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61.
John C. Meeks Carol S. Enderlin Keith L. Wycoff John S. Chapman Cecillia M. Joseph 《Planta》1983,158(5):384-391
The pathways of assimilation of ammonium by pure cultures of symbiont-free Anthoceros punctatus L. and the reconstituted Anthoceros-Nostoc symbiotic association were determined from time-course (5–300 s) and inhibitor experiments using 13NH 4 + . The major product of assimilation after all incubation times was glutamine, whether the tissues were cultured with excess ammonium or no combined nitrogen. The 13N in glutamine was predominantly in the amide-nitrogen position. Formation of glutamine and glutamate by Anthoceros-Nostoc was strongly inhibited by either 1mM methionine sulfoximine (MSX) or 1 mM exogenous ammonium. These data are consistent with the assimilation of 13NH 4 + and formation of glutamate by the glutamine synthetase (EC 6.3.1.2)-glutamate synthase (EC 1.4.7.1) pathway in dinitrogen-grown Anthoceros-Nostoc. However, in symbiont-free Anthoceros, grown with 2.5 mM ammonium, formation of glutamine, but not glutamate, was decreased by either MSX or exogenous ammonium. These results indicate that during short incubation times ammonium is assimilated in nitrogenreplete Anthoceros by the activities of both glutamine synthetase and glutamate dehydrogenase (EC 1.4.1.2). In-vitro activities of glutamine synthetase were similar in nitrogen-replete Anthoceros and Anthoceros-Nostoc, indicating that the differences in the routes of glutamate formation were not based upon regulation of synthesis of the initial enzyme of the glutamine synthetase-glutamate synthase pathway. When symbiont-free Anthoceros was cultured for 2 d in the absence of combined nitrogen, total 13NH 4 + assimilation, and glutamine and glutamate formation in the presence of inhibitors, were similar to dinitrogen-grown Anthoceros-Nostoc. The routes of immediate (within 2 min) glutamate formation and ammonium assimilation in Anthoceros were apparently determined by the intracellular levels of ammonium; at low levels the glutamine synthetase-glutamate synthase pathway was predominant, while at high levels independent activities of both glutamine synthetase and glutamate dehydrogenase were expressed. 相似文献
62.
Peggy Keeling Keith Johnson Daryl Sas Kathleen Klukas Peter Donahue Ross Johnson 《The Journal of membrane biology》1983,74(3):217-228
Summary The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component
of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional
membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents.
These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or
the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin,
papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed
to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides
were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped
with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage
obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily
penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of
the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being
evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer
to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing
no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the
primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the
C-terminus, appears to be exposed on the cytoplasmic side of the membrane. 相似文献
63.
64.
Keith A. Kvenvolden 《Origins of life and evolution of the biosphere》1974,5(1-2):71-86
Meteorites, particularly type II carbonaceous chondrites, provide natural, tangible evidence for chemical evolution, but they do not appear to contain any evidence for biological evolution. On the other hand, some of the oldest sedimentary rocks of the earth have yielded good evidence for early biological evolution; whatever evidence there may be for chemical evolution in these old rocks is generally obscure. Carbonaceous chondrites (types I, II, and III) have been examined for thier content of various kinds of organic compounds. Amino acids have been reported to be present in the three types, but only in type II carbonaceous chondrites (Murray and Murchison) has an indigenous suite of amino acids been found which is apparently free of most terrestrial contaminations. These indigenous compounds are thought to have resulted from extraterrestrial, abiotic, chemical syntheses, and the presence of the amino acids in meteorites provides strong support for the theory of chemical evolution. The geological record of the Swaziland Sequence and Bulawayan System of Southern Africa contains morphological and chemical fossils which indicate that early biological evolution was taking place at least 3.0 to 3.3 aeons ago. Interpretation of the significance of the chemical fossil record has proven to be difficult. At present the occurrence of simple compounds in these very ancient rocks is believed to have little or nothing to do with biochemical processes three aeons ago. The bulk of the reduced carbonaceous material in these rocks, however, probably represents the residue of three billion years old and older organic matter. Isotopic studies of this carbonaceous material may provide chemical evidence for early biological evolution. 相似文献
65.
Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat 总被引:7,自引:7,他引:0
1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action. 相似文献
66.
Pig liver pyruvate carboxylase. Purification, properties and cation specificity 总被引:4,自引:2,他引:2
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1. Pyruvate carboxylase was purified to apparent homogeneity from pig liver mitochondria and shown to be free of all kinetically contaminating enzymes. 2. The enzyme has a mol. wt. of 520000 and is composed of four subunits, each with a mol. wt. of 130000. 3. The enzyme can exist as the active tetramer, dimer and monomer, although the tetramer appears to be the form in which the enzyme is normally assayed. 4. For every 520000g of the enzyme there are 4mol of biotin, 3mol of zinc and 1mol of magnesium. No significant concentrations of manganese were detected. 5. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicates three polypeptide chains per monomer unit, each with a mol. wt. of 47000. 6. The amino acid analysis, stoicheiometry of the reaction and the activity of the enzyme as a function of pH are also presented. 7. The enzyme is activated by a variety of univalent cations but not by Tris(+) or triethanolamine(+). 8. The activity of the enzyme is dependent on the presence of acetyl-CoA; the low rate in the absence of added acetyl-CoA is not due to an enzyme-bound acyl-CoA. The dissociation constant for enzyme-bound acetyl-CoA is a marked function of pH. 相似文献
67.
Kinetics of the hydrolysis of N-benzoyl-l-serine methyl ester catalysed by bromelain and by papain. Analysis of modifier mechanisms by lattice nomography, computational methods of parameter evaluation for substrate-activated catalyses and consequences of postulated non-productive binding in bromelain- and papain-catalysed hydrolyses 总被引:8,自引:8,他引:0
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Christopher W. Wharton Athel Cornish-Bowden Keith Brocklehurst Eric M. Crook 《The Biochemical journal》1974,141(2):365-381
1. N-Benzoyl-l-serine methyl ester was synthesized and evaluated as a substrate for bromelain (EC 3.4.22.4) and for papain (EC 3.4.22.2). 2. For the bromelain-catalysed hydrolysis at pH7.0, plots of [S(0)]/v(i) (initial substrate concn./initial velocity) versus [S(0)] are markedly curved, concave downwards. 3. Analysis by lattice nomography of a modifier kinetic mechanism in which the modifier is substrate reveals that concave-down [S(0)]/v(i) versus [S(0)] plots can arise when the ratio of the rate constants that characterize the breakdown of the binary (ES) and ternary (SES) complexes is either less than or greater than 1. In the latter case, there are severe restrictions on the values that may be taken by the ratio of the dissociation constants of the productive and non-productive binary complexes. 4. Concave-down [S(0)]/v(i) versus [S(0)] plots cannot arise from compulsory substrate activation. 5. Computational methods, based on function minimization, for determination of the apparent parameters that characterize a non-compulsory substrate-activated catalysis are described. 6. In an attempt to interpret the catalysis by bromelain of the hydrolysis of N-benzoyl-l-serine methyl ester in terms of substrate activation, the general substrate-activation model was simplified to one in which only one binary ES complex (that which gives rise directly to products) can form. 7. In terms of this model, the bromelain-catalysed hydrolysis of N-benzoyl-l-serine methyl ester at pH7.0, I=0.1 and 25 degrees C is characterized by K(m) (1) (the dissociation constant of ES)=1.22+/-0.73mm, k (the rate constant for the breakdown of ES to E+products, P)=1.57x10(-2)+/-0.32x10(-2)s(-1), K(a) (2) (the dissociation constant that characterizes the breakdown of SES to ES and S)=0.38+/-0.06m, and k' (the rate constant for the breakdown of SES to E+P+S)=0.45+/-0.04s(-1). 8. These parameters are compared with those in the literature that characterize the bromelain-catalysed hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester and of alpha-N-benzoyl-l-arginine amide; K(m) (1) and k for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine amide hydrolysis and K(as) and k' for the serine ester hydrolysis are somewhat similar to K(m) and k(cat.) for the arginine ester hydrolysis. 9. A previous interpretation of the inter-relationships of the values of k(cat.) and K(m) for the bromelain-catalysed hydrolysis of the arginine ester and amide substrates is discussed critically and an alternative interpretation involving substantial non-productive binding of the arginine amide substrate to bromelain is suggested. 10. The parameters for the bromelain-catalysed hydrolysis of the serine ester substrate are tentatively interpreted in terms of non-productive binding in the binary complex and a decrease of this type of binding by ternary complex-formation. 11. The Michaelis parameters for the papain-catalysed hydrolysis of the serine ester substrate (K(m)=52+/-4mm, k(cat.)=2.80+/-0.1s(-1) at pH7.0, I=0.1, 25.0 degrees C) are similar to those for the papain-catalysed hydrolysis of methyl hippurate. 12. Urea and guanidine hydrochloride at concentrations of 1m have only small effects on the kinetic parameters for the hydrolysis of the serine ester substrate catalysed by bromelain and by papain. 相似文献
68.
69.
Rooting is shown to occur in excised cotyledons of Sinapis alba when grown in petri dishes on moist filter paper. Cotyledons were excised at intervals from 6 hours after the start of imbibition, when they were yellow, unexpanded and enclosed within the testa, to 27 days after sowing when the cotyledons were green and fully expanded and on plants possessing up to 3 foliage leaves. Rooting generally began 5 or 6 days after excision and was completed dining the following 5 days. The age of cotyledons at t ho time of excision had three effects on rooting: the lag period be-fore rooting began and the period during which rooting took place both increased with age: but the most marked effect was on the total number of cotyledons which were able to form roots, which increased until cotyledon expansion was almost complete, then decreased as the mature cotyledons became older. Optimal rooting was shown by cotyledons detached 8 days alter sowing, when they were half expanded. At this age S5 % of them formed roots between 6 days and 8 days after excision. 相似文献
70.
Keith Whaley W. Wilson Downie W. Carson Dick George Nuki C. B. S. Schofield John Anderson 《BMJ (Clinical research ed.)》1969,2(5654):421-422
A double-blind trial comparing lincomycin hydrochloride (Mycivin) and placebo in 22 patients with Reiter''s disease showed no significant difference in clinical or laboratory findings between the two groups. It is concluded that lincomycin hydrochloride is no more effective than placebo in the treatment of Reiter''s disease. 相似文献