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Biomedical subjects

J Ramwani

Publications and source records attributed to J Ramwani.

11 recordsLinked to original sources
PubMed1990

Phosphorylation of charge isomers (components) of human myelin basic protein: identification of phosphorylated sites.

Myelin basic protein isolated from normal human brain was resolved into its various components (charge isomers) by CM-52 column chromatography. Two of the components C-1 and C-4, were phosphorylated in vitro with a soluble preparation of brain protein kinase C. For each component, the peptides phosphorylated were identified. In both components a major site of phosphorylation was found at Ser7 in the N-terminal portion of the protein. Both the specific activity and the rate of phosphorylation were greatest at this site in both components when compared with the other sites. The rate of phosphorylation of peptide 5-13 was approximately 10 times greater than that of any of the other peptides derived from C-1, while the rate of phosphorylation of peptide 5-13 derived from C-4 was 10-20 times greater than that of any of the other peptides derived from C-4. In addition, peptide 5-13, which contained a major phosphorylation site in both C-1 and C-4, was phosphorylated at a faster rate in C-4 (460 cpm/nM/min) compared with C-1 (285 cpm/nM/min). Both the specific activity and the rate data presented in the present communication were correlated with the proportion of beta-structure in a previous study. In that study, C-1, which contained about 13% beta-structure before phosphorylation, increased to approximately 40% after phosphorylation. Construction of a model peptide of this N-terminal region, which included the phosphorylation site at Ser7, demonstrated that the beta-structure was stabilized by electrostatic interactions between the phosphate on Ser7 and the guanidyl groups of Arg5 and Arg9.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1988

Myelin basic protein binds GTP at a single site in the N-terminus.

Myelin basic protein from normal human brain was ADP-ribosylated with Cholera toxin, but not with Pertussis toxin. It bound azido-GTP at a single site in the N-terminal tetrapeptide at the Gln residue. The binding was considerably reduced when GppNHp was present during azido-GTP binding and totally inhibited when GTP gamma S was present. The relevance of this specific binding is not understood at this time.

Adenosine Diphosphate Ribose↗
PubMed1986

Purification of bovine striatal dopamine D-2 receptor by affinity chromatography.

Bovine striatal dopamine D-2 receptor has been purified approximately 2000-fold by affinity chromatography. The receptor, solubilized with cholic acid and sodium chloride, was adsorbed on haloperidol-linked Sepharose CL-6B and eluted with spiroperidol. The adsorption of receptor to the affinity matrix was biospecific as preincubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor. The process also displayed stereoselectivity with respect to (+)- and (-)-butaclamol. Nondopaminergic agents such as mianserin and propranolol failed to exhibit any effect on the adsorption process. Elution of the receptor was also biospecific, as dopaminergic drugs were most effective (spiroperidol greater than haloperidol greater than dopamine) in eluting the bound receptor; whereas other agents, e.g. propranolol, mianserin, and acetic acid, were only slightly effective. One-cycle affinity purification resulted in a recovery of 12% of the original membrane-bound dopamine D-2 receptor with a specific activity of 169,600 fmol/mg of protein as assayed with [3H]spiroperidol binding. The order of potency of D-2 agonists (N-propylnorapomorphine greater than NO434 greater than apomorphine greater than dopamine) and antagonists (spiroperidol greater than (+)-butaclamol greater than domperidone) with the purified preparation was found to be similar to that of the solubilized dopamine D-2 receptor.

Animals↗
PubMed1986

Characterization of high-affinity dopamine D2 receptors and modulation of affinity states by guanine nucleotides in cholate-solubilized bovine striatal preparations.

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.

Animals↗
PubMed1983

Factors affecting solubilized dopamine-sensitive adenylate cyclase.

Bovine striatal adenylate cyclase was solubilized with sodium cholate and assayed for its responsiveness to a variety of agents. Magnesium ions (0-20 mM), guanylyl-imidodiphosphate (10-50 microM), and striatal lipids were effective in increasing enzyme activity. The adenylate cyclase could be stimulated by dopamine, and neuroleptic drugs inhibited the effect of dopamine (50 microM) with potencies that paralleled their clinical potencies. The IC50 values for spiroperidol, haloperidol and chlorpromazine were 0.2, 3 and 500 nM respectively.

Adenylyl Cyclase Inhibitors↗
PubMed1983

The passive transfer of severe allergic neuritis in Lewis rats with lymphoid cells preincubated with P2 protein.

The passive transfer of both clinical signs and histologic lesions characteristic of allergic neuritis was successfully performed in Lewis rats using pooled spleen and lymph node cells, or T lymphocytes therefrom, if first preincubated in petri dishes with P2 protein for 72 hr. For passive transfer, cells were taken from donors 8-16 days after sensitization with P2 protein or myelin in Freund's complete adjuvant, and administered via the tail vein; clinical signs appeared 12-13 days later. This study supports the importance of cell-mediated immunity in EAN and the antigenic role of the P2 protein.

Animals↗
PubMed1983

A simple and efficient method for solubilization of the dopamine receptor.

The solubilization of dopamine receptors from bovine caudates was carried out with different detergents and detergent-salt combination. 0.25 M cholic acid: 1 M NaCl combination solubilized more effectively than the other detergents used. Solubilized receptors demonstrated the similar affinity and specificity as the membrane receptors. Solubilized receptors satisfied all the criteria of solubilization. Solubilized receptors were highly stable at 4 degrees C and freezing at -70 degrees C.

Animals↗
PubMed1981

Allergic neuritis: phospholipid requirement for the disease-inducing conformation of the P2 protein.

The P2 protein, a small, highly ordered basic protein of peripheral nerve myelin, is a potent inducer of allergic neuritis in rats when complexed with phospholipids such as phosphatidylserine. Isolated P2 protein administered without lipid is a poor neuritogen, and if first oxidized with performic acid, aminoethylated in 8 M urea or heat denatured, it loses nearly all activity. When the aminoethylated or oxidized forms are combined with phosphatidylserine, however, they recover essentially full neuritogenic activity. Complexing with lipid also greatly enhances the activity of the heart denatured form. Spleen cells sensitized to the aminoethylated and heated forms of P2 protein show a pronounced mitogenic response to either of these forms as well as to the P2 protein itself, but only when sensitization is initiated with the lipid complex. These data indicate that the lipid complex reverses the distortion acquired by chemical treatment or denaturation and converts the P2 molecule into a conformation approximating that of the native P2 protein in myelin. These studies imply that the neuritogenic domain, while highly sensitive to denaturing conditions, requires interaction with phospholipids in order to attain the most favourable conformation for inducing a cell-mediated response that leads to disease.

Animals↗
PubMed1981

Allergic neuritis: a neuritogenic peptide from the P2 protein that induces disease in rats.

Peptide CN1, a large 93 residue peptide, derived from residues 21-113 of the bovine and rabbit P2 protein of sciatic nerve myelin, induces severe allergic neuritis in Lewis rats. When complexed with phosphatidylserine and tested at 50 microgram dosage in Freund's complete adjuvant, it induces severe clinical and histologic signs (cellular infiltration and demyelination of the sciatic nerve) in most animals. It is as potent in disease induction as the P2 protein on a weight basis. In contrast, when not complexed with phosphatidylserine, Peptide CN1 induced only mild clinical signs and histologic lesions in 3 of 10 rats. CNBr peptides CN2 and CN3, derived from the carboxyl and amino terminal ends, respectively, were not active. Spleen and lymph node cells from rats sensitized to Peptide CN1 responded to both P2 and Peptide CN1 in culture in the mitogenic assay. These data show that the major neuritogenic domain for the rat resides in the CN1 region.

Animals↗