Untreated Enzyme Research Articles

Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide.

Anandamide (arachidonylethanolamide) is known as an endogenous agonist for cannabinoid receptors. An amidohydrolase, which hydrolyzed anandamide, was solubilized from the microsomal fraction of porcine brain with 1% Triton X-100. The enzyme was partially purified by Phenyl-5PW hydrophobic chromatography to a specific activity of approximately 0.37 mumol/min/mg of protein at 37 degrees C. As assayed with 14C-labeled substrates, the apparent Km value for anandamide was 60 microM, and anandamide was more active than ethanolamides of linoleic, oleic, and palmitic acids. Ceramidase and protease activities were not detected in our enzyme preparation. The purified enzyme also synthesized anandamide from free arachidonic acid in the presence of a high concentration of ethanolamine with a specific activity of about 0.16 mumol/min/mg of protein at 37 degrees C. On the basis of cochromatographies, pH dependence, heat inactivation, and effects of inhibitors such as arachidonyl trifluoromethyl ketone, p-chloromercuribenzoic acid, diisopropyl fluorophosphate, and phenylmethylsulfonyl fluoride, it was suggested that the anandamide amidohydrolase and synthase activities were attributable to a single enzyme protein.

Changes in the Iron Coordination Sphere of Fe(II) Lipoxygenase-1 from Soybeans upon Binding of Linoleate or Oleate

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.

Changes in the iron coordination sphere of Fe(II) lipoxygenase-1 from soybeans upon binding of linoleate or oleate.

Fe K-edge X-ray absorption spectra of the non-heme iron constituent of lipoxygenase-1 from soybeans were obtained. The spectrum of 2.5 mM Fe(II) lipoxygenase, mixed with 1.2 M linoleate in the absence of O2, was compared to the spectrum of the native (i.e. untreated) enzyme. In the lipoxygenase-linoleate complex, an edge shift to lower energy was observed. This indicated that the iron-ligand distances in this complex are slightly longer than those in the untreated enzyme species. The extended X-ray absorption fine structure spectrum of Fe(II) lipoxygenase, prepared by anaerobic reduction of 2.5 mM Fe(II) lipoxygenase with 1.2 M linoleate, was very similar to the spectrum of the anaerobic lipoxygenase-linoleate complex. We conclude that the conformational differences between the iron coordination spheres of native and cycled Fe(II) lipoxygenase must be ascribed to the presence of linoleate, and not to changes in the enzyme that occur only after one cycle of oxidation and reduction. Furthermore, spectra of 2.5 mM Fe(II) lipoxygenase mixed with 1.2 M oleate, either in the absence or in the presence of O2, were also identical to the spectrum of the Fe(II) lipoxygenase-linoleate complex. This finding is in agreement with our observation that oleate is a competitive inhibitor of the lipoxygenase reaction. Moreover, the similarity of the lipoxygenase-oleate complexes in the presence and absence of O2 excludes the possibility that O2 binding to the iron cofactor is induced upon binding of a fatty acid to lipoxygenase.

Glomerular dynamics and morphology of aged spontaneously hypertensive rats. Effects of angiotensin-converting enzyme inhibition.

Relationships between glomerular dynamics and renal injury, micropuncture and histological studies were assessed in 73 week-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats divided into untreated and angiotension-converting enzyme inhibitor-treated (quinapril; 3 mg/kg/day; for 3 weeks) groups. Urinary protein excretion (UPE) and histologic arteriolar (AIS) and glomerular (GIS) injury scores were determined. Mean arterial pressure (MAP) of untreated SHR was increased compared with WKY (200 +/- 6 vs 119 +/- 4 mm Hg; P < 0.01), effective renal plasma flow (ERPF) was reduced (1.47 +/- 0.21 vs 3.06 +/- 0.26 ml/min/per g; P > 0.01), and filtration fraction (FF) and total renal vascular resistance (RVR) of SHR were increased (P < 0.01). Single-nephron plasma flow (SNPF) of untreated SHR was decreased (174 +/- 17 vs 80 +/- 9 ml/min; P < 0.01), and single-nephron filtration fraction and afferent arteriolar resistance (RA) were increased (19.4 +/- 1.8 vs 30.0 +/- 2.5% and 1.90 +/- 0.25 vs 9.05 +/- 1.35 U, respectively; both P < 0.01). Despite reduced SNPF, glomerular capillary pressure (PG) increased (49.7 +/- 0.7 vs 53.8 +/- 1.3 mm Hg; P < 0.05), the result of efferent arteriolar constriction (1.15 +/- 0.18 vs 2.84 +/- 0.36 U; P < 0.01). Untreated SHR had higher UPE (13.9 +/- 1.5 vs 42.8 +/- 3.2; mg/100 g per day; P < 0.01) and GIS and AIS scores than WKY (4.3 +/- 1.1 vs 64.3 +/- 8.4 and 16.6 +/- 3.1 vs 96.3 +/- 14.4; both P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Mutants of d-aminopeptidase with increased thermal stability

Mutant d-aminopeptidases from Ochrobactrum anthropi with increased thermal stability were obtained by random mutagenesis. One of the mutants, no. 65, was derived from E. coli cells transformed with DNA treated with sodium nitrite. The remaining activity of the purified mutant ezyme no. 65 after heat treatment at 52°C for 10 min was 20% that of the untreated mutant enzyme no. 65, whereas the native enzyme showed 5% of the untreated native enzyme activity after the same treatment. The gene for the mutant enzyme no. 65 was sequenced and it was found that Gly155 and Gly279 in the native enzyme were replaced by Ser and Asp, respectively. Five mutants carrying one or two mutations were generated from the native gene by site-specific mutagenesis. The enhancement of the thermal stability of mutant enzyme no. 65 was attributed to the substitution of Gly155 to Ser.

Divalent Cations and Chelators as Regulators of Brain Fructose-1,6-bisphosphatase

Purified fish and rat brain FruP2ase(s) are stimulated by a number of chelators, viz., histidine, EDTA, citrate, imidazole, and a number of histidine analogues. These also impart 5′-AMP sensitivity to the otherwise insensitive enzyme. Beyond 3 mM concentration, histidine inhibits the enzyme activity, which can be prevented by Mn2+. Atomic absorption spectrophotometry showed the presence of 5-6 mol of Mn2+ and Zn2+ bound to both fish and rat brain FruP2ase, which can be removed by exhaustive EDTA-dialysis. The EDTA-dialyzed brain FruP2ase records an absolute Mn2+ requirement and 5′-AMP sensitivity without any chelator treatment. The 5′-AMP sensitivity of such enzyme is abolished by prior incubation with Zn2+. The Zn2+-treated brain FruP2ase fails to bind to a Blue-Sepharose column, in contrast to that seen using the untreated enzyme. These results suggest that rat and fish brain FruP2ase(s) are actually Mn2+- and Zn2+-containing proteins with Zn2+ bound at or near the nucleotide-binding site.

The role of eosinophil cationic protein (ECP) and histamine in vernal keratoconjunctivitis

(1) to measure levels of tear eosinophil cationic protein (ECP) in patients with vernal keratoconjunctivitis (VKC); (2) to verify any correlation between ECP and the severity of the disease; (3) to verify the effect of different topical therapies on the levels of tear ECP; (4) to investigate the activity of histamine degrading enzymes in tears and plasma of VKC patients. ECP was measured by RIA in tears from 23 VKC patients before and after therapy, and from seven controls. Tear cytology, ocular signs and symptoms were also evaluated before and after treatment. Histamine was measured by ELISA in both plasma and tears from 19 VKC patients and six age-matched controls, in both acid-treated (no enzyme activity) and untreated (enzyme activity) samples. (1) tear ECP levels were significantly higher in VKC patients than in healthy controls; (2) tear ECP levels showed a significant correlation to signs (p <0.005) and symptoms (p <0.001) of the disease; (3) tear ECP was reduced by both 2% cyclosporine and 0.1% dexamethasone; (4) tear and plasma histaminase activity, measured by the ratio of histamine in treated to untreated samples, were significantly lower in VKC patients compared to controls (p <0.0001 and p <0.001, respectively). Tear ECP levels reflect the clinical status of VKC patients. The measurement of ECP may prove to be a useful objective tool in evaluating allergic conjunctivitis and the effectiveness of specific treatments. Moreover, VKC patients show a significantly impaired histaminase capability, at both the local and systemic level. This dysfunction may exacerbate the effects of local histamine release in the course of the disease.

Prevention and induction of oxidative damage in E. coli cells by cationized proteins

The successful prevention of oxidative damage in E. coli B cells by cationized catalase (cCAT), and the induction of oxidative stress by cationized glucose oxidase (cGO) and cationized superoxide dismutase (cSOD) is presented in this study. Exposure of E. coli cells to hydrogen peroxide and hydroxyl radical resulted in a rapid killing of the cells. Measurements of biochemical markers: cellular potassium levels and uptake and accumulation of leucin indicated membrane damage in some of the oxidants employed. Following incubation with native CAT or SOD, the cells were washed and exposed to oxidative stress. The results of this procedure did not protect the cells against the oxidative damage. In contrast, incubation of the cells with pretreated CAT with poly- l-histidine, followed by washing of the cells and the subsequent introduction of oxidative stress inducers, resulted in a pronounced protection of the cells against the oxidative stress. Employment of pretreated SOD, and exposure, after washing the cells, to oxidative stress, resulted in an enhancement of the oxidative damage in some cases. Exposure of the cells to cGO resulted in a marked killing of the cells as compared to the untreated enzyme. The use of E. coli cells as a model system for studying the effect of cationized enzymes on cell surfaces is discussed.

Purification and characterization of an aminopeptidase from tuna ( Thunnus albacares) pyloric caeca

An aminopeptidase was purified from a water soluble fraction of tuna pyloric caeca by heat treatment, Zn 2+ fractionation, ion exchange on a DEAE cellulose column, gel filtration on Fractogel TSK-55, and immobilized metal ion affinity chromatography (IMAC) on IDA(Cu 2+/Zn 2+)-Sepharose 6B. The molecular mass of the enzyme was estimated to be 150 000 on Sephacyl S-300 HR, and was found to be near 72 000 by SDS-PAGE. The aminopeptidase, which is a glycoprotein rich in acidic amino acids, is optimally active at pH 8.8 and 65°C. The enzyme activity was not affected by Mg 2+, Zn 2+, Ca 2+, Mn 2+, Co 2+, PMSF, iPr 2 FP, 4-hydroxymercuribenzoic acid, iodoacetamide, puromycin, and cysteine but it was strongly inhibited by metal chelators (EDTA and o-phenanthroline), amastatin, Hg 2+, Cd 2+, and Cu 2+. The enzyme was also inhibited by some l-amino acids. Kinetic parameters of the enzyme were determined with some aminoacyl- p-nitroanilides and aminoacyl- β-naphthylamides. l-Alanine -p- nitroanilide and l-alanine-β-naphthylamide were hydrolyzed most rapidly while the highest hydrolytic coefficient ( k cat/ K m) value was obtained with l-methionine -p- nitroanilide . The apoaminopeptidase was prepared and reconstitution of an active enzyme was carried out using metal chelating interaction chromatography on an IDA-Sepharose 6B column charged with a metal ion. Full activity was restored with Zn 2+, Co 2+, Cu 2+ and Al 3+. Zn 2+-Enzyme was the most thermostable form of the aminopeptidase. Reversal inhibition by Cu 2+ and Cd 2+ was also examined. When the aminopeptidase was partially deglycosylated by a treatment with N-glycoside F some of its physical properties differed from that of the native enzyme: its electrophoretic mobility was reduced and its stability to denaturation by SDS and by ionic strength were lower than those of the untreated enzyme. All together, our results indicate that the tuna pyloric caeca aminopeptidase is distinct from the peptide hydrolases characterized in the literature.

The role of the carbohydrate chains of Galβ-1,4-GlcNAcα2,6-sialyltransferase for enzyme eactivity

Galβ-1,4-GlcNAcα2,6-sialyltransferase (CMP- N-acetylneuraminate : β-galactoside α2,6 sialyltransferase, EC 2.4.99.1) is a glycoprotein containing carbohydrate chains of the complex type (Jamieson, J.C. (1989) Life Sci. 43, 691–697). The carbohydrate chains may be important for controlling the expression of sialyltransferase catalytic activity during transit of the enzyme from the rough endoplasmic reticulum to the Golgi complex where it is active as a membrane bound enzyme anchored to the luminal face. To study the role of the carbohydrate chains of sialyltransferase for enzyme activity, conditions were established in which the native enzyme was deglycosylated with N-Glycanase and endo F. It was found that Glycanase removed the carbohydrate chains from native sialyltransferase, but methanol had to be present for rapid and complete deglycosylation. Presence of methanol or ethanol were not essential for removal of carbohydrate chains with endo F. There was a correlation between the loss of catalytic activity of sialyltransferase with increased deglycosylation. After deglycosylation with Glycanase for 18 h catalytic activity was largely eliminated and there was a reduction in molecular mass of about 5 kDa compared to the untreated enzyme when examined by immunoblot analysis; this reduction was identical to that found when the denatured enzyme was deglycosylated with Glycanase. At shorter times of incubation partially deglycosylated forms of the enzyme were detected. Complete deglycosylation of native or denatured sialyltransferase with endo F could not be achieved. However, incubation with endo F for 24 h resulted in a loss of catalytic activity of about 60%. Immunoblot analysis showed the presence of three forms of the enzyme corresponding in molecular mass to the native and deglycosylated enzyme and a third form corresponding to a partially deglycosylated enzyme. Sialyltransferase was also subjected to sequential treatment with exoglycosidases. Removal of NeuAc and Gal had little effect on catalytic activity, but subsequent removal of GlcNAc resulted in a significant loss in catalytic activity suggesting that the presence of the trimannose core with GlcNAc attached is important for the expression of catalytic activity. The presence of organic solvents during deglycosylation with Glycanase may be a useful method that can be applied to other glycoproteins.

The manganese center of oxygen-evolving and calcium-depleted photosystem II: a pulsed EPR spectroscopy study

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

Cytokine synthesis in human peripheral blood mononuclear cells after oral administration of polyenzyme preparations.

Pharmaceutical preparations containing mixtures of various proteolytic and nonproteolytic enzymes have been suggested for use in the treatment of malignant diseases. However, the mode of action of such preparations was not clear. We have shown before that intact bromelain, papain or amylase, which are components of a commercial polyenzyme preparation, induce cytokine production in peripheral blood mononuclear cells in vitro. IFN-alpha and IFN-gamma which had no effect alone, synergistically increased TNF production when applied together with the enzymes. Here we show that trypsin alone had only a small inducing effect. The tryptic but not the autolytic fragments of papain and bromelain have a higher (10- to 40-fold) inducing capacity for TNF production than the untreated enzyme. Additionally we demonstrate that after ingestion of milligram doses of the polyenzyme preparation (as recommended for clinical use), PBMNC of healthy donors acquire the ability to produce TNF-alpha, IL-1 beta and IL-6 when incubated ex vivo with IFN-gamma. Our results indicate that the biological effects observed after oral administration of polyenzyme preparations are related to their ability to induce cytokine production. This may explain the antitumor effects of such enzymes. Our results also suggest that polyenzyme preparations may have a stronger immunomodulary effect when used in combination with IFN-gamma.

The effect of amino acid substitution at position 219 of Citrobacter freundii cephalosporinase on extension of its substrate spectrum.

The cephalosporinase of Citrobacter freundii GN346 is a class-C beta-lactamase comprising 361 amino acids. The substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate specificity to unfavorable substrates such as oxyimino cephalosporins [Tsukamoto, K., Ohno, R. & Sawai, T. (1990) J. Bacteriol. 172, 4348-4351]. To investigate the cause of this phenomenon, Glu219 was changed to glutamine, cysteine or tryptophan. All the resultant enzymes showed higher cefuroxime-hydrolytic activities than the wild type, the order of increasing cefuroxime-hydrolytic activity being as follows: Trp greater than Lys greater than Cys greater than Gln greater than Glu. The rate of hydrolysis of cefuroxime by the Trp219 enzyme was approximately 3 x 10(4) times that of the wild-type enzyme. The order of increasing cefuroxime hydrolysis was approximately proportional to the molecular volume of the amino acid substituted and independent of the ionic character of the amino acids. The cysteine residue at position 219 in the Cys219 enzyme allowed its complete reaction with an SH-blocking reagent, 4-chloromercuriphenylsulfonic acid. The modified enzyme with the bulkier residue showed a 45% higher cefuroxime-hydrolytic activity than the untreated enzyme. These results suggested that extension of the substrate spectrum may be attributed to alteration in the configuration of the enzyme around position 219.

2,4,5-Trichlorophenoxyacetic acid influence on 2,6-dinitrotoluene-induced urine genotoxicity in Fischer 344 rats: Effect on gastrointestinal microflora and enzyme activity

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) and 2,6-dinitrotoluene (2,6-DNT) are hazardous chemicals that have potential harmful effects. 2,6-DNT is recognized as a hepatotoxicant while 2,4,5-T, a component of Agent Orange, is also suspect. 2,6-DNT requires both oxidative and reductive metabolism to elicit genotoxic effects. To determine what effect 2,4,5-T had on 2,6-DNT metabolism, intestinal enzymes, microbial populations, and urine mutagenicity were examined during 2,4,5-T treatment. Weanling Fischer 344 male rats were treated daily with 54.4 mg/kg 2,4,5-T by gavage for 4 weeks. One, two, and four weeks after the initial 2,4,5-T dose, rats were administered (po) 2,6-DNT (75 mg/kg) and urine was collected for 24 hr in metabolism cages. Azo reductase, nitroreductase, β-glucuronidase, dechlorinase, and dehydrochlorinase activities were examined concurrently. Treatment of rats for 1 week reduced the transformation of 2,6-DNT to mutagenic urinary metabolites. This was accompanied by a decrease in the fecal anaerobic microorganisms. The elimination of Lactobacillus fermentum from the small intestine and cecum of treated animals accompanied a significant increase in oxygen-tolerant lactobacilli and other unidentified aerobic microorganisms. However, there were no significant alterations in the intestinal enzyme activities examined. By 2 weeks of 2,4,5-T treatment, microbiota and urine genotoxicity returned to the levels observed in control animals. This trend continued for the duration of the experiment. After 2 weeks, while cecal nitroreductase and azo reductase activities increased, small intestinal β-glucuronidase activity decreased. By 4 weeks, treated and untreated animal intestinal enzyme activities were indistinguishable. The transient increase in azo reductase and nitroreductase after treatment with 2,4,5-T for 2 weeks may have been counteracted by the reduced β-glucuronidase activity, thus resulting in no change in 2,6-DNT-derived urine mutagenicity. However, other enviromental chemicals, unaffectd by β-glucuronidase, potentially could be activated by 2,4,5-T exposure.

Bovine pancreatic asparagine synthetase explored with substrate analogs and specific monoclonal antibodies

Several substrate analogs were tested for their ability to inhibit bovine pancreatic asparagine synthetase. Of the substrate analogs tested both 6-diazo-5-oxo- l-norleucine (DON) and 5-chloro-4-oxo- l-norvaline (CONV) were shown to inhibit the enzyme strongly. DON inhibited the glutaminase and glutamine-dependent asparagine synthetase activities and CONV inhibited the ammonia-dependent activity as well. Both of these inhibitors appeared to be relatively tight binding since desalting failed to remove the inhibition. The inactivation of bovine pancreatic asparagine synthetase by DON is accompanied by a shift from a 47,000 molecular weight monomer to a 96,000 molecular weight dimer as observed by HPLC gel filtration chromatography. This DON-induced shift is prevented by the presence of the substrate glutamine. A monoclonal antibody known to inhibit specifically the ammonia-dependent and glutamine-dependent asparagine synthetase activities but not glutaminase (monoclonal antibody 2B4) binds to both the monomer and the dimer forms of untreated enzyme, as well as to the dimer form of the DON-inactivated enzyme. On the other hand, a monoclonal antibody known to inhibit specifically the glutaminase and glutamine-dependent activities and not the ammonia-dependent asparagine synthetase (monoclonal antibody 5A6) binds to both forms of untreated enzyme but cannot bind to the DON-inactivated enzyme. These data are used to describe the relation of regions of the active site of asparagine synthetase in relation to antibody binding sites.

Effect of dipicolinate, a chelator of zinc, on bone protein synthesis in tissue culture: The essential role of zinc

The present investigation was undertaken to clarify the essential role of zinc on bone protein synthesis in tissue culture. Calvariae were removed from 3-week-old male rats and cultured for periods up to 72 hr in Dulbecco's Modified Eagle Medium (high glucose, 4500 mg/dl) supplemented with antibiotics and bovine serum albumin. The calvariae were incubated for 24 hr at 37° in 5% CO 2/95% air in medium containing 10 −6–10 −3 M diplcolinate, a chelator of zinc, and then the bones were transferred into medium containing either 10 −4 M zinc sulfate or vehicle without diplcolinate. Zinc content in bone tissues was decreased when the culture was treated with 10 −4 and 10 −3 M dipieolinate for 24 hr. When calvariae treated with 10 −4 M dipicolinate for 24 hr were further cultured in medium without dipicolinate for 24 and 48 hr, bone alkaline phosphatase activity was decreased by about 40% (P < 0.01) of untreated bone enzyme activity. The decreased alkaline phosphatase activity was increased markedly by the presence of 10 −4 M zinc (about 2.5-fold of control value). This effect of zinc was blocked completely by the presence of 10 −7 M cycloheximide, but 10 −8 M actinomycin D caused only a partial inhibition. When calvariae treated with 10 −4 M dipicolinate were pulsed with [ 3H]proline, the incorporation of [ 3H]proline into the acid-insoluble residues of bone tissue was decreased by about 40% (P < 0.01) of the value obtained from calvariae not treated with dipieolinate. The presence of 10 −4 M zinc caused an increase of about 2-fold in [ 3H]proline incorporation. Bone DNA content was not altered significantly by treatment with 10 −4 M dipicolinate or 10 −4 M zinc. These results clearly indicate that endogenous zinc induces the stimulation of protein synthesis at the translational process in bone cells. The present study further supports the view that zinc plays an essential role for protein synthesis in bone cells.

Isolation, purification, and properties of Penicillium charlesii alkaline protease.

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.

Shift to the Na+ from of Na+/K+‐transporting ATPase due to modification of the low‐affinity ATP‐binding site by Co(NH3)4ATP

1. Inactivation of purified Na+/K+-transporting ATPase by the MgATP complex analogue Co(NH3)4ATP, which binds to the low-affinity ATP-binding site, results in the concomitant inhibition of the K+-activated p-nitrophenylphosphatase, which is considered to be a partial reaction catalyzed by the enzyme in the E2 conformational state. 2. Complete inactivation of Na+/K+-transporting ATPase by Co(NH3)4ATP does not alter the ADP/ATP exchange reaction which is considered to be part of the catalytic activity in the E1 conformation. 3. The enzyme binds eosin at the high-affinity ATP-binding site as measured by the change in eosin fluorescence. Eosin binding to the Co(NH3)4ATP-inactivated enzyme is, in contrast to the untreated enzyme, not stimulated by Na1. Inactivation by Co(NH3)4ATP increased the half-maximal opposing effect of K+ on eosin binding from 1.1 mM in the control to 43.2 mM in the almost completely inactive enzyme. No eosin fluorescence changes were observed when the Co(NH3)4ATP-inactivated enzyme was treated subsequently with CrATP. This MgATP complex analogue forms a stable complex at the high-affinity ATP-binding site. CrATP thus abolishes eosin binding. 4. It is concluded, that Co(NH3)4ATP interacts with Na+/K+-transporting ATPase in the E2 conformation and arrests it there. This affects eosin binding to the high-affinity ATP-binding site, since the K+ sensitivity is lost. A possible interpretation of these differing effects of Co(NH3)4ATP on partial reactions of Na+/K+-transporting ATPase is that the sodium pump works as an (alpha,beta)2 diprotomer. It is likely that the arrest of one alpha,beta promoter in the E2 conformational state by occupancy of the low-affinity ATP-binding site with Co(NH3)4ATP induces the Na+ form (E1 form) in the corresponding alpha,beta promoter, as is indicated by the unaffected ADP/ATP exchange and the response of the eosin fluorescence on Na+ and K+.

Activation of rat liver microsomal glutathione transferase by limited proteolysis.

The activity of rat liver microsomal glutathione transferase is increased by limited tryptic proteolysis; the membrane-bound and purified forms of the enzyme are activated about 5- and 10-fold respectively. The cleavage sites that correlate with this activation were determined by amino acid sequence analysis to be located after Lys-4 and Lys-41. Differences in the relative extent of cleavage at these two sites did not consistently affect the degree of activation. Thus the data support the conclusion that cleavage at either site results in activation. The trypsin-activated enzyme was compared with the form activated with N-ethylmaleimide, which modifies Cys-49. These two differently activated forms were found to have similar kinetic parameters, which differ from those of the unactivated enzyme. The relatedness of the two types of activation is also demonstrated by the observation that microsomal glutathione transferase fully activated by N-ethylmaleimide is virtually resistant to further activation by trypsin. This is the case despite the fact that the N-ethylmaleimide-activated enzyme is much more susceptible to trypsin cleavage at Lys-41 than is the untreated enzyme. The latter observation indicates that activation with N-ethylmaleimide is accompanied by a conformational change involving Lys-41.

Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.