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Biomedical subjects

T Saitoh

Publications and source records attributed to T Saitoh.

At least 451 records · Page 25Linked to original sources
PubMed1981

Dissection of the 66 000-dalton subunit of the acetylcholine receptor.

The 66 000-dalton or delta subunit of the acetylcholine receptor from Torpedo marmorata was covalently labeled in the presence of carbamoylcholine by 5-azido [3H]trimethisoquin (5-A[3H]T), a photoaffinity derivative of the local anesthetic trimethisoquin. After the attack of purified receptor with increasing concentrations of trypsin, the delta chain successively yielded fragments with apparent molecular weights of 50 000 (distinct from the beta subunit and referred to as the 50 000-bis (fragment), 49 000, and 47 000. With nondenatured (sodium cholate solubilized or membrane-bound) receptor, the 47 000-dalton fragment was not sensitive to trypsin and contained all of the covalent 5-A[3H]T label. This fragment was still glycosylated and had the same amino acid N terminus, valine, as the intact delta chain. A specific in vitro phosphorylation site of the delta subunit was located between the 49 000- and 50 000-dalton trypsin cleavage fragment and most likely is exposed to the cytoplasmic side of the membrane. A 16 000-dalton fragment of the delta chain was identified, which carriers a disulfide bond (or bonds) capable of cross-linking nonreduced receptor 9S monomerse into 12S dimers. The fragment did not remain associated with the receptor molecule after trypsin treatment.

Animals↗
PubMed1981

Dietary therapy in two patients with vitamin B12-unresponsive methylmalonic acidemia.

The biochemical and therapeutic responses to dietary therapy were studied in a 25-month-old girl and a 1-month-old girl with methylmalonic acidemia (MMA-emia), which was unresponsive to vitamin B12. The minimum daily intake of protein which patients could tolerate and display a good development was between 1.0 and 1.2 g per kg body weight. Supplementation with amino acid mixture devoid of toxic amino acids was required to prevent protein malnutrition when daily protein intake was restricted to 0.6 g per kg body weight. Caloric intake should be sufficient, not only to promote growth but also to prevent a rise in MMA level, especially when a patient has ketoacidosis. It was found that MMA excretion per mg creatinine in random urine specimens correlated significantly with serum MMA and twenty four-hour output of MMA per kg body weight. Therefore measurement of MMA in a single urine specimen is useful for evaluating the in vivo accumulation of MMA.

Acidosis↗
PubMed1981

Change in state of phosphorylation of acetylcholine receptor during maturation of the electromotor synapse in Torpedo marmorata electric organ.

Two populations of membrane fragments, both rich in acetylcholine receptor (AcChoR), appeared during subcellular fractionation by ultracentrifugation of neonatal Torpedo marmorata electric organs. One of these equilibrated at 38.5% (wt/wt) sucrose, as did AcChoR-rich membranes from adult fish; the other equilibrated at 36.8% sucrose. AcChoR purified from these light membrane fractions gave the same subunit profile as adult AcChoR (after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) but was more susceptible to heat inactivation and focused at an isoelectric point more alkaline by 0.1 pH unit. Treatment of adult AcChoR with Escherichia coli alkaline phosphatase decreased its thermal stability and shifted its isoelectric point towards alkaline pH. However, identical treatment did not affect AcChoR purified from neonatal light membrane fractions. The gamma and delta chains of AcChoR can be phosphorylated in vitro by endogenous protein kinases, which copurify with AcChoR-rich membranes. Treatment of AcChoR from neonatal light membranes by E. coli alkaline phosphatase enhanced the phosphorylation of the gamma and delta chains but did so to a smaller extent than in the case of adult AcChoR. In conclusion, adult AcChoR appears to be more phosphorylated than AcChoR from neonatal light membranes, indicating that its state of phosphorylation changes during development.

Alkaline Phosphatase↗
PubMed1980

Phosphorylation in vitro of membrane fragments from Torpedo marmorata electric organ. Effect on membrane solubilization by detergents.

Acetylcholine receptor-rich membrane fragments purified from Torpedo marmorata electric organ were phosphorylated, in vitro, by endogenous protein kinases. The 40 000-Mr chain, which carries the acetylcholine receptor site, was never labelled; on the other hand, protein bands of apparent molecular weights 43 000, 50 000 and 66 000, which are present in the acetylcholine receptor-rich membranes, were repeatedly phosphorylated. The phosphorylation of these three peptides required the presence of divalent cations, such as Mg2+ or Mn2+, and was, in addition, stimulated up to 3--5-fold by K+. The effect of Na+ ions appeared less specific since Na+ ions reduced the labelling of all the polypeptides susceptible to phosphorylation. Cholinergic agonists and antagonists, local anesthetics and cyclic nucleotides did not affect the phosphorylation of the receptor-rich membranes. Phosphorylation selectively modified the solubilization of several polypeptides by nondenaturing detergents: phosphorylated 43 000-Mr, 50 000-Mr and 66 000-Mr polypeptides were solubilized at lower concentrations of detergent than their non-phosphorylated counterparts. Two-dimensional gels revealed the existence of a charge heterogeneity of the 40 000-Mr and 43 000-Mr chains. The microheterogeneity of the 43 000-Mr chain, but not that of the 40 000-Mr chain, might result from a selective phosphorylation of this particular chain.

Acetylcholine↗
PubMed1979

Subunits of RNA polymerase in function and structure. 7. Structure of premature core enzyme.

The structure of premature core enzyme, an obligatory intermediate in both in vivo and in vitro assembly of Escherichia coli DNA-dependent RNA polymerase, was compared with that of native core enzyme. Though this assembled but inactive form of core enzyme harbors the gross conformation similar to that of native enzyme, minor and presumably local differences exist, which were identified by near-ultraviolet circular dichroism spectra, tritium-hydrogen exchange rate, protease sensitivity, intersubunit cross-linking rate by bifunctional reagents, sedimentation behavior, and elution profile from phosphocellulose. Taken together these results indicate that the core enzyme subunits are loosely associated in the premature core. The temperature-dependent maturation is required for the core subunits to be tightly associated, leading to the formation of structurally stable and functionally active RNA polymerase.

Circular Dichroism↗
PubMed1979

Subunits of RNA polymerase in function and structure. 8. Catalytic properties of self-reactivated core enzyme.

As an attempt to identify the maturation pathway of Escherichia coli RNA polymerase, the catalytic properties of core enzyme reactivated in the absence of maturation-promoting factors (sigma subunit or DNA) (that is, of self-reactivated core enzyme) were compared with those of native core enzyme. Differences have been found in the intrinsic activities such as in the template specificity, Km value of DNA template for the polymerase, activation energy for RNA synthesis, and increment of enzyme activity by sigma subunit. These observations imply that the transcription initiation by self-reactivated core enzyme is inaccurate and, therefore, more strict conditions including the presence of maturation-promoting factors are required for premature core be activated to the genuine function with the transcription specificity of native core enzyme.

DNA-Directed RNA Polymerases↗