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.

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.

Fixed Speed Release System: Huperzine a Loaded Microspheres in Thermosensitive Methylcellulose Based Hydrogel

2011 ◽  
Vol 236-238 ◽  
pp. 2491-2494
Author(s):  
Rui Sun ◽  
Si Hao Chen ◽  
Chen Chen Xing
Keyword(s):  
In Vitro Release ◽  
Huperzine A ◽  
Injection System ◽  
Release System ◽  
In Situ Hydrogel ◽  
The Difference ◽  
Vitro Release

Huperzine A loaded microspheres are prepared using a W/O solvent evaporation method. The fixed speed release system is that microspheres composed with thermosensitive methylcellulose based hydrogel. SEM is used to analysis the surface morphology of Huperzine A loaded microspheres, which is shown that the difference of the size of microspheres. The size less than 200µm can be used as injection. Gelation experiment shows that the methylcellulose based solution can be transformed into in-situ hydrogel in 15 minutes at body temperature. The result of in vitro release shows that Huperzine A released in a nearly fixed speed. All the meterials of experiment can be biodegrated completely without operating to remove the gel. This is a promising long-term injection system, which is security and controlable for clinic.

AMP deaminase binding in contracting rat skeletal muscle

1992 ◽  
Vol 263 (2) ◽  
pp. C287-C293 ◽  
Author(s):  
K. W. Rundell ◽  
P. C. Tullson ◽  
R. L. Terjung
Keyword(s):  
Skeletal Muscle ◽  
Iodoacetic Acid ◽  
Amp Deaminase ◽  
Energy Demands ◽  
Slow Twitch Muscle ◽  
Fast Twitch ◽  
Resting Muscle

AMP deaminase, which hydrolyses AMP to inosine 5'-monophosphate (IMP) and NH3 at high rates during excessive energy demands in skeletal muscle, is activated when bound to myosin in vitro. We evaluated AMP deaminase binding in vivo during muscle contractions to assess whether binding 1) is inherent to deamination and found only with high rates of IMP production or simply coincident with the contractile process and 2) requires cellular acidosis. AMP deaminase activity (mumol.min-1.g-1) was measured in the supernatant (free) and 10(4)-g pellet (bound) homogenate fractions of muscle of anesthetized rats after in situ contractions to determine the percent bound. In resting muscle, nearly all (approximately 90%) AMP deaminase is free (cytosolic). During contractions when energy balance was well maintained, binding did not significantly differ from resting values. However, during intense contraction conditions that lead to increased IMP concentration, binding increased to approximately 60% (P less than 0.001) in fast-twitch and approximately 50% in slow-twitch muscle. Binding increased in an apparent first-order manner and preceded initiation of IMP formation. Further, binding rapidly declined within 1 min after cessation of intense stimulation, even though the cell remained extremely acidotic. Extensive binding during contractions was also evident without cellular acidosis (iodoacetic acid-treated muscle). Thus the in vivo AMP deaminase-myosin complex association/dissociation is not coupled to changes in cellular acidosis. Interestingly, binding remained elevated after contractions, if energy recovery was limited by ischemia. Our results are consistent with myosin binding having a role in AMP deaminase activation and subsequent IMP formation in contracting muscle.

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.

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion

1998 ◽  
Vol 274 (2) ◽  
pp. C465-C471 ◽  
Author(s):  
James W. E. Rush ◽  
Peter C. Tullson ◽  
Ronald L. Terjung
Keyword(s):  
Gastrocnemius Muscle ◽  
Amp Deaminase ◽  
Soluble Cell ◽  
Fast Twitch Muscle ◽  
Fast Twitch ◽  
Menten Constant ◽  
Soluble Cell Fraction

We examined a possible mechanism to account for the maintenance of peak AMP deamination rate in fast-twitch muscle of rats fed the creatine analog β-guanidinopropionic acid (β-GPA), in spite of reduced abundance of the enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined at saturating AMP concentration) and AMPD protein abundance (Western blot) were coordinately reduced ∼80% in fast-twitch white gastrocnemius muscle by β-GPA feeding over 7 wk. Kinetic analysis of AMPD in the soluble cell fraction demonstrated a single Michaelis-Menten constant ( K m; ∼1.5 mM) in control muscle extracts. An additional high-affinity K m (∼0.03 mM) was revealed at low AMP concentrations in extracts of β-GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high rates of deamination in β-GPA-treated muscle in situ, despite the loss of AMPD enzyme protein. The elimination of this kinetic effect by treatment of β-GPA-treated muscle extracts with acid phosphatase in vitro suggests that phosphorylation is involved in the kinetic control of skeletal muscle AMPD in vivo.

Sex distribution in arrested precompacted human embryos

Zygote ◽  
1993 ◽  
Vol 1 (2) ◽  
pp. 155-162 ◽  
Author(s):  
Santiago Munné ◽  
Ya Xu Tang ◽  
Heinz-Ulrich G. Weier ◽  
Jonathan Stein ◽  
Michelle Finkelstein ◽  
...  
Keyword(s):  
Development Rate ◽  
Human Embryos ◽  
Gonadal Differentiation ◽  
Sex Distribution ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
The Difference ◽  
Developmental Block

Evidence of sexual dimorphism before fetal gonadal differentiation in mammals has been accumulating, suggesting that male embryos develop faster than female ones. The current investigation was performed to evaluate whether the development rate of precompacted human embryos is controlled by sex chromosomes. Sex was determined by polymerase chain reaction and fluorescence in situ hybridisation in 172 arrested embryos derived from in vitro fertilisation. The sex ratio (1.02:0.98) did not differ significantly from 1:1. Although more males appeared to have greater fragmentation, the difference between the sex ratios of highly fragmented and normal embryos (1.08:0.92) was not significant. Arrested female embryos had a tendency to exhibit more than five nuclei and less than 10% fragmentation, but the trend was not statistically significant. The current results suggest that the first developmental block in human embryos occurs prior to and shortly after genomic activation and is not determined by the presence of the Y chromosome.

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