Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41.

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Potentiometric study of cytochrome c1aa3 from Thermus thermophilus

We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus. In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE). Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution). The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB. The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration. It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.     

Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.     

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO₃⁻-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg²⁺, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.     

Binding of 8-bromoguanylic acid to ribonuclease T1 as studied by absorption and circular dichroism spectroscopy

8-Bromoguanosine 2'- and 3'-phosphates have been shown to bind to RNase T1 with the same affinity as the corresponding guanosine phosphates, inducing difference absorption and circular dichroism spectra similar to those induced by the guanosine phosphates. Since the brominated ligands have reduced electron density on N-7 of the guanine ring and syn-fixed conformation due to a bulky, electron-withdrawing Br substituent on C-8, the difference spectra are not attributable to the protonation on N-7 and to the restriction of the ligand to syn-conformation as proposed previously.     

Cell-free synthesis of mitochondrial ATPase inhibitor precursor and its transport into yeast mitochondria

ATPase inhibitor protein, which blocks mitochondrial ATPase activity by forming an enzyme-inhibitor complex, was found to be synthesized as a larger precursor in a cell-free translation system directed by yeast mRNA. Other protein factors, which stabilize latent ATPase by binding to the enzyme-inhibitor complex, were also found to be formed as larger precursors. The precursor of ATPase inhibitor protein was transported into isolated yeast mitochondria and was cleaved to the mature peptide in the mitochondria. Impaired mitochondria lacking phosphorylation activity could not convert the precursor to the mature form. Neither antimycin A nor oligomycin alone exhibited a marked effect on the transport-processing of the precursor by intact mitochondria. However, when antimycin A was added with oligomycin, the transport-processing was markedly inhibited. The processing was also strongly inhibited by an uncoupler, carbonylcyanide p-trifluoro-methoxyphenyl hydrazone. The inhibition by the uncoupler was not relieved by ATP added externally. It is concluded that the transport-processing of precursor proteins requires intact mitochondria with a potential difference across the inner membrane.     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

The effect of 2-deoxy-D-glucose on insulin release by neonatal rat B cells in culture

The culture for 7 days in medium with 5.5 mM glucose and 1 mM 2-deoxy-D-glucose enhanced the glucose sensitivity of neonatal rat B cells, and even stimulated their growth in vitro. Also, 2-deoxy-D-glucose supplementation maintained insulin release evoked by leucine and 2-ketoisocaproate from B cells at day 7 at levels several times higher than at day 1. The effect of leucine was greatly augmented by glutamine, whereas that of the 2-keto acid remained almost unchanged irrespective of the presence of glutamine. These results suggest an increase in oxidative catabolism of medium nutrients in B cells grown in medium with 2-deoxy-D-glucose for 7 days, and such metabolic changes may promote the growth of B cells in vitro.