scholarly journals Reaction of cyanide with cytochrome aa3 in isolated perfused rat head in situ

1986 ◽  
Vol 233 (1) ◽  
pp. 187-191 ◽  
Author(s):  
K Kariman ◽  
D S Burkhart
Keyword(s):  
Mitochondrial Function ◽  
Spectral Characteristics ◽  
Difference Spectrum ◽  
Cytochrome Aa3 ◽  
Bilateral Carotid ◽  
Anaesthetized Rats ◽  
The Difference ◽  
Kinetics Of

The reaction of cyanide with cytochrome aa3 in intact mitochondria is known to differ significantly from the reaction with the isolated enzyme. To examine the cyanide reaction with cytochrome aa3 in situ, we studied the spectral characteristics and the reaction kinetics of cyanide with reduced brain cytochrome aa3 in an isolated perfused rat head preparation. Anaesthetized rats underwent bilateral carotid-arterial cannulation. The head (skull intact, muscle removed) was perfused with a crystalloid solution containing Na2S2O4, and the animal was then decapitated. By means of reflectance spectrophotometry the reaction of cyanide with cytochrome aa3 was continuously monitored with the use of the 590 nm-575 nm, 610 nm-575 nm and 590 nm-610 nm wavelength pairs. We found that: the kinetics of the absorbance change at 590 nm and 610 nm were similar, with almost identical apparent rate constants, suggesting that these spectral changes are the results of the formation of a single complex; the difference spectrum obtained on addition of cyanide to the fully reduced preparation showed a peak at 588 nm and a trough at 610 nm, consistent with spectral characteristics of the cyanide-ferrocytochrome aa3 complex in isolated enzyme and isolated mitochondria in vitro; this observation underscores the accuracy of monitoring the effects of inhibitors of mitochondrial function on cytochrome redox reactions in situ; the half-maximal (K0.5) effect was approx. 50 microM, significantly lower than that in vitro. The lower apparent K0.5 for cyanide in this preparation in situ may be due to a difference in the pH of the two systems. This approach provides the means to study the inhibitors of mitochondrial function in intact brain under a physiological environment.

Absorption of beta-casomorphins from autoperfused lamb and piglet small intestine

1990 ◽  
Vol 259 (3) ◽  
pp. G443-G452 ◽  
Author(s):  
L. C. Read ◽  
A. P. Lord ◽  
V. Brantl ◽  
G. Koch
Keyword(s):  
Small Intestine ◽  
Intestinal Lumen ◽  
Physiological Conditions ◽  
Naturally Occurring ◽  
Gut Epithelium ◽  
Measured Absorption ◽  
Beta Casein ◽  
Kinetics Of

beta-Casomorphins (beta-CMs) derived from milk beta-casein may exert various opiate activities in milk-fed infants. To assess the physiological significance of beta-CMs as a source of circulating opioids in infants, we measured absorption rates of several beta-CMs under near-physiological conditions using in situ autoperfused lamb intestine. The naturally occurring beta-CMs, beta-CM-7 and beta-CM-4-amide, were absorbed readily into blood with no transfer into lymph. Uptake peaked within several minutes of the luminal infusion of peptide but then declined sharply and stopped within a further 10-15 min. The recovery in blood, intestinal contents, and tissue at the end of the 30-min experiment was less than 1% of the infused dose. The low recovery was due to rapid proteolysis based on in vitro studies that demonstrated half-lives of less than 5 min in lamb blood, luminal contents, and lymph. The synthetic dipeptidyl peptidase IV-resistant analogue beta-[D-Ala2]CM- 4-amide was stable during incubation in blood, lymph, or luminal contents and was absorbed into blood at rates that were maximal within several minutes and remained steady for the 30-min period. We conclude that although natural beta-CMs are transferred across the lamb small intestine, rapid degradation within the intestinal lumen, gut epithelium, and blood would prevent entry into the circulation under normal conditions. Val-beta-CM-7, a putative stable precursor, had similar stability and kinetics of absorption to beta-CM-7, results that exclude Val-beta-CM-7 as a stable precursor for delivery of beta-CMs to the circulation. Essentially identical results to those in lambs were obtained in 7-day-old piglets.

Changes in lipophosphoglycan and gene expression associated with the development ofLeishmania majorinPhlebotomus papatasi

Parasitology ◽  
1995 ◽  
Vol 111 (3) ◽  
pp. 275-287 ◽  
Author(s):  
E. M. B. Saraiva ◽  
P. F. P. Pimenta ◽  
T. N. Brodin ◽  
E. Rowton ◽  
G. B. Modi ◽  
...  
Keyword(s):  
Differential Expression ◽  
Fold Increase ◽  
Surface Expression ◽  
Side Chain ◽  
Surface Coat ◽  
Natural Vector ◽  
Metacyclic Promastigotes ◽  
Kinetics Of

SUMMARYStage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course ofL. majorinfection in a natural vector,Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics invitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promatigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesisin vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression invitro, also showed selective expression by promastigotes in the fly, and was used inin situhybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

Minimum intracellular PO2 for maximum cytochrome turnover in red muscle in situ

1987 ◽  
Vol 252 (5) ◽  
pp. H906-H915 ◽  
Author(s):  
T. E. Gayeski ◽  
R. J. Connett ◽  
C. R. Honig
Keyword(s):  
Energy Demand ◽  
Concentration Ratio ◽  
Probability Distributions ◽  
Michaelis Constant ◽  
Cytochrome Aa3 ◽  
Wide Range ◽  
Red Muscle

Probability distributions of myoglobin (Mb) saturation and intracellular PO2 were determined with subcellular spatial resolution in dog gracilis muscles during steady-state twitch contraction at 5-100% of maximal rate of O2 consumption (VO2). Calculations (Clark, A., and P. A.A. Clark. Biophys. J. 48: 931-938, 1985) and measurements (Gayeski, T. E. J., and C. R. Honig. Adv. Exp. Med. Biol. 200: 487-494, 1986) indicate that the PO2 in equilibrium with Mb is virtually identical to the PO2 at cytochrome aa3. Median intracellular PO2 and PO2 in the lower tails of probability distributions were poorly correlated with VO2. The variability of cell PO2 was greatly diminished when median PO2 was less than the PO2 for half saturation of MB, since Mb acts as a PO2 buffer. The lower tails of PO2 distributions contained almost no anoxic loci even when median PO2 was less than 1 Torr. VO2 was well correlated with the concentration ratio of phosphocreatine to free creatine (PCr/Crf) over a wide range of PO2. PO2 greater than or equal to 0.5 Torr supported maximal VO2 and energy demand. We conclude that 1) the mechanism of action of cytochrome aa3 is the same in red muscle in vivo as in mitochondria in vitro, and 2) an upper bound on the apparent Michaelis constant for maximal VO2 of red muscle is approximately 0.06 Torr.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

Electroretinographic Determination of Spectral Sensitivity in Albino and Pigmented Brook Trout (Salvelinus fontinalis, Mitchill)

1971 ◽  
Vol 49 (12) ◽  
pp. 1030-1037 ◽  
Author(s):  
H. Kobayashi ◽  
M. A. Ali
Keyword(s):  
Absorption Spectrum ◽  
Spectral Sensitivity ◽  
Brook Trout ◽  
Salvelinus Fontinalis ◽  
Narrow Band ◽  
Visible Spectrum ◽  
The Difference

A technique for recording electroretinograms from the unpunctured eyes in situ of living, anesthetized fish is described. This technique permits the use of the same fish in a number of experiments over a period of weeks, months, or years. Using this technique the spectral sensitivity of dark-adapted (scotopic) and light-adapted (photopic) fish was measured at 13 bands of the visible spectrum. The scotopic curves of albino and pigmented trout thus obtained in the winter have their maxima around 525 nm which differ from that of the absorption spectrum of the scotopic pigment in situ and in vitro of older fish obtained in the summer. The photopic curve of the pigmented fish is a broad one with humps around 425 nm, 545 nm, and 595 nm. The albino's curve has a relatively narrow band with a peak around 630 nm and a shoulder at about 550 nm. The difference between the shapes of the two curves may be ascribed to the increase in the intensity of light of longer wavelengths within the eyeball of the albino, due to reflection from blood vessels and sclera caused by the absence of pigmentation.

Potential of solubility, enzymatic methods and NIRS to predict in situ rumen escape protein

1997 ◽  
Vol 45 (2) ◽  
pp. 291-306 ◽  
Author(s):  
J.L. De Boever ◽  
B.G. Cottyn ◽  
J.M. Vanacker ◽  
C.V. Boucque
Keyword(s):  
Phosphate Buffer ◽  
Near Infrared ◽  
Streptomyces Griseus ◽  
Maize Silage ◽  
Near Infrared Reflectance ◽  
In Vitro Method ◽  
Rumen Degradation ◽  
The Difference

The percentage of feed protein escaping rumen degradation was measured by the in situ method (%EPsitu) for 29 compound feeds, untreated and formaldehyde-treated soyabean meal and 12 forages: 3 grass silages, 2 maize silages, fresh grass, grass hay, fodder beets, fresh potatoes, ensiled beet pulp, chopped ear-maize silage and brewers' grains. Loss of particles through bag pores was determined by the difference between the washable fraction (W) and the fraction soluble in borate-phosphate buffer at pH 6.7 (S). W - S was most pronounced for compound feeds (on average 14.4 percentage units), for brewers' grains and maize silages. A correction of %EPsitu, assuming that W - S degrades like the potentially degradable fraction, was not appropriate. Solubility in borate-phosphate buffer after 1 h, enzymic degradability by protease from Streptomyces griseus or ficin after 1, 6 and 24 h and near infrared reflectance spectroscopy (NIRS) (for compound feeds alone) were examined as a routine method to predict %EPsitu. With the buffer and S. griseus the effect of pH (6.7 vs. 8.0) and at pH 8.0 the effect of amount of substrate (500-mg sample vs. 20 mg N) were tested. With ficin, 500-mg samples were incubated at pH 6.7. Predictions were better when compound feeds and forages were considered separately. However, the best in vitro method was different for the 2 feed categories, being solubility in buffer for the compound feeds and enzymic degradation of a constant amount of protein with S. griseus at pH 8.0 for forages. NIRS showed potential to predict %EPsitu of compound feeds, but needs more reference samples. The Dutch feed tables appeared more accurate than the best in vitro method for compound feeds, but was too inaccurate for some forages like fodder beets, maize silage and ear-maize silage.

scholarly journals A comparative study of ruminal activity in Churra and Merino sheep offered alfalfa hay

1997 ◽  
Vol 65 (1) ◽  
pp. 121-128 ◽  
Author(s):  
M. J. Ranilla ◽  
M. D. Carro ◽  
C. Valdés ◽  
F. J. Giráldez ◽  
S. López
Keyword(s):  
Cell Wall ◽  
Rumen Fluid ◽  
Merino Sheep ◽  
Rumen Ph ◽  
Sheep Rumen ◽  
Fermentation Parameters ◽  
Micro Organisms ◽  
Kinetics Of

AbstractA study was carried out to compare the fermentation parameters and kinetics of digestion of a range of different foods in the rumen of two breeds of sheep (Churra and Merino). Ten mature sheep (five Churra and five Merino), each fitted with a rumen cannula, were used in this study. In situ rumen degradability of both dry matter (DM) and cell wall was greater in Churra than in Merino sheep, the breed differences being significant for most of the foods used in the study (P < 0·05). These differences were greater when the foods had a higher cell wall concentration and this could be related to differences in the ruminal environment. However, when the foods were incubated with rumen fluid their in vitro organic matter (OM) degradability was similar in both breeds. Rumen pH was higher (P < 0·05) and ammonia concentrations were lower (P < 0·05) in Churra than in Merino sheep. Rumen volatile fatty acid concentrations tended to be higher in Merino than in Churra sheep, though differences were only significant just before feeding (P < 0·05). The ratio acetate: propionate was higher in the Churra than Merino breed before and 12 h after feeding (P < 0·05). Protozoa numbers in rumen liquid were similar for both genotypes. The greater degradation of forages in the rumen of Churra sheep is discussed in relation to the possible higher activity of fibre-degrading micro-organisms and the greater buffering capacity of the rumen contents against fermentation acids, which could result in more favourable conditions for the microbial degradation of foods in the rumen.

scholarly journals Regulation of platelet AMP deaminase activity in situ

1990 ◽  
Vol 265 (1) ◽  
pp. 267-275 ◽  
Author(s):  
A J M Verhoeven ◽  
J Marszalek ◽  
H Holmsen
Keyword(s):  
Human Platelets ◽  
Partial Purification ◽  
Amp Deaminase ◽  
H2o2 Treatment ◽  
Half Saturation Constant ◽  
Intracellular Phosphate ◽  
The Difference ◽  
Main Regulator

The regulation of platelet AMP deaminase activity by ATP, GTP and phosphate was studied in human platelets in situ, and in vitro after partial purification. In intact platelets, a similar 50% decrease in cytosolic ATP was induced by either glucose starvation or treatment with H2O2. During starvation, AMP deaminase was in the inhibited state, as ATP consumption was mostly balanced by the accumulation of AMP. During H2O2 treatment, however, the enzyme was in the stimulated state, as the AMP formed was almost completely deaminated to IMP. Cytosolic GTP fell by 40-50% in both starvation and H2O2 treatment. In contrast, intracellular phosphate was 4-5-fold higher in starved than in H2O2-treated cells. These data point to phosphate as the main regulator of AMP deaminase activity in situ. This conclusion was verified by kinetic analysis of partially purified AMP deaminase. At near-physiological concentrations of MgATP, MgGTP and phosphate, the S0.5 (substrate half-saturation constant) for AMP was 0.35 mM. Half-maximal stimulation by MgATP occurred at a concn. between 2 and 3 mM. This stimulation was antagonized by the inhibitory effects of phosphate (IC50 = 2.0 mM) and MgGTP (IC50 = 0.2-0.3 mM), which acted in synergism (IC50 is the concentration causing 50% inhibition). We conclude that the difference in adenylate catabolism between starved and H2O2-treated platelets is due to the distinct phosphate concentrations. During starvation, refeeding and H2O2 treatment, the values of the adenylate charge and the phosphorylation potential were kept closely co-ordinated, which may be effected by AMP deaminase.

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