摘要:

The effect of purified staphylococcal δ-lysin and bee venom melittin on the permeability of large unilamellar vesicles (liposomes) composed of structurally defined lipids was investigated by following release of sequestered ATP using a bioluminescent assay. ATP release was linearly related to the logarithm of the peptide concentration. Sensitivity to the lytic peptides, as measured by the release of ATP and the slope of the dose–response curve, depended on the chain length and the degree of saturation of the hydrocarbon substituents of the phospholipid in the vesicles. Neither the sign of the surface charge nor the presence of sterol had any significant effect on the sensitivity of liposomes to peptide-induced lysis. Release of ATP and haemoglobin from intact sheep and cod erythrocytes was also determined after their exposure to δ-lysin and melittin. Both cell types showed similar degrees of sensitivity to each lytic peptide when ATP release was followed, but the release of haemoglobin from sheep erythrocytes by δ-lysin was much less than from cod erythrocytes. The implications of these results on the mode of action of the two agents are discussed.

ISSN:0714-7511    

DOI:10.1139/o85-001

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

The divalent cations Mg2+and Mn2+in excess of the concentrations required to complex with ATP (excess Mg2+or Mn2+) modulate the activity of adenylate cyclase. As a substrate, Mn∙ATP was at least as effective as Mg∙ATP in supporting adenylate cyclase activity in white and brown adipose tissue membranes. Both excess Mg2+and Mn2+had quantitatively different effects on the enzyme of lean and ob/ob mice and qualitatively different effects in white and brown adipose tissue. Inwhite adipocyte membranesexcess Mg2+increased basal activity, as well as activity owing to guanylylimidodiphosphate (Gpp(NH)p) (with or without isoproterenol) and NaF. Maximal activation by Gpp(NH)p + isoproterenol required a higher concentration of Mg2+in tissue from ob/ob than lean mice. Excess Mn2+prevented the activation of the enzyme by Gpp(NH)p or Gpp(NH)p + isoproterenol in a dose-dependent manner. Mn2+inhibited even in the presence of maximally effective Mg2+concentrations. The enzyme of the ob/ob mouse membrane required a significantly higher dose of Mn2+to achieve 50% inhibition. Inbrown adipose tissue, specific activities of the isoproterenol + Gpp(NH)p and NaF stimulated enzyme were significantly lower in the obese mice under all conditions studied. Except that NaF-stimulated activity was increased significantly more in the membranes of lean mice by the combination of Mg2++ Mn2+, these cations did not produce significantly different dose-dependent effects in membranes from lean and ob/ob mice. Maximal activation occurred at lower concentrations of MgCl2(3–5 versus 10–20 mM) and required higher concentrations of MnCl2(3–5 versus 1 mM) in brown than in white adipose tissue membranes. Furthermore, Mn2+in excess of the concentration required to activate maximally produced little or no inhibitory effect on the brown adipose tissue enzyme. These studies illustrate the diversity of adenylate cyclase modulation in different tissues. Further studies with bacterial toxins will be necessary to verify whether there are differences in the equilibrium of association of the stimulatory and inhibitory components of guanine nucleotide regulatory proteins in tissues of lean and obese m

ISSN:0714-7511    

DOI:10.1139/o85-002

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

The metallothionein (MT) content of fetal rat liver was measured daily during the final week of gestation, by both cadmium saturation and polarographic methods. MT levels rise sharply at day 18 of gestation and continue to increase into the neonatal period. In late gestation, MT serves to bind Cu and Zn from the preexisting hepatic pools of these metals, as well as to accumulate additional amounts of both metals The fetal MT is similar to the adult rat protein both in terms of its protein composition and metal-binding properties. Perinatally the Zn/MT ratio remains constant for several days suggesting a carefully regulated process. At birth, most of the hepatic Zn and a significant amount of hepatic Cu are bound to MT. Immunohistochemical localization of MT shows a progressive increase in cytoplasmic MT with the appearance of nuclear MT by day 20 of gestation in fetal rat liver. The results are discussed in terms of a model for regulation of MT synthesis and for the metal storage role of MT in perinatal development.

ISSN:0714-7511    

DOI:10.1139/o85-003

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Radioisotopic, pH 6.8 assays were designed to measure hepatic cortisol sulfation in chickens, gerbils, and hamsters of both sexes. Enzyme levels with 40 μMcortisol were similar in males of all three species and due mostly to lowKmenzymes with 10–30 μMcortisolKm's. Maximum enzyme activity in male chickens required 40 μMCortisol. In the other species, the much higher maximum enzyme activity observed required 500 μMcortisol owing to sulfotransferases withKm's for the hormone near 300 μM. Coenzyme 3′-phosphoadenosine-5′-phosphosulfate requirements also varied between species. Sex differences of the enzyme levels were found only in hamsters. There, males possessed only 24–33% of the enzyme levels found in females. Cortisol 21-sulfate was the reaction product in all of the species. Sexual dimorphism in hamsters appeared to be due to repressive effects of androgens. pH optima of enzyme activities in the three species ranged from pH 6 to 7. Routine use of pH 6.8 assays allowed representative interspecies comparisons. DEAE-Sephadex fractionation of cytosol showed that chicken liver contained mostly two enzymes with different pH optima that catalyzed cortisol sulfation. These differed from the enzymes that catalyzed dehydroepiandrosterone and estradiol sulfation. In the gerbil four enzymes with similar pH optima catalyzed cortisol sulfation. The second of these to elute from DEAE-Sephadex columns was the lowKmform. In hamsters most glucocorticoid sulfotransferase activity appeared to be due to one enzyme. The molecular weights of the lowKmgerbil enzyme and the main hamster enzyme were 98 300 ± 6100 and 105 000 ± 8100. Hamsters and gerbils responded to injection of cortisol by hepatic tyrosine aminotransferase induction.

ISSN:0714-7511    

DOI:10.1139/o85-004

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Nonactivated (8.5S) rabbit uterine progestin receptor was enriched 10- to 30-fold by chromatography on columns of spheroidal hydroxylapatite and DEAE-cellulose. A total of`~25 μg of receptor (purity ~1%) was injected at multiple sites into a BALB/c mouse. After several injections, splenic lymphocytes were fused with P3x63Ag8.653 mouse myeloma cells. This fusion produced in excess of 240 hybridomas, which were screened by an enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, and sucrose gradient centrifugation. One colony (KN 382/EC1) produced a mouse immunoglobulin G1which bound rabbit 8.5S uterine progestin receptor. The cell line has been repeatedly cloned under conditions of limiting dilution and expanded in mice as ascitic tumors. Antibody was purified by (NH4)2SO4precipitation, DEAE-cellulose chromatography, and affinity chromatography with protein A – Sepharose CL-4B. Specificity of the antibody was determined by sucrose gradient centrifugation and solid-phase radioimmunoassay. The antibody bound to progestin receptors from rabbit uterus and MCF-7 breast cancer cells. It did not react with progestin receptors from rat uterus, guinea pig uterus, or chick oviduct, nor did it bind to estrogen receptors from any of the tissues we tested.

ISSN:0714-7511    

DOI:10.1139/o85-005

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Sucrose gradient analysis of the binding of our monoclonal antibody (secreted by cell line KN 382/ECl) to [17 α-methyl-3H] R5020-labeled rabbit uterine progestin receptors revealed that the antibody bound only to the 8.5S form (Kd = 0.86 nM) and not to the 7S and smaller complexes.125I-labeled antibody, on the other hand, bound to both the 8.5S complex and a component of dissociated receptor. Calculation of the relative mass (Mr) of the125I-labeled immunoglobulin G1(lgG1) – protein complex indicated the addition of a 60 000Mrpeptide. Electrophoretic analysis of immunoaffinity purified receptor substantiated this by revealing two protein bands. Sequential washing of adsorbed receptor was accompanied by dissociation of the bound steroid and theMr92 000 peptide. Thepeptide could only be completely eluted from the immunoadsorbent under denaturing conditions. Autofluorography of receptor complexes covalently bound with [17α-methyl-3H]R5020 revealed two bands, one with aand the second with a. Upon immunoprecipitation both peptides precipitated with theandpeptides. Gel electrophoresis demonstrated that thepeptide and thedid not comigrate. We have concluded that the nondissociated 8.5S rabbit uterine progestin receptor complex containsandnonsteroid-binding components, in addition to theandsteroid binders and that the antibody preferentially recognizes thenonsteroid-binding peptide.

ISSN:0714-7511    

DOI:10.1139/o85-006

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Controlled proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides that are designated II and IV. After 5 h of proteolysis the incubation mixture containing these peptides retains all of the component enzyme activities of the fatty acid synthetase complex. The two peptides are then separated by chromatography on an Affi-Gel Blue column. Gel filtration of the fraction containing peptide II yields a homogeneous peptide as shown by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of this peptide has been estimated to be 130 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, size exclusion chromatography, and amino acid analysis. The sedimentation coefficient for peptide II is approximately 7.4S. Peptide II contains the domains for the β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase activities of the fatty acid synthetase complex.

ISSN:0714-7511    

DOI:10.1139/o85-007

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Succinyl-CoA synthetase fromEscherichia colihas an α2β2subunit structure and is known to display half-of-the-sites reactivity with respect to its phosphorylation by ATP. The studies reported herein are a component of our attempts to rationalize the heterologous tetrameric structure in terms of catalytic function. The isolated refolded β subunit interacts specifically with an affinity column of agarose–hexane–CoA, consistent with the idea that the CoA-binding subsite of the active center is located on the β subunit. The enzyme is inactivated by phenylglyoxal according to biphasic kinetics; saturating levels of the substrates CoA and ATP, alone or in combination, give only partial protection against such inactivation. Treatment of the enzyme with the sulfhydryl reagent 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) resulted in rapid inactivation, accompanied by the reaction of three to four -SH groups per molecule; prolonged incubation with NBD-Cl eventually results in reaction of 16 of the 24 sulfhydryl groups of the tetramer. Hybrid enzyme preparations have been constructed by refolding mixtures containing β and various ratios of native and NBD-Cl-modified a subunits. The loss of activity associated with the incorporation of chemically modified α is not that predicted by a simple model based on binomial distribution, but is complex and consistent with the kind of intersubunit communication that may be expected for catalytic cooperativity between the two active sites of the enzyme molecule.

ISSN:0714-7511    

DOI:10.1139/o85-008

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Under physiological conditions, concanavalin A interacts with the surface of phospholipid liposomes through two distinct classes of binding sites, a relatively small number of high affinity sites and a much larger number of lower affinity sites. Addition of bovine serum albumin induces extensive additional binding of concanavalin A to liposomal membranes and this binding is saturable and "specific" (α-methyl mannoside inhibitable). Fraction V and high purity albumin both induce almost identical levels of concanavalin A binding to liposomes. Scatchard plots of the binding data demonstrate the induction of a large number of new, relatively high affinity lectin-binding sites on addition of albumin. Albumin-induced binding of concanavalin A to the bilayer surface shows a broad pH optimum and is not inhibited by 40% (w/v) ethylene glycol, suggesting that hydrophobic forces are relatively unimportant. In contrast, divalent succinyl-concanavalin A shows very little tendency to bind to liposomes, either in the absence or presence of albumin. Passage of high purity albumin down a concanavalin A affinity column or treatment with periodate completely eliminates the additional lectin binding. It thus seems likely that albumin-induced concanavalin A binding to liposomes is related to the presence of a concanavalin-A-binding component. This phenomenon may have important implications for lectin-binding studies carried out on membranes which have been exposed to serum proteins.

ISSN:0714-7511    

DOI:10.1139/o85-009

出版商:NRC Research Press    

年代:1985

数据来源: NRC

摘要:

Using a partially purified enzyme preparation obtained from hamster epididymis, a simple assay has been developed to measure the sulfurylation of dehydroisoandrosterone (DHA) and desmosterol in the presence of 3′-phosphoadenosine 5′-phospho[35S]sulfate ([35S]PAPS). After stopping the enzymatic reaction with methanol and KCl, the35S-labelled steroid sulfates are readily extracted into an organic phase. Optimal conditions for the sulfurylation of the two steroids were compared; optimum pH is 8.7 for DHA and 9.8 for desmosterol. Sulfoconjugation of desmosterol increases with magnesium concentrations up to 6 mM, while 40 mMconcentrations of the divalent ion are required for the optimal sulfurylation of DHA. Maximum sulfurylation of these steroids requires the presence of 15 mMcysteine. Michaelis–Menten kinetics are observed with DHA which has an apparentKmof 32 μM, while desmosterol inhibits sulfotransferase activity at high concentrations. Saturation of the enzyme with PAPS results in an allosteric behaviour. Only the 3β-hydroxyl function of the steroid nucleus appears to be an appropriate sulfate acceptor for the epididymal hydroxysteroid sulfotransferase.

ISSN:0714-7511    

DOI:10.1139/o85-010

出版商:NRC Research Press    

年代:1985

数据来源: NRC