Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Interactions of phenyl isocyanide (PheNC) with purified engineered cytochrome P450d wild type and putative distal mutants, Glu318Asp and Glu318Ala, were studied with optical absorption spectra. The wild type and the mutant Glu318Asp were purified as the high-spin state, while the mutant Glu318Ala was purified as the oxygen-bound low-spin form. Thus, it is suggested that Glu318 is important to make the appropriate heme environment of P450d. Spectral dissociation constants (0.19-0.39 mM) of the ligand for the ferric mutants were lower than that (0.74 mM) of the wild type. These dissociation constants were changed by adding a substrate, 7-ethoxycoumarin. The reduced wild type-PheNC complex showed a Soret peak at 451 nm, while the reduced mutant-PheNC complexes showed two peaks at 451 and 423 nm. The 451-nm peak of the complexes decreased with the concomitant increase of a new peak at 433 nm at room temperature. Thus, it was suggested that P450d can take two conformationally different forms from the characteristic spectral features. The Soret spectral conversions which followed the first-order kinetics were analyzed by changing the temperature. The activation energy (69 kcal/mol) for the conversion for the wild type was higher than those (37-50 kcal/mol) for the mutants. The activation energy for the wild type further increased (by 55%) by adding the substrate, while those for the mutants were essentially unchanged by adding the substrate. We discuss the important role of Glu318 at the putative distal site of P450d in the packing or the conformational stability of the putative distal site of the P450d molecule.     

Development and distribution of the early life stages of the longfin pearleye <Emphasis Type="Italic">Benthalbella linguidens</Emphasis> (Aulopiformes: Scopelarchidae) in the western North Pacific

The early life history and development of the scopelarchid Benthalbella linguidens was studied, based on 203 specimens (from 5.3 to 89.7mm in body length: BL) collected from Kuroshio, Oyashio waters and transition waters of the western North Pacific. The early life stages of B. linguidens are distinguished from those of other species of Benthalbella that inhabit the North Pacific by the characters of 62–64 myomeres in the larval stage and 26–28 anal fin rays in juvenile and transforming specimen. Larvae are elongate; notochord flexion begins at ca. 12mm BL and is completed at ca. 15mm BL. The fin ray complements are established at ca. 40mm BL. The single transforming specimen (89.7mm BL) that has peritoneal pigment was collected from transition waters. All larvae were collected from Kuroshio and transition waters from winter to early summer; however, the size of larvae in Kuroshio waters was apparently smaller than that in transition waters, with ranges of 5.3–32.4mm BL (mean 17.1) and 15.3–35.3mm BL (mean 27.5), respectively. Juveniles were distributed in transition and Oyashio waters and were absent in Kuroshio waters, where adults are commonly distributed. These occurrences of larvae and juveniles in the western North Pacific indicate that B. linguidens spawns in Kuroshio waters in winter and uses transition waters as nursery grounds.     

Biochemical characterization of pantoate kinase, a novel enzyme necessary for coenzyme a biosynthesis in the archaea

Although bacteria and eukaryotes share a pathway for coenzyme A (CoA) biosynthesis, we previously clarified that most archaea utilize a distinct pathway for the conversion of pantoate to 4'-phosphopantothenate. Whereas bacteria/eukaryotes use pantothenate synthetase and pantothenate kinase (PanK), the hyperthermophilic archaeon Thermococcus kodakarensis utilizes two novel enzymes: pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). Here, we report a detailed biochemical examination of PoK from T. kodakarensis. Kinetic analyses revealed that the PoK reaction displayed Michaelis-Menten kinetics toward ATP, whereas substrate inhibition was observed with pantoate. PoK activity was not affected by the addition of CoA/acetyl-CoA. Interestingly, PoK displayed broad nucleotide specificity and utilized ATP, GTP, UTP, and CTP with comparable k(cat)/K(m) values. Sequence alignment of 27 PoK homologs revealed seven conserved residues with reactive side chains, and variant proteins were constructed for each residue. Activity was not detected when mutations were introduced to Ser104, Glu134, and Asp143, suggesting that these residues play vital roles in PoK catalysis. Kinetic analysis of the other variant proteins, with mutations S28A, H131A, R155A, and T186A, indicated that all four residues are involved in pantoate recognition and that Arg155 and Thr186 play important roles in PoK catalysis. Gel filtration analyses of the variant proteins indicated that Thr186 is also involved in dimer assembly. A sequence comparison between PoK and other members of the GHMP kinase family suggests that Ser104 and Glu134 are involved in binding with phosphate and Mg(2+), respectively, while Asp143 is the base responsible for proton abstraction from the pantoate hydroxy group.     

Analysis of anchor residues in a naturally processed HLA-DR53 ligand

 The peptide motif of the HLA-DR53 (DRB4^*0101) molecule, which is associated with autoimmune diseases including Vogt-Koyanagi-Harada’s syndrome, was determined by peptide binding assay using human L plastin p581 – 595 peptide and its substituted analogues. L plastin p581 – 595 peptide is one of the naturally processed peptides bound to HLA-DR9/DR53 (DRB1^*0901/DRB4^*0101) molecules. The binding affinity of each peptide to the HLA-DR53 molecule was measured by fluorescence intensity of biotinylated peptides to L cell transfectants expressing HLA-DR53 molecules, followed by treatment with avidin-fluorescence. Binding of biotinylated peptides to HLA-DR53 molecules was not inhibited by all single-alanine-substituted nonbiotinylated peptides, indicating that the replaced position was important for binding to the HLA-DR53 moleule. The inhibitory motif is considered to be an HLA-DR53-specific binding motif, composed of a positively charged residue (K) at position 1, a hydrophobic residue (I) at position 4, positively charged residue (R or K) at position 8 or 9, and another hydrophobic residue (I) at position 10. This predicted motif is different from the binding motifs of other HLA-DR molecules. Received: 29 April 1996 / Revised: 16 June 1996     

Expression and function of proteins during development of the basal region in rice seedlings

A differential display of proteins with a two-dimensional polyacrylamide gel electrophoresis approach was used to analyze protein expression changes during development of the basal region in rice seedlings (Oryza sativa L. cv. Nipponbare). The proteins were detected as 700 Coomassie Brilliant Blue-stained spots with pI values from around 3.5 to 9.0. A proteome reference map was established for the basal region of two-week-old seedlings. The basal region proteome map was used to analyze quantitative variations in the tissue during development from 2-, 4-, 6-, 8-, and 10-week-old seedlings. During development, 31 proteins were up-regulated, and 30 proteins were down-regulated compared with the 2-week-old basal region proteome map. The main functions of these proteins were primary metabolism and protein synthesis or maintenance. Calreticulin precursor, enolase, and voltage-dependent anion channel were identified among the up- and down-regulated proteins. The twin spots of calreticulin precursor and enolase with different pI values are possibly due to post-translational modifications such as phosphorylation. In addition, seven proteins showed developmental stage-specific expression. All of the developmentally regulated proteins of the basal region were clustered by the S-system, a differential equation that fit to time course of cluster and analyzed for cluster relationships. Proteins with unknown functions were tentatively assigned to functional groups based on cluster relationships. Basal region development proteome data will be valuable for resolving questions in functional genomics. In addition, cluster analysis of the basal region proteome during development will be useful for the assessment of functional proteins.     

Exercise-induced left bundle branch block and subsequent mechanical left ventricular dyssynchrony--resolved with pharmacological therapy

A 53-year-old man with depressed ejection fraction (EF) of 35% and QRS width of 88 ms at rest was admitted to our institution with a complaint of exertional chest discomfort and dyspnea. During treadmill exercise, left bundle-branch block (LBBB) with a QRS width of 152 ms occurred at a heart rate of 100 bpm. During LBBB, the patient showed significant mechanical dyssynchrony as evidenced by a two-dimensional speckle tracking radial strain of 260 ms (≥ 130 ms), defined as the time difference between anterior-septum and posterior wall. Five-month after carvedilol and candesartan administration, EF had improved to 49% and LBBB did not occur until a heart rate of 126 bpm was attained during treadmill exercise. It appears that pharmacological therapy may be useful for patients with heart failure and exercise-induced LBBB.     

A comparison of the annual changes in testicular activity and serum androgen levels between the early and delayed maturing groups of male Cottus hangiongensis

Synopsis Annual changes in testicular activity and concentration of two serum androgens were monitored in two groups of the river-sculpin Cottus hangiongensis collected from the upper and lower reaches of a river at southern Hokkaido, Japan. One of them (early maturing group) underwent testicular maturation with aberrant spermatids and spermatid masses produced during the reproductive cycle. Moreover, regular seasonal changes in serum testosterone and 11-ketotestosterone concentrations were observed. On the other hand, in the other group (delayed maturing group), although body size of the fish was large enough to undergo reproduction, annual changes in gonadosomatic index and testicular activity did not vary much. During the months of active testicular development in the early maturing group, spermatogenesis was observed to begin in some regions of the testes of delayed maturing fish, but always resulted in the formation of aberrant spermatids and spermatid masses. Moreover, concentration of serum androgens did not significantly vary throughout the year. Results suggest that low androgen production is a proximal factor for delayed sexual maturity in the delayed maturing group, and that the occurrence of aberrant spermatids and spermatid masses during spermatogenesis is not linked to the delayed maturity.     

Effects of pulsed electrical stimulation on growth factor gene expression and proliferation in human dermal fibroblasts

Molecular and Cellular Biochemistry - Human dermal fibroblast proliferation plays an important role in skin wound healing, and electrical stimulation (ES) promotes skin wound healing. Although the...     

Synthesis of betulin derivatives and their protective effects against the cytotoxicity of cadmium

The protecting effect of betulin against cadmium toxicity was investigated using 11 kinds of analogues. It was elucidated by analyzing the analogue activities that both hydroxyl groups on C-3 and C-28 and the isopropenyl group on C-19 played important roles for expressing efficient activities. In addition, the cytotoxicity of betulin was also reduced by being functionalized using the above functional group.