Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0-8.0. Addition of tryptophan also decreased the Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.     

Enhancement of inositol phospholipid metabolism and activation of protein kinase C in ras-transformed rat fibroblasts

The inositol phospholipid metabolism is one of the main pathways of signal transduction in cells. We measured the activities of its key enzymes in v-Ha-ras-transformed 208F rat fibroblasts. In the ras-transformed clones, incorporation of [32P]Pi into intermediates of the inositol phospholipid metabolism was stimulated. The activities of phosphatidylinositol and phosphatidylinositol-4-phosphate kinases in the transformed clones were about 35-50% more than in untransformed cells, indicating increased inositol phospholipid metabolism. However, the activity of diacylglycerol kinase in their membrane fraction was 25-35% less than that of untransformed cells, although the total diacylglycerol kinase activity did not change. The imbalance of these kinases could constitute one of the main reasons leading to the increased level of inositol phosphates and the accumulation of diacylglycerol to 2-2.2 times that in control 208F cells. Phosphatidylinositol-4,5-bisphosphate-phospholipase C activity did not change on the transformation when assayed under various conditions. The increased level of diacylglycerol caused intracellular translocation, activation, and down-regulation of protein kinase C changes which may be one of the essential events in transformation by the v-Ha-ras gene.     

Identification of new bile alcohols, 5 beta-cholestane-3 alpha,7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha,7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha,7 alpha,26,27-tetrol in human gallbladder bile

The nature of cholestanetetrols present as the glucurono-conjugates in human gallbladder bile was studied. Glucurono-conjugated bile alcohols were isolated by ion exchange chromatography and, after enzymatic hydrolysis, were fractionated by reversed phase partition chromatography to give a fraction containing tetrahydroxy bile alcohols which was analyzed by gas-liquid chromatography and mass spectrometry. Along with the three previously identified bile alcohols, 5 alpha- and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,24-tetrols, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha,26-tetrol, three new cholestanetetrols, possessing two hydroxyl groups in the ring system and two in the side chain, were detected in the tetrahydroxy bile alcohol fraction. These new bile alcohols were identified as 5 beta-cholestane-3 alpha, 7 alpha,24,26-tetrol, 5 beta-cholestane-3 alpha, 7 alpha,25,26-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha,26,27-tetrol by direct comparison of their gas-liquid chromatographic behaviors and mass spectral data with those of authentic standards prepared from chenodeoxycholic acid by partial synthesis.     

Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sex differences in gallbladder bile acid composition and hepatic steroid 12 alpha-hydroxylase activity in hamsters

The gallbladder bile acid composition and the activity of the hepatic steroid 12 alpha-hydroxylase were determined in male and female hamsters. Cholic acid, chenodeoxycholic acid, and deoxycholic acid were the major bile acids in both sexes; in addition, 7-ketodeoxycholic acid and lithocholic acid were present. A sex-linked difference in the ratio of cholic acid (plus its metabolites) to chenodeoxycholic acid (plus its metabolite) was observed. The ratio was 1.93 +/- 0.39 in males and 2.74 +/- 0.54 in females. Another sex-linked difference was found in the activity of the 12 alpha-hydroxylase. The extent of the 12 alpha-hydroxylation of 7 alpha-hydroxycholest-4-en-3-one to yield 7 alpha, 12 alpha-dihydroxycholest-4-en-3-one was about two times greater in the microsomal suspension obtained from the liver of female hamsters than in that of male hamsters. A positive correlation between the 12 alpha-hydroxylase activity and the ratio of cholic acid/chenodeoxycholic acid was also observed. These results strongly support the proposal that the activity of the 12 alpha-hydroxylase is the major factor in determining the relative proportion of cholic acid and chenodeoxycholic acid formed from cholesterol in the liver.     

Intramolecular cross-linkage of lysozyme. Imidazole catalysis of the formation of the cross-link between lysine-13 (epsilon-amino) and leucine-129 (alpha-carboxyl) by carbodiimide reaction

The salt bridge between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme was converted to an amide bond by 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) reaction in the presence of imidazole (0.3-1 M) at pH 5 and room temperature, followed by dialysis at pH 10. Absence of imidazole under a similar condition did not give this intramolecularly cross-linked lysozyme derivative (CL-lysozyme) but resulted in the formation of intermolecularly cross-linked lysozyme oligomers. From the mechanistic studies on the formation of CL-lysozyme, imidazole was suggested to play the following three roles. (1) Some carboxyl groups activated by EDC in lysozyme were converted to acylimidazole groups which protected them from the reaction with amino groups in other lysozyme molecules at pH 5. These could be hydrolyzed at pH 10 to regenerate free carboxyls. (2) High concentrations of imidazole (pH 5) increased the ionic strength of the solution which weakened the salt bridge in lysozyme and facilitated the activation of the alpha-carboxyl group by EDC. (3) The alpha-carboxyl group activated by EDC was converted to an acylimidazole group which could react with the epsilon-amino group of Lys-13 in the same molecule to form an amide bond. The last step may involve some conformational change of the backbone of lysozyme and be slower than the hydrolysis reaction of the alpha-carboxyl group activated by EDC itself. However, acylimidazole groups are stable against hydrolysis at pH 5. This may afford enough time to allow the epsilon-amino group of Lys-13 to attack the acylimidazole group of Leu-129.     

The reaction to c-banding of c-banded constituents during mitotic cycle ofCrepis capillaris

The reaction to C-banding was investigated throughout the mitotic cycle ofCrepis capillaris (2n=6): (1) 18–22 C-bodies or C-bands were found during mid telophase and interphase to prophase and metaphase, and also 12–14 at late anaphase to early telophase in the mitotic cycle. Fewer C-bands in late anaphase to early telophase were due to the absence of minute bands; (2) large and medium sized C-bands were strongly stained by Giemsa, while small and minute bands stained palely. It is suggested that inCrepis capillaris the difference of color in C-banded segments following Giemsa staining is referable to the amount of constitutive heterochromatin rather than to the difference in the condensation and decondensation; (3) the size of C-bodies changed during telophase to interphase and prophase. It is inferred that the extent of C-bodies is regulated by both the length of DNA sequences of constitutive heterochromatin and the amount of proteins combined with C-banded DNA. It was shown that the reaction to C-banding is neither due to the differential condensation of chromatin nor to a higher concentration of DNA in the C-banded regions, in the C-banding mechanism as has been suggested so far at least.     

Esterified and total 7 alpha-hydroxycholesterol in human serum as an indicator for hepatic bile acid synthesis

Serum levels of 7 alpha-hydroxycholesterol and activities of hepatic microsomal cholesterol 7 alpha-hydroxylase in surgical patients were analyzed by capillary gas-liquid chromatography-selected ion monitoring technique using a new internal standard, 5 alpha-cholestane-3 beta, 7 beta-diol. We found that concentrations of 7 alpha-hydroxycholesterol obtained after alkaline hydrolysis were higher than those without alkaline hydrolysis, indicating that a considerable amount of 7 alpha-hydroxycholesterol in human serum is present in esterified form. Esterified 7 alpha-hydroxycholesterol could also be quantitatively hydrolyzed with cholesterol esterase, suggesting that fatty acid is bound at the 3 beta-position of the cholestenediol. The serum levels of esterified and free 7 alpha-hydroxycholesterol in patients with cholelithiasis were 198.0 +/- 90.3 and 48.3 +/- 19.8 pmol/ml (mean +/- SD), respectively, and were similar to those in patients without hepatobiliary diseases. After treatment with chenodeoxycholic acid (300 mg per day) for 7 to 10 days, esterified and free 7 alpha-hydroxycholesterol levels decreased to 64.9 +/- 33.6 and 20.5 +/- 11.1 pmol/ml, respectively. Activity of cholesterol 7 alpha-hydroxylase was also inhibited. Treatment with ursodeoxycholic acid (600 mg per day) for 7 to 10 days had no inhibitory effect on serum 7 alpha-hydroxycholesterol levels and the enzyme activity. In all groups, high correlations were found between the activity of cholesterol 7 alpha-hydroxylase and serum levels of 7 alpha-hydroxycholesterol: free (r = 0.71, n = 38, P less than 0.001); esterified (r = 0.87, n = 38, P less than 0.001); total (r = 0.87, n = 38, P less than 0.001). Esterified and total 7 alpha-hydroxycholesterol was more highly correlated with the enzyme activity than the free form. We conclude that a significant amount of 3 beta-acyl esters of 7 alpha-hydroxycholesterol is present in human serum and that serum levels of esterified and/or total 7 alpha-hydroxycholesterol are likely to reflect the activity of hepatic cholesterol 7 alpha-hydroxylase and thus the amount of primary bile acids synthesized in the liver.     

Carbohydrate structures of nonspecific cross-reacting antigen-2, a glycoprotein purified from meconium as an antigen cross-reacting with anticarcinoembryonic antigen antibody. Occurrence of complex-type sugar chains with the Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----outer chains

Nonspecific cross-reacting antigen-2 (NCA-2) is a glycoprotein purified from meconium as a closely correlated entity with carcinoembryonic antigen (CEA). As in the case of CEA, only asparagine-linked sugar chains are included in NCA-2. In order to elucidate the structural characteristics of the sugar chains of NCA-2, they were quantitatively released from the polypeptide backbone by hydrazinolysis and reduced with NaB3H4 after N-acetylation. The radioactive oligosaccharides were fractionated by paper electrophoresis, serial chromatography on immobilized lectin columns, and Bio-Gel P-4 (under 400 mesh) column chromatography. Structures of the oligosaccharides were estimated from the data of the binding specificities of immobilized lectin columns and the effective size of each oligosaccharide determined by passing through a Bio-Gel P-4 column and were then confirmed by endo-beta-galactosidase digestion, sequential digestion with exoglycosidases with different aglycon specificities, and methylation analysis. NCA-2 contains a similar number (27 mol) of sugar chains in one molecule compared with CEA (24-26 mol). However, all sugar chains of NCA-2 were complex-type in contrast to CEA, approximately 8% of the sugar chains of which were high mannose-type (Yamashita, K., Totani, K., Kuroki, M., Matsuoka, Y., Ueda, I., and Kobata, A. (1987) Cancer Res. 47, 3451-3459). About 80% of the oligosaccharides from NCA-2 contain bisecting N-acetylglucosamine residues, and the percent molar ratio of mono-, bi, tri, and tetraantennary oligosaccharides was 2:14:57:27. (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----4(+/- Fuc alpha 1----3)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, (+/- Fuc alpha 1----2)Gal beta 1----3(+/- Fuc alpha 1----4)GlcNAc beta 1---- 3Gal beta 1----4GlcNAc, and GalNAc beta 1----3Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4GlcNAc were found as their outer chain moieties. Approximately 60% of the oligosaccharides from NCA-2 contain the Gal beta 1----4 or 3GlcNAc beta 1----3Gal beta 1----4GlcNAc beta 1----group in their outer chains.