Ck-G and pp107 were separated by spermine-agarose affinity chromatography; ppl07 is eluted with a stepped gradient at 250 mM NaCl and ck-G is eluted at 500 mM NaCl. Ck-G was subsequently purified by casein-agarose and GTP-agarose affinity chromatography. The 107 kDa protein was purified using heparin-agarose affinity chromatography. Phosphorylation of purified pp107 by ck-G was stimulated by spermine (ED₅₀ = 0.2 mM) and inhibited by low concentrations of heparin (0.1–5 µg/ml). The Km and Vmax for the reaction were 1.46 µM and 32.2 nmoles P transferred/20 min/mg protein, respectively; 1 mole pp107 incorporated 0.81 mole phosphorus. pp107 was found to be an acidic substrate with a pI of 3.87 and was absorbed to wheat-germ agglutinin-agarose. The specificity of pp107 phosphorylation was studied using diacylglycerol-activated calcium/phospholipid-dependent protein kinase C, calcium-activated calmodulin-dependent protein kinase, and the catalytic subunit of cAMP-dependent protein kinase A. Phosphorylation of pp107 by the other protein kinases tested never exceeded 4% of that of ck-G. Our data show that pp107 is an acidic glycoprotein which may serve as a high-affinity and specific substrate for ck-G.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis - ck-G Casein Kinase G (casein kinase II) - PK-C Diacylglycerol-Activated Calcium/phospholipid-Dependent Protein Kinase - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - EGTA Ethylene Glycol Bis (B-aminoethylether)-N,N,N,N-tetraacetic Acid - PMSF Phenylmethyl Sulfonyl Floride - TCA Trichloroacetic Acid     

Equilibria in the fibrinogen-fibrin conversion. IX. Effects of calcium ions on the reversible polymerization of fibrin monomer

An investigation was made of the role of calcium ions in the reversible stage of fibrin polymerization, using a direct and relatively simple approach. Purified fibrin monomer in solution (7.5 mg/ml) in 1.0 m NaBr (pH 5.3) was polymerized by raising the pH to 5.7–7.7 by the addition of aliquots of standard NaOH solution and the rate and total extent of proton release during polymerization were measured potentiometrically. In the presence of added CaCl₂ (10⁻⁵-10⁻²m) the rate of proton release was increased and the clotting time was decreased. The profile of equilibrium proton release vs pH of polymerization was also shifted, the maximum being increased and occurring at a lower pH. Sedimentation velocity studies in the intermediate pH range (5.7–6.0) showed that the altered profile of equilibrium proton release was due to a broadening of the pH range of polymerization, and that polymerization remained reversible in the presence of CaCl₂. At pH 5.3, where fibrin is essentially monomeric, addition of CaCl₂ resulted in the release of protons and small increases in sedimentation coefficient and reduced viscosity. Under the same conditions, a similar release of protons was observed from fibrinogen, but there was no effect on its sedimentation coefficient. It was concluded that the proton release at pH 5.3 was due mainly to binding of calcium ions to fibrinogen and fibrin monomer. The effect of CaCl₂ on the sedimentation coefficient of fibrin at pH 5.3 was found to decrease with decreasing protein concentration, indicating that it was the result of a small extent of polymerization, rather than a conformational change. Added MgCl₂ had no effect on fibrin monomer at pH 5.3 and no significant effect on the rate or extent of proton release during polymerization at higher pH, indicating that there are specific binding sites for calcium ions in fibrinogen and fibrin. The observed effects of bound calcium ions on reversible fibrin polymerization are explained most simply in electrostatic terms.     

Novel fibrinolytic enzymes from Virgibacillus halodenitrificans SK1-3-7 isolated from fish sauce fermentation

Virgibacillus sp. SK1-3-7 exhibited the highest fibrinolytic activity among 25 bacterial isolates obtained from fish sauce fermentation. Results of 16S rRNA gene sequence analysis showed 99% homology to Virgibacillus halodenitrificans ATCC 49067. It was, therefore, identified as V. halodenitrificans SK1-3-7. Fibrinolytic enzymes from V. halodenitrificans SK1-3-7 were partially purified using ammonium sulfate fractionation, hydrophobic and ion-exchange chromatographies. The enzymes with molecular weight of 20- and 36-kDa showed fibrinolytic activity on a fibrin zymogram. The enzymes were stable between pH 4 and 10 and below 60 °C. The enzymes were activated by 20 mM CaCl₂ and 0.15 M NaCl. The activity increased with CaCl₂ up to 100 mM and increased with NaCl concentration up to 2 M. In addition, the residual fibrinolytic activity of 61% was found at 4 M NaCl. The enzymes were completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-pNA, suggesting a subtilisin-like serine proteinase. V. halodenitrificans SK1-3-7 enzymes hydrolyzed fibrin to a greater extent than did plasmin. In addition, the enzymes were resistant to pepsin and trypsin digestion. The de novo peptide homology analysis of a 20- and 36-kDa proteinase revealed no matches to bacilli serine proteinases, suggesting that both were novel fibrinolytic enzymes.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

Production and purification of a calcium-dependent protease from Bacillus cereus BG1

The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg²⁺ and Ba²⁺, which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L^–1) starch 5, CaCl₂ 2, yeast extract 2, K₂HPO₄ 0.2 and KH₂PO₄ 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60°C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl₂.