Purification of retinol dehydrogenase from bovine retinal rod outer segments

We purified retinol dehydrogenase from bovine rod outer segments using polyethylene glycol precipitation and hydroxylapatite, concanavalin A-Sepharose CL-4B, and Sepharose CL-6B column chromatography in the presence of NADP. We obtained 13-fold purification of retinol dehydrogenase with specific activity of 61.8 nmol/min/mg and 3.8% recovery. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that retinol dehydrogenase had a molecular mass of 37,000 daltons. The Km values of purified retinol dehydrogenase for all-trans retinol and all-trans retinal were 6.6 mM and 0.085 mM, respectively. The purified enzyme reacted with the all-trans retinal but not with 13-, 11-, and 9-cis compounds. In addition, we prepared antibody to retinol dehydrogenase using rat. The anti-retinol dehydrogenase antibody precipitated retinol dehydrogenase activity and was confirmed to bind to 37-kDa protein by Western blotting. We also found that anti-retinol dehydrogenase antibody bound to bovine rod outer segments specifically by immunohistochemical technique. The molar ratio of retinol dehydrogenase to opsin in rod outer segments estimated by enzyme-linked immunosorbent assay was 1:140.     

Tau protein kinase I converts normal tau protein into A68-like component of paired helical filaments.

From bovine brain microtubules we purified tau protein kinase I (TPKI, Mr 45,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and tau protein kinase II (TPKII) whose activity was attributed to a 30-kDa protein on SDS-PAGE by affinity-labeling using an ATP analog. Both kinases were activated by tubulin. TPKII, but not TPKI, phosphorylated tau fragment peptides previously used for detection of a Ser/ThrPro kinase activity. Therefore, TPKII was considered to be the Ser/ThrPro kinase. TPKI was more effective than TPKII for producing the decrease of tau-1 immunoreactivity and mobility shift of tau on SDS-PAGE. Moreover, TPKI, but not TPKII nor other well-known protein kinases, generated an epitope present on paired helical filaments. These findings suggested that tau phosphorylated by TPKI resembled A-68, a component of paired helical filaments.     

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on insulin release from rat pancreatic islets.

The role of protein phosphatases in the regulation of insulin release from rat pancreatic islets was studied with protein phosphatase inhibitors, okadaic acid and calyculin A. Okadaic acid inhibited glucose- and glyceraldehyde-induced insulin release dose-dependently and also inhibited the potentiation of glucose-induced release either by adding forskolin, an activator of adenylate cyclase or by increasing K+ concentration to 25 mM. At a non-stimulatory concentration of 3 mM glucose, a high concentration (2 microM) of okadaic acid inhibited insulin release induced by high K+ or 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, but a low concentration (1 microM) of okadaic acid did not significantly inhibit TPA-induced insulin release. Calyculin A also inhibited glucose-induced insulin release, and the effect was greater than that of okadaic acid. The data suggest that protein phosphatases may play an important role in the regulation of insulin release.     

Molecular cloning and sequence analysis of bovine T-cell receptor γ and δ chain genes

Nine bovine T-cell receptor (Tcr) chain (Tcrg) and three Tcr chain (Tcrd) cDNA clones were isolated from the cDNA libraries constructed from peripheral blood lymphocytes and thymocytes. Of nine Tcrg cDNA clones, only four were rearranged and contained specific V, J, and C gene segments, but the remaining five contained specific J and C or only C gene segments without the V gene segment. Three kinds of Tcrg-C, which were highly related at the nucleotide and amino acid levels, were found and designated as Tcrg-C1, Tcrg-C2, and Tcrg-C3. Compared with human and mouse Tcrg-C, bovine Tcrg-C sequences are much longer, with about 27–55 amino acids corresponding to the hinge and connector regions, where the characteristic repetitive 5-amino acid motif (TTEPP or TTKPP) exists in sheep Tcrg-C as previously reported. From three Tcrd cDNA clones, two Tcrd-V and three Tcrd-J segments were isolated. The nucleotide and deduced amino acid sequences of bovine Tcrd-C, especially the transmembrane and cytoplasmic domains, are well conserved among species. As in bovine Tcrg-C, diversity of amino acid residues in the Tcrd-C region is concentrated in the hinge regions. Southern blot analysis showed that there are at least three Tcrg-C genes and one Tcrd-C gene in the bovine genome. The analysis also revealed the presence of Tcrg-C- and Tcrd-C-associated restriction fragment length polymorphisms among bovine breeds.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D90409-20.     

Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Two new mutant loci (smhB and lytD) in Escherichia coli which confer temperature-sensitive growth and lysis phenotypes

A temperature-sensitive mutation in the murH gene of Escherichia coli confers a lysis phenotype at the restrictive temperature. An extragenic suppressor of murH apparently representing a new locus at 12.5 min on the linkage map and designated smhB is described. The smhB mutation by itself also conferred a temperature-sensitive lysis phenotype. A mutation in another new locus designated lytD which arose spontaneously in the smhB mutant was mapped close to smhB at 12.7 min on the linkage map. The lytD mutation by itself conferred a temperature-sensitive lysis phenotype indistinguishable from that of the murH mutant. Thus, the suppression of lysis in the smhB murH and the smhB lytD double mutants suggests a mechanism involving the reciprocal suppression of the two individual lysis-causing mutant alleles. The suppressor activity of smhB was apparently relatively specific in that smhB failed to prevent lysis induced by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. This suggests that murH, smhB, and lytD may be functionally related.     

Regulation of D-alanine carboxypeptidase and peptidoglycan cross-linkage in amino acid-deprived Escherichia coli.

The effect of amino acid deprivation on the activities of D-alanine carboxypeptidase (CPase) and peptidoglycan transpeptidase in Escherichia coli was determined. Enzymes were assayed in ether-treated bacteria (ETB) which were permeable to peptidoglycan nucleotide precursors. ETB were prepared at intervals from cultures grown in the presence and absence of a required amino acid. The specific activity of CPase in ETB decreased 50 to 85% during amino acid deprivation. This was paralleled by a 60 to 70% decrease in the specific activity of peptidoglycan transpeptidase. Both enzymes reached their lowest level of activity about 40 min after the onset of amino acid deprivation. The decrease in CPase activity apparently was not due to degradation of the enzyme, since full activity was restored after disruption of ETB by sonication. A decrease in CPase activity was associated with an enhancement of transpeptidation. The peptidoglycan synthesized in vitro by amino acid-deprived ETB was 1.7 times more cross-linked than the peptidoglycan synthesized by control ETB These results support the proposal that CPase may be involved in regulating transpeptidation in E. coli.     

Isolation of calmodulin from the protozoan, Tetrahymena pyriformis, by the use of a tubulin-Sepharose 4B affinity column

We developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.