摘要:

Fetal calf liver cells were cultured in a serum-free medium in the presence of dibutyryl cAMP (dbcAMP), dibutyryl cGMP (dbcGMP), or cGMP. After a 20-h incubation with the nucleotides the synthesis of the fetal hemoglobins F0and F1was measured by the incorporation of [3H]leucine. The three cyclic nucleotides stimulated hemoglobin synthesis at a concentration of 1 μM. dbcGMP (10 nM) inhibited the synthesis of F0and F1but at a concentration of 1 μMsignificantly stimulated the synthesis of both hemoglobins. dbcAMP (10 μM) preferentially stimulated the synthesis of the transient hemoglobin F1whereas cCMP (1 μM)stimulated specifically the synthesis of the definitive hemoglobin F0. Prostaglandin E1(100 nM) stimulated the synthesis of F1in a manner similar to that observed for dbcAMP. AMP, GMP, CMP, and sodium butyrate did not stimulate hemoglobin synthesis at the concentrations indicated above. The analysis of the α- and γ-globin chains by high pressure liquid chromatography indicated that the synthesis of both chains are stimulated by the three cyclic nucleotides. These changes in hemoglobin synthesis taking place at different concentrations of cyclic nucleotides may be of importance in the maturation of erythroid cells, because in these cells differentiation is normally coupled with cell proliferation. Furthermore, some of these cyclic nucleotides may be second messengers of erythropoietic hormones. We have found that the intracellular levels of cGMP were increased 15 min after addition of step III sheep plasma erythropoietin to the cultures, whereas the amounts of cAMP remained unchanged. It is then possible that erythropoietin itself or a factor present in the erythropoietin preparation may act by a cGMP-dependent mechanism.

ISSN:0008-4018    

DOI:10.1139/o82-001

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

Acrosin from human spermatozoa was required for studies of immunological interference with fertilization, but no detailed purification scheme was available for the human enzyme. Since human semen samples cannot be obtained cheaply or in large numbers and contain relatively small amounts of acrosin, development of purification procedures was carried out with bovine semen. Bovine acrosin had not previously been fully purified, and over 1 mg of pure acrosin was obtained from 100 mL of bovine semen, by a process of saline and Triton X-100 washes of the spermatozoa, 1 mMHCl extraction, gel filtration, and ion-exchange and affinity chromatography. The bovine acrosin had a molecular weight (MW) of 39 000 and a specific activity of 93 U/mg, measured with 0.5 mMbenzoyl arginine ethyl ester. The same extraction procedure could be followed for human acrosin, but better yields were obtained in the purification if the ion-exchange step was omitted. The human acrosin had a MW of 49 000, and traces of a 38 000 MW component were sometimes observed. From 14 human semen samples, containing initially 7–10 U of acrosin activity, about 2.5 U (approximately 20 μg of protein) could be obtained in a pure state.

ISSN:0008-4018    

DOI:10.1139/o82-002

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

Ergosterol inhibited germ-tube formation and the membrane-bound enzyme chitin synthase inCandida albicans. The sterol solvents methanol and ethanol stimulated chitin synthase activity but inhibited germination. Methanol increased both the rate of protease activation of the chitin synthase proenzyme and theVmaxof the enzyme. The activation was not due to a change in the protease action on the proenzyme. The methanol activation was reversible and therefore the extraction of inhibitory components from the membrane was not causing the activation. Methanol had no effect on theN-acetylglucosamine activation sites. Analysis of theN-acetylglucosamine activation by an iteration programme gaveKavalues of 0.3 mMfor the high affinity sites and 20 mMfor the low affinity sites. The degree of cooperativity with respect to the substrate uridine diphosphateN-acetylglucosamine was not changed by the presence of methanol and therefore the solvent did not affect the subunit–subunit interactions of the enzyme. Arrhenius plots of chitin synthase have discontinuities and methanol did not affect the transition temperature indicating that the structure of the membrane in the immediate vicinity of the enzyme was unchanged.

ISSN:0008-4018    

DOI:10.1139/o82-003

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

Nuclei and chromatin isolated in the presence of calcium or magnesium fromRana catesbeianaliver tissue exhibit considerable endogenous deoxyribonuclease activity. This activity is present in liver nuclei isolated from froglets as well as in liver nuclei isolated from untreated and thyroid hormone treated premetamorphic tadpoles. Nuclei and chromatin isolated in the absence of divalent cations and in the presence of spermine exhibit no detectable expression of the endogenous deoxyribonuclease activity. The endogenous deoxyribonuclease present, but not expressed, in spermine-isolated tadpole liver nuclei or chromatin is salt extractable. Once dialyzed, the salt-extracted deoxyribonuclease is activated by calcium or magnesium. This deoxyribonuclease shows maximal enzymic activity in 15 mMcalcium at pH 8.0 or in 15 mMmagnesium at pH 7.4. After Ca2+activation, deoxyribonuclease activity is maximally inhibited by amounts of spermine similar to that required to completely inhibit DNase I. Destruction of the salt-extracted deoxyribonuclease activity by treatment with proteinase K or heat suggests that it is of a proteinaceous nature. The localization and nature of this enzymic activity established that it is associated with the salt-soluble proteins affiliated with tadpole and froglet liver chromatin.

ISSN:0008-4018    

DOI:10.1139/o82-004

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

α1-Globulin-type protease inhibitors were isolated from goat serum by two methods, namely preparative isoelectric focusing and preparative electrophoresis in polyacrylamide gel. The fractions obtained by the first method showed varying isoprotein compositions by analytical isoelectric focusing. Sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed the presence of one protein in the fractions with the same velocity of migration as purified human α1-antitrypsin and a second protein with a slightly higher migration velocity. The ratios of trypsin-inhibiting to chymotrypsin-inhibiting capacities in all the fractions were the same and both inhibitors were stable upon storage. The reaction of the inhibitors with trypsin and chymotrypsin was also demonstrated by analytical isoelectric focusing.The fractions obtained by preparative gel electrophoresis (the second method) contained the same proteins but their proportions varied widely in different fractions as demonstrated by analytical electrofocusing in the presence of urea and by SDS–PAGE. The early fractions, which consisted predominantly of α1-antitrypsin, showed a high inhibiting capacity for trypsin and none or only negligible capacity for chymotrypsin. Conversely, in the late fractions, the proportions of the proteins and inhibiting capacities were reversed. At 4 °C the trypsin-inhibiting capacity was stable for weeks but the chymotrypsin-inhibiting capacity of the preparation rapidly decreased.These observations indicate that the inhibition of proteases by goat α1-globulins is due to at least two closely associated but distinguishable proteins. One of these, corresponding to human α1-antitrypsin, would have an appreciable capacity to inhibit trypsin, but unlike the latter, little or no capacity for chymotrypsin inhibition. The inhibition of chymotrypsin is due to the second, unidentified α1-globulin.

ISSN:0008-4018    

DOI:10.1139/o82-005

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

Microsomes were isolated from rat liver and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER) components, and the purity of these fractions was determined. The dolichol content of each of the three fractions was estimated, using high-pressure liquid chromatography. Although highest concentrations (1940 ng/mg protein) were found in Golgi, the RER contained the largest absolute amounts. The presence of large quantities of dolichol in RER is consistent with the role of dolichol as an intermediate in asparagine-linked glycoprotein synthesis. RER and SER fractions contained high specific activities for dolichol phosphokinase, while the activity in Golgi was quite low. High concentrations of dolichol in Golgi and high dolichol phosphokinase activity in SER suggest that dolichol (and dolichyl phosphate) may be utilized in Golgi for glycoprotein processing and in the transmembrane movement of sugars such as galactose.

ISSN:0008-4018    

DOI:10.1139/o82-006

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

The adsorption of Br-X537A and its effect on the surface potential of monoolein lipid bilayers was measured using the nonactin conductance as a probe to determine the surface charge density. Because of the pH dependence of this adsorption, it was concluded that not only the negatively charged molecules X−could induce a surface charge but also a dimer HX2−made from X−and the neutral molecule HX. Also an important bilayer conductance was induced by Br-X537A. From the Br-X537A concentration dependence of this conductance, the effect of pH, and the induced surface potential, it was found that two charged complexes are transported across the bilayer depending on pH. At pH ≥ 7 the conducting molecule is X−and at pH ≤ 5 the complex is H2X3−. A quantitive model is obtained to calculate both the induced surface potential and the conductance.

ISSN:0008-4018    

DOI:10.1139/o82-007

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

The cation conductance induced by Br-X537A was measured for K+and Ca2+at different pH and different Br-X537A concentrations. The K+-concentration dependence was different depending on the pH of the solution. At pH 4.3, the conductance depended linearly on [K+] while at pH 7 it depended on [K+]2. About the same dependence on [Br-XS37A] was found at these pH values. From these results it was concluded that two complexes were transporting K+; at pH ≥ 7 the complex is mostly K2X3−and at pH ≤ 5 the complex is mostly KHX3−. From the conductance dependence on [Ca2+], pH, and [Br-X537A] it was found that the only conducting complex is CaX3−. It was also observed that Br-X537A could transport tetracaine. Taking into account the surface charge induced by tetracaine, the conductance depended linearly on the tetracaine concentration. Correcting for the surface charge induced by Br-X537A, the tetracaine conductance was found to depend on [Br-X537A]2. Consequently, the conducting complex is TX2−at pH ≥ 7. No other conducting complexes are formed as the pH is decreased and at pH 4 tetracaine is not conducted. It was also found that procaine was not conducted between pH 8 and 4.

ISSN:0008-4018    

DOI:10.1139/o82-008

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

The reproducibility of preparation, stability at 4 °C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found mat 2% (w/v) Nonidet P-40 solubilized 50–60% and 2% (w/v) sodium deoxycholate solubilized 60–70% of the total membrane protein. As judged by125I-labelled lentil lectin staining of the sodium dodecyl sulfate – polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet P-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found mat very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 MKCl, or by incubation with DNase I.

ISSN:0008-4018    

DOI:10.1139/o82-009

出版商:NRC Research Press    

年代:1982

数据来源: NRC

摘要:

Tubulin was reacted with a monofunctional imidoester, ethyl acetimidate, and three bifunctional imidoesters ranging in extension from 5 to 10 Å. Extensive cross-linking was found to occur with the three bifunctional reagents resulting in the formation of dimers, trimers, tetramers, pentamers, and hexamers. In addition, a similar cross-linking pattern was observed with me monofunctional reagent. This type of cross-linking is rarely seen when proteins are reacted with imidoesters. Given that the cross-linking span of ethyl acetimidate is only 3 Å it is reasonable to infer that there are nucleophilic groups in close proximity within the tubulin dimer. Amidinated tubulin is still capable of assembling into microtubules.

ISSN:0008-4018    

DOI:10.1139/o82-010

出版商:NRC Research Press    

年代:1982

数据来源: NRC