scholarly journals A 1H n.m.r. study of the kinetic properties expressed by glyceraldehyde phosphate dehydrogenase in the intact human erythrocyte

1982 ◽  
Vol 208 (3) ◽  
pp. 583-592 ◽  
Author(s):  
K M Brindle ◽  
I D Campbell ◽  
R J Simpson
Keyword(s):  
Human Erythrocyte ◽  
Intact Cell ◽  
Spin Echo ◽  
Glycolytic Flux ◽  
Kinetic Properties ◽  
Exchange Equilibrium ◽  
Glyceraldehyde Phosphate Dehydrogenase ◽  
Rate Limiting

Glyceraldehyde phosphate dehydrogenase is one of four glycolytic enzymes in the human erythrocyte that together can catalyse exchange of isotope between the C-2 position of lactate and solvent. Detailed measurements of the exchange can be made by using a non-invasive spin-echo p.m.r. method that has been described previously [Brindle, Brown, Campbell, Foxall & Simpson (1982) Biochem. J. 202, 589-602]. By studying the dependence of the exchange on the activity of an individual enzyme, the specific isotope-exchange equilibrium velocity of the enzyme can be measured. Suggestions that glyceraldehyde phosphate dehydrogenase is bound to the membrane in the intact cell, and that it may, under certain conditions, be rate-limiting for glycolytic flux, were examined in the present study by comparing the exchange properties expressed by the enzyme in situ and in vitro. It is concluded that glyceraldehyde phosphate dehydrogenase is not rate-limiting for glycolytic flux and that it is unlikely that a significant fraction of the enzyme is bound to the erythrocyte membrane in situ.

scholarly journals A .1H n.m.r. study of isotope exchange catalysed by glycolytic enzymes in the human erythrocyte

1982 ◽  
Vol 202 (3) ◽  
pp. 589-602 ◽  
Author(s):  
K M Brindle ◽  
F F Brown ◽  
I D Campbell ◽  
D L Foxall ◽  
R J Simpson
Keyword(s):  
Lactate Dehydrogenase ◽  
Human Erythrocyte ◽  
Isotope Exchange ◽  
Spin Echo ◽  
Glycolytic Enzymes ◽  
Exchange Equilibrium ◽  
Continuous Assessment ◽  
The Individual ◽  
Fructose 1,6 Bisphosphate

The exchange of hydrogen and deuterium atoms between the C-2 position of lactate and solvent was monitored in suspensions of human erythrocytes by using a non-invasive spin-echo p.m.r. method that permits continuous assessment of the rate and the extent of exchange. Exchange rates were measured in cells suspended in buffers made in 2H2O and 1H2O after the addition of L-[2-1H]lactate and L-[2-2H]lactate respectively. The rate of exchange is dependent on the activities of four glycolytic enzymes (fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and lactate dehydrogenase) and on the concentrations of their substrates. The dependence of the exchange on the following substrates was studied: (1) lactate, (2) the triose phosphates and fructose 1,6-bisphosphate and (3) pyruvate. Observation of the exchange in vitro, in a system produced by mixing the isolated enzymes, permits determination of the individual isotope-exchange equilibrium velocities of the enzymes. The dependence of the equilibrium velocity of human erythrocyte lactate dehydrogenase on NAD+ + NADH concentration was measured. Possible applications of these methods are discussed.

1H n.m.r. studies of the kinetic properties expressed by erythrocyte enzymes in situ and in vitro

1983 ◽  
Vol 11 (3) ◽  
pp. 280-281 ◽  
Author(s):  
KEVIN M. BRINDLE ◽  
IAIN D. CAMPBELL ◽  
ROBERT J. SIMPSON
Keyword(s):  
Kinetic Properties ◽  
Erythrocyte Enzymes

scholarly journals Uric Acid Is a Genuine Metabolite of Penicillium cyclopium and Stimulates the Expression of Alkaloid Biosynthesis in This Fungus

2002 ◽  
Vol 68 (4) ◽  
pp. 1524-1533 ◽  
Author(s):  
Florian Helbig ◽  
Jörg Steighardt ◽  
Werner Roos
Keyword(s):  
Uric Acid ◽  
Low Molecular Weight ◽  
Alkaloid Production ◽  
Alkaloid Biosynthesis ◽  
Penicillium Cyclopium ◽  
Measurable Activity ◽  
Reduced Rate ◽  
Rate Limiting

ABSTRACT On searching for endogenous, low-molecular-weight effectors of benzodiazepine alkaloid biosynthesis in Penicillium cyclopium uric acid was isolated from ethanolic or autoclaved mycelial extracts of this fungus. The isolation was based on a three-step high-pressure liquid chromatography procedure guided by a microplate bioassay, and uric acid was identified by mass spectrometry and the uricase reaction. Conidiospore suspensions that were treated with this compound during the early phase of outgrowth developed emerged cultures with an enhanced rate of alkaloid production. Uric acid treatment did not increase the in vitro measurable activity of the rate-limiting biosynthetic enzyme, cyclopeptine synthetase. However, these cultures displayed a reduced rate of uptake of the alkaloid precursor l-phenylalanine into the vacuoles of the hyphal cells as assayed in situ. It is suggested that the depressed capacity of vacuolar uptake caused by the contact of outgrowing spores with uric acid liberated from hyphal cells results in an enhanced availability of the precursor l-phenylalanine in the cytoplasm and thus accounts at least in part for the increase in alkaloid production.

scholarly journals Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin

1978 ◽  
Vol 173 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J Fischer ◽  
T R Kohler ◽  
L G Lipson ◽  
J Flores ◽  
P A Witkum ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Time Course ◽  
Intact Cell ◽  
Intact Cells ◽  
Maximal Stimulation ◽  
Rate Limiting

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

scholarly journals Arg-Vasotocin Directly Activates Isotocin Receptors and Induces COX2 Expression in Ovoviviparous Guppies

2021 ◽  
Vol 12 ◽  
Author(s):  
Li Kang Lyu ◽  
Jian Shuang Li ◽  
Xiao Jie Wang ◽  
Yi Jia Yao ◽  
Ji Fang Li ◽  
...  
Keyword(s):  
Poecilia Reticulata ◽  
Intraperitoneal Administration ◽  
Gene Expression Measurement ◽  
Reproductive Behaviors ◽  
Prostaglandin F ◽  
Rate Limiting ◽  
Cox2 Expression ◽  
Expression Measurement

Oxytocin (OT) is a crucial regulator of reproductive behaviors, including parturition in mammals. Arg-vasopressin (AVP) is a nonapeptide homologous to Arg-vasotocin (AVT) in teleosts that has comparable affinity for the OT receptor. In the present study, ovoviviparous guppies (Poecilia reticulata) were used to study the effect of AVT on delivery mediated by the activation of prostaglandin (PG) biosynthesis via isotocin (IT) receptors (ITRs). One copy each of it and avt and two copies of itrs were identified in guppies. The results of the affinity assay showed that various concentrations of AVT and IT (10−6, 10−7, and 10−8 mol/L) significantly activated itr1 (P < 0.05). In vitro experiments revealed significant upregulation (P < 0.05) of cyclooxygenase 2 (cox2), which is the rate-limiting enzyme involved in PG biosynthesis, and itr1 by AVT and IT. Furthermore, dual in situ hybridization detected positive signals for itr1 and cox2 at the same site, implying that ITR1 may regulate cox2 gene expression. Measurement of prostaglandin F2a (PGF2a) concentrations showed that AVT induced PGF2a synthesis (P < 0.05) and that the effect of IT was not significant. Finally, intraperitoneal administration of PGF2a significantly induced premature parturition of guppies. This study is the first to identify and characterize AVT and ITRs in guppies. The findings suggest that AVT promotes PG biosynthesis via ITR and that PGF2a induces delivery behavior in ovoviviparous guppies.

scholarly journals A BIOCHEMICAL AND HISTOCHEMICAL STUDY OF GLUTAMIC OXALACETIC TRANSAMINASE ACTIVITY OF RAT HEPATIC MITOCHONDRIA FIXED IN SITU AND IN VITRO

1968 ◽  
Vol 39 (3) ◽  
pp. 725-732 ◽  
Author(s):  
Sin Hang Lee ◽  
Richard M. Torack
Keyword(s):  
Histochemical Study ◽  
Intact Cell ◽  
Mitochondrial Fraction ◽  
Tissue Homogenate ◽  
Transaminase Activity ◽  
Glutamic Oxalacetic Transaminase ◽  
Histochemical Methods ◽  
Liver Tissue Homogenate

Rat liver perfused in situ briefly with a glutaraldehyde-formaldehyde mixture was homogenized in isotonic sucrose. The mitochondria, isolated from a homogenate of the perfused liver by differential centrifugation, assumed a slender and compact appearance similar to those often seen in an intact cell. The glutamic oxalacetic transaminase (GOT) activity of this mitochondrial fraction survived an additional formaldehyde fixation and was studied by biochemical and histochemical methods. The biochemical assay of the enzyme activity revealed that the activity was only slightly less than that of an unfixed mitochondrial fraction. The reaction product due to mitochondrial GOT activity was found to be localized to the cristae, as had been demonstrated in an intact liver cell. GOT activity of the mitochondrial fraction isolated from fresh liver tissue homogenate in 0.25 M sucrose was inactivated readily by either glutaraldehyde or formaldehyde and was no longer demonstrable by biochemical and histochemical methods after fixation.

scholarly journals Uridine kinase activities and pyrimidine nucleoside phosphorylation in fluoropyrimidine-sensitive and -resistant cell lines of the Novikoff hepatoma

1977 ◽  
Vol 164 (2) ◽  
pp. 379-387 ◽  
Author(s):  
N Greenberg ◽  
D E Schumm ◽  
T E Webb
Keyword(s):  
Intact Cell ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Human Carcinoma ◽  
Novikoff Hepatoma ◽  
Uridine Kinase ◽  
Cell Extracts ◽  
Rate Limiting

Uridine kinase, the rate-limiting enzyme in the activation (phosphorylation) of uridine and the corresponding chemotherapeutic analogues, is present as two isoenzymes localized exclusively in the cytosol of rapidly growing neoplasms, including the S-37 sarcoma, EL-4 leukaemia, HeLa cells (a human carcinoma) and the Novikoff hepatoma. The activities of the isolated isoenzymes are markedly decreased when the concentrations of ATP, phosphate or Mg2+ that are optimum in vitro are replaced by concentrations of ATP, phosphate or Mg2+ that are optimum in vitro are replaced by concentrations approximating to those found in vivo. Further, comparisons of the Km values of isolated uridine kinases with those for cellular uptake of pyrimidine nucleosides and their rate of intracellular phosphorylation suggest that nucleoside-transport systems play a rate-limiting role in nucleoside analogue activation and consequently that it is impossible to estimate the Km of uridine kinase in the intact cell. During the development of tumour-cell resistance to 5-fluorouracil or 5-fluorouridine in vivo there was an early differential increase in the activity of a low-affinity (high-Km) uridine kinase isoenzyme, as measured in cell extracts, and a 7-fold increase in the Km values for the uptake of both uridine and 5-fluorouridine into the intact resistant cells.

scholarly journals Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro

1976 ◽  
Vol 153 (2) ◽  
pp. 499-501 ◽  
Author(s):  
M Swissa ◽  
H Weinhouse ◽  
M Benziman
Keyword(s):  
Carbohydrate Metabolism ◽  
Citrate Synthase ◽  
Acetobacter Xylinum ◽  
Kinetic Properties ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Permeabilized Cells

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

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