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1.
Rabbit liver phosphofructo-1-kinase, designated isozyme B, and rabbit brain phosphofructokinase, which contains all three isozymes as heteropolymers, have been modified by [14C]fluorosulfonylbenzoyladenosine (FSBAdo). Several lines of evidence supported modification at the binding site for AMP. The modification proceeded to the extent of 2 to 4 mol of reagent incorporated per mol of tetramer, and AMP protected against the reaction. The kinetic properties of modified isozymes A and B and of modified brain phosphofructokinase were examined and compared to their unmodified forms. It was observed that modification greatly diminished ATP inhibition of all of the isozymes. Furthermore, equilibrium binding studies of modified phosphofructokinase B showed a greatly diminished capacity and affinity for cyclic AMP. Cyclic AMP had little or no influence on the properties of modified A isozyme or brain phosphofructokinase, but was capable of further deinhibiting modified B isozyme, apparently at sites remaining unmodified by FSBAdo. Phosphofructokinase B, modified by radiolabeled FSBAdo, was digested by trypsin, and the digest separated by high-pressure liquid chromatography. The labeled peptide was isolated and sequenced to provide the sequence: Asn-Tyr-Gly-Thr-Lys-Leu-Gly-Val-Lys, with the lysine in the fifth position being the site of modification. To isolate isozyme C, a monoclonal antibody to this isozyme was produced by injecting purified rabbit brain phosphofructokinase into mice, and subsequently selecting for those clones that recognized brain phosphofructokinase but not purified phosphofructokinases A and B. The selected monoclonal was specific for native rabbit isozyme C and would not recognize mouse or rat brain phosphofructokinases. Linking the antibody to an inert phase provided an efficient means of purifying rabbit isozyme C from rabbit brain. The enzyme so recovered retained little of its original activity, but the method provided a simple technique for the preparation of enzyme for protein chemistry studies. The modified C isozyme was isolated on the immuno-affinity column and digested with trypsin. A tryptic peptide bearing the label was isolated and sequenced to provide the structure: Asn-Phe-Gly-Thr-Lys-Ile-Ser-Ala-Arg, with position 5 being the site of modification. The sequences of isozymes B and C are homologous to the site of modification of the A isozyme by FSBAdo. 相似文献
2.
COMPARISON OF THE BEHAVIOUR OF A SOLUBLE AND A MEMBRANE-BOUND ENZYME IN TRANSECTED PERIPHERAL NERVES
Jolanta Skangiel-Kramska Stella Niemierko Liliana Lubiska 《Journal of neurochemistry》1969,16(6):921-926
Phosphoglucoisomerase (PGI), a soluble enzyme, and AChE, a membrane-bound enzyme were studied in transected peroneal nerves of dog and in isolated segments of these nerves. Although activities of both enzymes increased at the ends of transected nerves, marked differences in their behaviour were observed. The increment in AChE activity was much sharper than that of PGI and continued to grow with time whereas the increase in PGI developed fully within the initial hours after transection and did not change thereafter. In an isolated nerve segment AChE accumulated at both ends with a concomitant decrease in the middle part, whereas changes in PGI activity appeared only in the terminal parts, the rest of the nerve remaining at the normal level. The terminal increase of PGI did not, contrary to that of AChE, depend on the length of the isolated segment. The changes in PGI activity may be features of a local peritraumatic reaction whereas those of AChE indicate involvement of the whole segment along which the enzyme containing organelles are transported. 相似文献
3.
Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells3H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis. 相似文献
4.
Summary Using light microscopic autoradiography it was found that in the middle S and G2 phases of the cell cycle in antheridial filaments (S + G2 + M type) ofChara vulgaris L., an intensified RNA synthesis took place. The process was correlated with enhanced binding of3H actinomycin D to the DNA template, increased RNA polymerase activity, and enhanced chromatin decondensation.This work was supported by the Polish Academy of Sciences within the project 09.7.3.1.4. 相似文献
5.
Summary The nervous system of the most primitive fresh-water Turbellaria of the order Catenulida was investigated. Neurosecretory
cells were found to occur in it. These cells have a structure similar to that of nerve cells, they only differ by the presence
of neurosecretion granules and a more developed golgi complex. In the simplest Catenulida,Stenostomun, neurosecretory granules are found both in the brain cells and in the trunks in the nerve fibres where they are rather randomly
distributed. Another representative of this family, the more highly organizedCatenula, exhibits specialized brain and nerve trunk regions containing neurosecretion granules.
This work was supported in part by the Committee on Cytobiology of the Polish Academy of Sciences 相似文献
6.
Actin-binding proteins involved in the capping of epidermal growth factor receptors in A431 cells. 总被引:3,自引:0,他引:3
K Kwiatkowska I A Khrebtukova D A Gudkova G P Pinaev A Sobota 《Experimental cell research》1991,196(2):255-263
A capping process of epidermal growth factor receptors (EGF-Rs) was used for the study of the relation between the receptors and the actin-binding proteins (spectrin, vinculin, annexin I) that may be involved in EGF-R-cytoskeleton interaction. In intact, adherent A431 cells, EGF-Rs were diffusively distributed on the cell surface. Spectrin, vinculin, and annexin I were located beneath the plasma membrane. An abundance of EGF-Rs as well as submembrane proteins was observed in regions of membrane ruffles and cell-cell contacts. Annexin I was localized also in cytoplasm being attached to filamentous structures surrounding the nucleus and extending to the cell periphery. Under polyvalent ligand treatment, EGF-Rs of adherent cells were aggregated on one side of the cell. Spectrin, vinculin, and annexin I dislocated together with EGF-Rs and were concentrated under plasma membrane at regions where cap formation took place. In suspended A431 cells only spectrin was located under the plasma membrane whereas annexin I and vinculin were diffusively distributed through the cells. During cap formation only spectrin was colocalized with EGF-Rs. The results confirmed the major role of spectrin as a receptor-microfilament linking protein. 相似文献
7.
Jerzy Jurka Jolanta Walichiewicz Aleksandar Milosavljevic 《Journal of molecular evolution》1992,35(4):286-291
Summary We report a collection of 53 prototypic sequences representing known families of repetitive elements from the human genome. The prototypic sequences are either consensus sequences or selected examples of repetitive sequences. The collection includes: prototypes for high and medium reiteration frequency interspersed repeats, long terminal repeats of endogenous retroviruses, alphoid repeats, telomere-associated repeats, and some miscellaneous repeats. The collection is annotated and available electronically.[/ap ]Offprint requests to: J. Jurka 相似文献
8.
Suzanne Desjardins-Giasson Jolanta Gutkowska Raul Garcia Jacques Genest 《Prostaglandins & other lipid mediators》1982,24(1):105-114
Prostaglandin E2 (PGE2) and 6 keto-PGF1α, the stable metabolite of prostacyclin (PGI2), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE2 and 6-keto-PGF1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE2 and 6-keto-PGF1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI2 is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE2 and 6-keto-PGF1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone. 相似文献
9.
10.
Nowak R Baranowska-Bosiacka I Stefańska B Machaliński B Hłyńczak AJ Tarasiuk J 《Acta biochimica Polonica》2005,52(4):953-957
Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers. 相似文献