Peter Kostka

Instability of malondialdehyde in the presence of H2O2: Implications for the thiobarbituric acid test

The content of thiobarbituric acid-reactive material (primarily malondialdehyde) is frequently used to estimate the extent of lipid peroxide formation. How-ever, malondialdehyde is unstable in the presence of millimolar concentrations of hydrogen peroxide. This observation considerably limits the applicability of the thiobarbituric acid test, as hydrogen peroxide is known to be formed in a number of lipid peroxidation-promoting systems. The instability of malondialdehyde in the presence of hydrogen peroxide seems to account for the inconsistent outcomes in studies relating the manipulations of intermediate H2O2 levels to the initiation of lipid peroxidation.

Sub cellular Fractionation of the Longitudinal Smooth Muscle/Myenteric Plexus of Dog Ileum: Dissociation of the Distribution of Two Plasma Membrane Marker Enzymes

Abstract: The distribution of plasma membrane markers, the sodium pump [evaluated as ouabain‐sensitive, potassium‐stimulated p‐nitrophenyl phosphatase (K+‐pNPPase)], [3H]saxitoxin binding, and 5′‐AMPase, was studied in the subcellular fractions prepared from the ho‐mogenates of the longitudinal smooth muscle/myenteric plexus of dog ileum. The K+‐pNPPase activity and [3H]‐saxitoxin binding were found to be predominantly associated with the synaptosomal fraction as indicated by the high level of these activities in the crude synaptosomal fraction and by the copurification of K+‐pNPPase and [3H]saxitoxin binding, but not 5′‐AMPase, with several synaptosomal markers during the fractionation of the crude synaptosomal fraction on density gradients. In contrast to the K+‐pNPPase activity and [3H]saxitoxin binding, the 5′‐AMPase activity was found to be concentrated in the microsomal pellet. Further fractionation of microsomes on a density gradient resulted in copurification of 5′‐AMPase, but not K+‐pNPPase or [3H]saxitoxin binding, with other smooth muscle plasma membrane‐bound enzymes, such as high‐affinity Ca2+‐ATPase, Mg2+‐ATPase, and Ca2+‐ATP‐ase. It was concluded that in the longitudinal smooth muscle/myenteric plexus, the sodium pump activity is present in higher density in the neuronal plasma membranes whereas 5′‐AMPase activity is concentrated in the smooth muscle plasma membranes.

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+.

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.

Presynaptic and postjunctional muscarinic receptors in dog ileum: binding studies.

[3H]N-Methylscopolamine identified two distinct populations of muscarinic receptors in membranes derived from the longitudinal smooth muscle/myenteric plexus of dog ileum. In isolated axonal varicosities, the half-maximal saturation of binding sites occurred at 2.38 +/- 0.39 nM [3H]N-methylscopolamine, with maximal binding capacity 140 +/- 35 fmol/mg protein (mean +/- S.D., n = 8). In purified smooth muscle plasma membranes, the Kd value was 16 +/- 3 nM with Bmax 1960 +/- 494 fmol/mg. The displacement potencies of subtype-selective muscarinic antagonists in the fraction of axonal varicosities followed the order 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methiodide much greater than pirenzepine = methoctramine greater than AF-DX 116 with pKi values 7.38, 5.67, 5.70 and 5.13, respectively. Both 4-DAMP methiodide and pirenzepine were approximately 4-fold less potent in displacing the ligand from the receptors in smooth muscle plasma membranes as compared to varicose receptors. The potency ratios of cardioselective antagonists methoctramine and AF-DX 116 on varicose and smooth muscle receptors were 1 and 1.7. It is concluded that presynaptic receptors located on isolated axonal varicosities have pharmacological properties similar to glandular (M3) subtype of muscarinic receptors. The binding properties of receptors present in smooth muscle plasma membranes were found incompatible with those of any of the M1, M2 or M3 subtypes.

Identification and characterization of high-affinity Ca2(+)-ATPase associated with axonal plasma membranes of dog mesenteric nerves.

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca2+-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with Km for Ca ions of 0.4 μM and Vmax=12.5±2.4 μmol Pi.mg−1h−1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive3H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with Ki=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >Zn≥Co>Ni. In contrast to low affinity (high Km) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 μM) and N-ethylmaleinimide (100 μM). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.

Calmodulin Stimulation of Plasmalemmal Ca2+-Pump of Canine Aortic Smooth Muscle

Plasma-membrane-enriched fractions of canine aortic smooth muscle possess an ATP-supported Ca2+ accumulation which has an absolute requirement for Mg2+ and a high affinity for Ca2+ (Km approximately 0.5 microM). The rate of ATP-supported Ca2+ transport is not affected by several calmodulin antagonists, but is stimulated by exogenously added calmodulin. The maximal effect of calmodulin on the rate of ATP-dependent Ca2+ transport (at 5.0 microM Ca2+) occurs at 10 micrograms/ml calmodulin and represents an approximately 3-fold stimulation. This calmodulin stimulation of Ca2+ transport does not require pretreatment of the membranes by EGTA and is an intrinsic property of the plasma membranes. A high-affinity Ca2+-ATPase (Km for Ca2+ approximately 0.5 microM) is also present in the aortic smooth muscle plasma membrane. This high-affinity Ca2+-ATPase does not require Mg2+ for catalytic activity, but is in fact inhibited by increasing Mg2+ concentrations. Calmodulin at concentrations effective for the stimulation of the ATP-dependent Ca2+ transport has no effect on the high-affinity Ca2+-ATPase activity or on the basal ATPase activity stimulated by 5 mM Mg2+ or Ca2+. Our results indicate that isolated plasma membranes of canine aortic smooth muscle contain no endogenous calmodulin. The ability of exogenously added calmodulin to stimulate the rate of ATP-dependent Ca2+ transport by vascular smooth muscle plasma membranes suggests that calmodulin may play a role in lowering the cytoplasmic concentration of ionized calcium during vasodilatation. An Mg2+-independent, but not an Mg2+-dependent high-affinity Ca2+-ATPase, was identified in the plasma membranes. This may be separate from the plasmalemmal Ca2+-pump.

Properties of potassium activatedp-nitrophenyl phosphatase of plasma membranes isolated from rat stomach muscle

The plasma membrane-enriched fraction isolated from smooth muscle of rat gastric fundus was found to contain a substantial level of potassium-stimulatedp-nitrophenylphosphatase activity (K-pNPPase), and its subcellular distribution closely resembled that of other plasma membrane enzyme markers. The kinetic profile of K-pNPPase and its sensitivity toward ouabain and vanadate confirmed the identification of this activity with the partial reaction of the sodium pump. The specific activity of K-pNPPase and its sensitivity to ouabain was significantly increased in the presence of saponin, indicating that part of this activity is latent when assayed on native membrane preparation. K-pNNPase was sensitive to the presence of calcium ions in the assay medium. The Ca2+-inhibition of K-pNNPase was accompanied by increased sensitivity of the enzyme to ouabain. On the other hand, calmodulin and Ca antagonists had no effect on K-pNPPase activity nor its sensitivity to calcium.