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Biomedical subjects

H C Pant

Publications and source records attributed to H C Pant.

At least 91 records · Page 5Linked to original sources
PubMed1984

Effect of ethanol on calcium metabolism in rat erythrocyte ghosts.

A metallochromic indicator dye Arsenazo III (Az) was employed to study the effect of ethanol on calcium metabolism in rat erythrocyte ghosts. Addition of 10-150 mM ethanol to a ghost suspension induced a concentration dependent increase in calcium-Arsenazo III (Ca-Az) complex absorbance. Similar results were obtained when ghost suspensions contained (1-10 mM) theophylline. The effect of both, ethanol and theophylline was not observed if the ghosts were pre-treated with 50 microM dantrolene. Theophylline has been reported to stimulate the intracellular release of calcium in a number of cell types and the drug dantrolene is known to inhibit the intracellular release of calcium from the skeletal muscle and nerve cells. These results suggest that ethanol may release calcium from intracellular sites in these ghosts.

Animals↗
PubMed1983

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes.

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.

Animals↗
PubMed1983

Temperature dependent conformational changes in calmodulin.

Calcium bound calmodulin undergoes a reversible conformational change in the temperature range (22-28 degrees C). The transition temperature depends upon the concentration of calcium bound to calmodulin. The transition occurs at higher temperature (28 degrees C) in fully Ca2+ bound calmodulin as compared to those with lower Ca2+ concentrations (22 degrees C). The sequence of filling the four Ca2+ binding sites is different for the two temperature dependent conformers.

Binding Sites↗
PubMed1983

Calcium-dependent 4-aminopyridine stimulation of protein phosphorylation in squid optic lobe synaptosomes.

When intact synaptosomes were incubated with [gamma-32P]ATP, maximal protein phosphorylation was attained 2 min after the start of incubation. Protein phosphorylation under basal conditions was dependent on external Ca2+, and the dominant peak of phosphorylation was a 50-kd protein. Incubation of intact synaptosomes in the presence of 3-6 mM 4-aminopyridine (4-AP) caused a markedly enhanced phosphorylation of high molecular weight proteins of 90, 100, 130, and 180 kd, with no increase in the 50 or 38 kd proteins. This effect of 4-AP was dependent on external calcium ions in the incubation medium. The 4-AP effect on the high molecular weight proteins was also found in synaptosomal plasma membranes isolated from the synaptosomes. Tetraethylammonium (TEA) ions did not produce this enhancement of phosphorylation.

4-Aminopyridine↗
PubMed1982

Distribution of calcium-activated protease activity and endogenous substrates in the squid nervous system.

Calcium-activated protease (CAP) activity was studied in various neural tissues of the squid using endogenous (neurofilament protein) and exogenous ([14C]casein) substrate assays. Both assays demonstrated a significant CAP activity in perikarya from stellate ganglia, in axoplasm extruded from the giant axon, and in squid retinal fibers. The endogenous protein substrates in the perikarya and axons were 60,000 and 200,000 daltons, respectively. The Km for the CAP degradation of [14C] casein in axoplasm was about 2 x10-6 m. In contrast, both assays detected no CAP activity nor endogenous substrate in nerve terminals (synaptosomes from squid optic lobe). The absence of both CAP activity and endogenous substrate in nerve endings suggests that the axonal neurofilaments are degraded by CAP at the axon-nerve ending junction, followed by an autoinactivation of the CAP. Consistent with this hypothesis is that exposure of axoplasmic CAP to calcium leads to a rapid degradation of axonal neurofilament protein (t 1/2 less than 2 min) and a slower inactivation of the CAP (t 1/2 = 90 min). Axonal CAP requires a relatively high concentration of CA2+ sensitivity form of CAP found in other tissues.

Animals↗
PubMed1982

Changes in erythrocyte ghost protein phosphorylation associated with physical dependence upon ethanol.

The effects of ethanol on phosphorylation in the erythrocyte ghost membranes was studied. Four groups of rats were used: controls; prodromal phase; overt withdrawal syndrome; and treated with a single dose (6g/kg) of ethanol. Spectrin (Band 2) phosphorylation was enhanced during prodromal phase (200 +/- 12%) and during overt ethanol withdrawal syndrome (150 +/- 18%), while a single dose of ethanol produced no significant change. Addition of micromolar calcium chloride solution to the phosphorylating medium produced pronounced decrease in spectrin phosphorylation in the ghost membranes isolated from controls and those treated with a single dose of ethanol, while this effect of calcium was less pronounced in the ghost membranes isolated from rats in the prodromal phase and in the withdrawal syndrome. This suggests that due to prolonged ethanol treatment membrane phosphorylating properties were less sensitive to the change in calcium concentration. The membrane polypeptide composition was unaffected upon ethanol treatment.

Alcoholism↗
PubMed1980

Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins.

Axoplasm extruded from the giant axon of the squid contains Ca2+-activated proteases. The protease in the 100,000 x g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF200), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca2+ ion, and no other divalent cation will substitute. The protease requires Ca2+ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF200 appears to proceed through a 100,000 MW and possibly a 47,000--50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu2+ and Fe2+, sulphydryl inhibitors, and leupeptin. This specific Ca2+-activated protease in squid axoplasm has identical properties to Ca2+-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca2+-activated protease purified from human platelets effectively degrades squid NF200, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.

Animals↗
PubMed1979

Divalent cation dependent phosphorylation of proteins in squid giant axon.

In vitro and in situ (after intracellular infusion) incubation of axoplasm from the squid giant axon with [gamma-32P]ATP produces a phosphorylation of primarily two proteins (of mol.wt. 200,000 and greater than 400,000). The phosphorylation of these proteins is stimulated by Mg2+, inhibited by Ca2+, and unaffected by 10(-7) to 10(-5) M cyclic nucleotides. The 200 kdalton and greater than 400 kdalton phosphorylated peaks appear to be neurofilament proteins, and phosphorylation of these peaks in situ is decreased by electrical stimulation of the axon.

Adenosine Triphosphate↗
PubMed1979

Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.

Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.

Adenosine Triphosphate↗
PubMed1979

Phosphorylation of specific, distinct proteins in synaptosomes and axons from squid nervous system.

Synaptosomes and axons from squid were incubated with [gamma-(32)P]ATP or [(32)P]orthophosphate and specific, distinct proteins were found to be labeled in each preparation. In axoplasm, only the major 200,000 M(r) neurofilament protein and a specific protein of approximately 400,000 M(r) were labeled, as reported previously [Pant, H. C., Shecket, G., Gainer, H. & Lasek, R. J. (1978) J. Cell Biol. 78, R23-R27]. These results were independent of whether the cosubstrates were (32)PO(4) (2-) or [gamma-(32)P]ATP. However, synaptosomes lacked the 200,000 M(r) neurofilament protein and several lower molecular weight proteins were labeled instead, the most prominent being a 47,000 M(r) species. [gamma-(32)P]ATP was much more effective in labeling the 47,000 M(r) species than (32)PO(4) (2-). Synaptosomes also contained a distinct 250,000 M(r) protein species which, however, was not labeled. The protein kinase activity in synaptosomes was sensitive to various pharmacological agents, depending on whether the labeled phosphate came directly from ATP or orthophosphate. Carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, a mitochondrial H(+) uncoupler, almost completely inhibited incorporation of (32)P into protein with (32)PO(4) (2-) as cosubstrate, as expected, but produced only 32% inhibition with [gamma-(32)P]ATP as cosubstrate. The activity could be augmented by incubating synaptosomes in a calcium-free medium and could be suppressed by increasing intrasynaptosomal Ca(2+) with A23187, a Ca(2+) ionophore. The latter effect was more prominent with (32)PO(4) (2-) than with [gamma-(32)P]ATP as cosubstrate. Depolarizing agents such as veratridine and high K(+) also suppressed activity, and the veratridine effect was completely reversed by tetrodotoxin or by omission of Ca(2+) when [gamma-(32)P]ATP was used, and partially reversed when (32)PO(4) (2-) was used. We conclude that the morphological transformation of an axon into a terminal is accompanied by significant changes in protein and phospho-protein composition that may be related to synaptic transmission.

Animals↗
PubMed1978

Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization.

The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive 125-I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-aminopyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, aparticular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.

Action Potentials↗
PubMed1978

An approach to the study of intracellular proteins related to the excitability of the squid giant axon.

The technique for covalently labeling proteins with 125I-labelled Bolton-Hunter reagent was used to determine the quantities of proteins released from the axoplasmic side of the squid axon membrane. The reagent could be introduced into the interior of the axon by the technique of intracellular perfusion, the radioiodination reaction being carried out in situ. Alternatively, the reaction could be carried out in vitro, i.e., by mixing the reagent with samples of proteins dissolved in the intracellular perfusion fluid collected from the axon. This technique was found to be sensitive enough to permit analysis of a large number of protein samples collected from a single axon. By the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis, it was found that proteins of approx. 56 000 daltons were released into the perfusate when a solution of potassium chloride or potassium bromide was introduced into the interior of an axon. Suppression of axonal excitability was associated with this release of proteins. The significance of these findings in relation to the structure and function of the axon is discussed.

Action Potentials↗
PubMed1978

Neurofilament protein is phosphorylated in the squid giant axon.

We have observed the phosphorylation of neurofilament protein from squid axoplasm. Phosphorylation is demonstrated by 32P labeling of protein during incubation of axoplasm with [gamma-32P]ATP. When the labeled proteins are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two bands, at 2.0 x 10(5) daltons and greater than 4 x 10(5) daltons, contain the bulk of the 32P. The 2.0 x 10(5)-dalton phosphorylated polypeptide comigrates on SDS-PAGE with one of the subunits of squid neurofilament protein. Both major phosphorylated polypeptides co-fractionate with neurofilaments in discontinuous sucrose gradient centrifugation and on gel filtration chromatography on Sepharose 4B. The protein-phosphate bond behaves like a phospho-ester, and labeled phospho-serine is identified in an acid hydrolysate of the protein. The generality of this phenomenon in various species and its possible physiological significance are discussed.

Axons↗
PubMed1978

Effect of active immunization against oestrogens on plasma gonadotrophins in the ewe and the response to synthetic oestrogen or LH.

Immunization against oestrogen resulted in elevated LH concentrations, in the form of a pulsatile release, which rose from 3 to as much as 41 ng/ml approximately every 1.5 h, even in the presence of high plasma progesterone concentrations. Elevated FSH concentrations showed only minor oscillations without consistent synchrony to the LH pulses. Injection of 250 microgram stilboestrol did not abolish the LH pulses but in 2 out of 8 ewes FSH was initially lowered. Injection of 1 mg stilboestrol abolished the LH pulses within 11 h and decreased FSH values in 2 out of 4 ewes. Between 16 and 35 h after injection there was a large increase in LH and FSH concentrations. Thus, in the ewe, the tonic secretion of LH and FSH is controlled by a negative feedback action of oestrogen, and diethylstilboestrol will exert both dose. Infusion of LH was unable to alter the frequency or height of the LH pulses, thereby excluding regulation of the LH pulses by a short feedback mechanism.

Animals↗
PubMed1978

The effect of passive immunization against oestrogens on the onset of parturition in the ewe.

Six ewes were treated twice daily with intravenous injections of antiserum to oestrogens and 3 ewes received normal sheep serum from Day 140 of gestation until parturition. All 9 ewes experienced normal parturitions and produced healthy active lambs after similar gestation lengths. Progesterone and prostaglandin F concentrations in maternal jugular venous plasma were similar in the immunized and control ewes, but total unconjugated oestrogen concentrations were significantly higher in the immunized ewes (P less than 0.005). It is concluded that immunization of the ewe against total unconjugated oestrogens did not affect the normal occurrence of parturition.

Animals↗