Tetraborate concentration on Morgan-Elson reaction and an improved method for hexosamine determination.

Effect of tetraborate concentration on the determination of hexosamine by Morgan-Elson color reaction according to Levvy and McAllan [(1959) Biochem. J., 73, 127–232] was studied using the three different preparations of the tetraborate solution. The concentration of the previous report during the chromogen formation (0.29 m) was too high comparing to the optimum concentration of the present experiment (0.175 m). An improved method is now proposed which is excellent on the ground of convenience and reproducibility; same readings were obtained with the tetraborate solution from various preparations which were stable at least for two years. It showed same molar extinction coefficient as obtained before.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

The dissociation of aggregate forms of dextransucrase

Aggregate forms of dextransucrase obtained from Streptococcus sanguis dissociate in the presence of sodium dodecyl sulfate. However, the enzyme was unstable under these conditions. Nonionic detergents such as Triton X-100 stabilize the enzyme (A. W. Miller and J. R. Robyt, (1981) Fed. Proc. Fed. Amer. Soc. Exp. Biol.40, 1656), but do not cause disaggregation. The combination of both detergents, at concentrations below their critical micellar concentrations, is effective in dissociating and in stabilizing the enzyme. Polyacrylamide gel electrophoresis of the enzyme in the presence of the mixed detergents produced five major active enzyme forms, and two minor ones. The molecular weights of these forms were established by a modification of the procedure of Hedrick and Smith ((1968) Arch. Biochem. Biophys.113, 675–683).     

G.L.C. of the O-trimethylsilyl derivatives of hexuronic acids

The free acids, sodium salts, and lactones of several hexuronic acids have been studied as their O-trimethylsilyl derivatives by gas-liquid chromatography using SE-30 and XE-60 liquid phases. Silylation was best performed in methyl sulphoxide. The equilibrium between the various forms of a hexuronic acid in methyl sulphoxide was also studied by g.l.c. following silylation. The hexamethyldisilazane used in the silylation disturbed the equilibrium attained in the solvent, but this was overcome by premixing the hexamethyldisilazane with chlorotrimethylsilane. Methyl sulphoxide and the silylating reagents gave a two-phase system in which the derivative was favourably partitioned into the upper layer. Partition coefficients and stabilities of the derivatives were measured, and a g.l.c. method for the analysis of the hexuronic acids was thereby developed. The oximes of the hexuronic acids were studied as alternative derivatives for g.l.c., and their equilibrium compositions and g.l.c. retention times are recorded.     

Radioenzymatic assay for the acetohydroxy acid synthase-catalyzed synthesis of alpha-aceto-alpha-hydroxybutyrate

A method has been developed for radiolabeling small amounts of ribosomal proteins extracted from polyacrylamide gels with potassium [¹²⁵]Iiodide. The procedure was used to label even those proteins which lack tyrosine and histidine residues by the modification of proteins with methyl p-hydroxybenzimidate. Specific radioactivities obtained range from 20,000 to 200,000 cpm/μg. The method has been used in the identification of eukaryotic ribosomal proteins from rabbit reticulocytes separated by polyacrylamide/sodium dodecyl sulfate gel electrophoresis.     

Effect of carboxyl-reduced heparin on the growth inhibition of bovine pulmonary artery smooth muscle cells

Heparin (HP) inhibits the proliferation of bovine pulmonary artery smooth muscle cells (BPASMC’s), among other cell types in vitro. In order to develop a potential therapeutic agent to reverse vascular remodeling, we are involved in deciphering the relationship between the native HP structure and its antiproliferative potency. We have previously reported the influence of the molecular size and the effects of various O-sulfo and N-acetyl groups of HP on growth-inhibitory activity. In this study, to understand the influence of carboxyl groups in the HP structure required for endogenous activity, a chemically modified derivative of native HP was prepared by converting the carboxyl groups of hexuronic acid residues in HP to primary hydroxyl groups. This modification procedure involves the treatment of HP with N-(3-dimethylaminopropyl)-N-ethylcarbodiimide followed by reduction with NaBH₄ to yield carboxyl-reduced heparin (CR-HP). When compared to the antiproliferative potency of native HP on cultured BPASMC’s at three dose levels (1, 10, and 100 μg/mL), the CR-HP showed significantly less potency at all the doses. These results suggest that hexuronic acid residues in both major and variable sequences in HP are essential for the antiproliferative properties of native HP.     

Improvements on the Prescott-Jones method for the colorimetric analysis of ureido compounds

The colorimetric assay procedure of Prescott and Jones ((1969) Anal. Biochem.32, 408–419) has been modified to make the standard curve more consistently linear. Improved results are obtained when the assay tubes are exposed to yellow light at an intensity of 45–50 fc (480–540 lx) during color development. Somewhat longer incubation times are required for color development with yellow light.     

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase nucleases, (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowex 1-X2 (Cl^?) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2–0.3 μg hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (or C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.     

Synthesis and evaluation of novel substituted 1,2,3-triazolyldihydroquinolines as promising antitubercular agents

A series of novel substituted 1,2,3-triazolyldihydroquinolines 6a–o was designed and synthesized from 2-acetylthiophene in five-step reaction sequence involving modified Boltzmann-Rahtz reaction of β-Enaminone; Vilsmeier-Haack chloroformylation using DMF/POCl₃; Ohira-Bestmann homologation of aldehyde to alkyne as key steps. The reaction of alkyne 4 with various aryl azides in the presence of copper sulfate and sodium ascorbate resulted desired new 1,2,3-triazolyldihydroquinolines 6a–o in excellent yields. In vitro screening of new compounds for anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv (Mtb), resulted in three derivatives 6a (MIC:1.56?µg/mL) and 6d, 6l (MIC:3.12?µg/mL) as promising antitubercular agents with lower cytotoxicity profiles.     

Identification of proteins according to biological activity following separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis: analysis of human exocrine pancreatic proteins

A novel procedure is described whereby proteins can be identified according to their biological activity after their separation in two dimensions using isoelectric focusing and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (G. Scheele, 1975, J. Biol. Chem., 250, 5375–5385). This procedure includes an optimal staining method for the visualization of two-dimensional gel spots, which avoids the use of chemical fixatives, and a one-step method for elution and renaturation of proteins. Fifteen out of the twenty discrete proteins separated from human pancreatic juice by the two-dimensional gel method were successfully identified by this procedure.     

Design,synthesis and evaluation of 1,2,3-triazole-adamantylacetamide hybrids as potent inhibitors of Mycobacterium tuberculosis

A series of novel 1,2,3-triazole-adamantylacetamide hybrids 5a–u, designed by combining bioactive fragments from antitubercular I-A09 and substituted adamantyl urea, were synthesized using copper catalyzed click chemistry. N-(1-Adamantyl)-2-azido acetamide 3 prepared from 1-adamantylamine was reacted with a series of alkyl/aryl acetylenes in the presence of copper sulfate and sodium ascorbate to give new analogues 5a–u in very good yields. Evaluation of all new compounds for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv (ATCC27294), resulted N-(1-adamantan-1-yl)-2-(4-(phenanthren-2-yl)-1H-1,2,3-triazol-1-yl)acetamide (5t) as most promising lead MIC: 3.12 μg/mL) with selectivity index >15.     

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids

Laser-induced native fluorescence detection with a KrF excimer laser (λ=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-μm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3σ) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; α-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7–170 nM.     

Amine boranes as alternative reducing agents for reductive alkylation of proteins

The use of several commercially available amine-borane complexes was investigated for the reductive methylation of amino groups of several proteins. An earlier study in our laboratory, using turkey ovomucoid as the model protein, showed that dimethylamine borane is a slightly weaker reducing agent, but a good, less toxic substitute for sodium cyanoborohydride (K. F. Geoghegan, J. C. Cabacungan, H. B. F. Dixon, and R. E. Feeney, 1981, Int. J. Peptide Protein Res.17, 345–352). N-α-Acetyl-l-lysine, poly-l-lysine, turkey ovomucoid, bovine serum albumin, chicken ovalbumin, β-lactoglobulin, casein, and soybean protein were reductively methylated with dimethylamine borane and trimethylamine borane. The latter produced a consistently lower degree of modification even in the presence of sodium dodecyl sulfate. In a comparison that included the boranes triethylamine, t-butylamine, morpholine, and pyridine, pyridine borane was found to be slightly stronger than sodium cyanoborohydride. In a pH 7 solution containing 2 mmN-α-acetyl-l-lysine and 20 mm formaldehyde, complete dimethylation was achieved with about 10 mm pyridine borane after 2 h incubation at 22°C, while more than 15 mm was necessary with sodium cyanoborohydride. Like dimethylamine borane, both pyridine borane and triethylamine borane showed a reducing capacity at pH 7 which was as high as that at pH 9. Reductive alkylation under neutral to mild acid conditions allows modification of alkaline labile proteins and also limits the side reactions between proteins and formaldehyde.     

Specificity of the chlorophyll-to-protein binding in the chlorophyll-protein complexes of the thylakoid

We tried to establish whether the chlorophyll-protein complexes of the thylakoid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, originate from real entities existing in vivo, or are mere artifacts of the sodium dodecyl sulfate solubilization procedure. Making use of the finding that etiolated leaves exposed to periodic light form selectively the chlorophyll-protein complexes CPI and CPa, while after transfer to continuous light they form in addition the light-harvesting complexes (J. H. Argyroudi-Akoyunoglou, Z. Feleki, and G. Akoyunoglou, 1971, Biochem. Biophys. Res. Commun.45, 606–614; J. H. Argyroudi-Akoyunoglou and G. Akoyunoglou 1979, FEBS Lett.104, 78–84) we tried to see whether the latter complexes contain newly formed chlorophyll. We labeled the chlorophyll a formed in periodic light with δ-[¹⁴C]aminolevulinic acid, and determined the specific radioactivity of chlorophyll in the complexes formed before or after transfer to continuous light. We found that the light-harvesting complexes contain primarily newly formed and nonradioactive chlorophyll. The results suggest that (i) the chlorophyll a of CPI and CPa formed in periodic light does not exchange with that of the light-harvesting complexes formed after transfer to continuous light. (ii) The light-harvesting complexes formed after transfer to continuous light contain primarily newly formed chlorophylls a and b. (iii) The binding of chlorophyll to protein in the complexes is specific and not an artifact of the sodium dodecyl sulfate action. (iv) As the thylakoid membrane grows and differentiates, the chlorophyll synthesized binds on the apoproteins of the complexes in a stepwise manner.     

Nonidentical subunits of pyrocatechase from Pseudomonas arvilla C-1.

Pyrocatechase [catechol:oxygen, 1,2-oxidoreductase (decyclizing), EC 1.13.11.1] from Pseudomonas arvilla C-1 has been reported to contain 2 g atoms of iron/mol of enzyme, based on a molecular weight of 90,000, determined by sedimentation and diffusion constants (Y. Kojima, H. Fujisawa, A. Nakazawa, T. Nakazawa, F. Kanetsuna, H. Taniuchi, M. Nozaki, and O. Hayaishi, 1967, J. Biol. Chem., 242, 3270–3278). The molecular weight was estimated again by sedimentation equilibrium and Sephadex G-200 gel filtration and found to be 63,000 and 60,000, respectively. The enzyme was also found to contain 1 g atom of iron/mol of enzyme, based on a molecular weight of 63,000. The enzyme was dissociated into two bands on polyarcylamide gel electrophoresis in the presence of either sodium dodecyl sulfate or 8 m urea, and was separated into two subunits, α and β, by CM-cellulose chromatography using a buffer solution containing 8 m urea. The molecular weights of the α and β subunits were determined to be 30,000 and 32,000, respectively, by sodium dodecyl sulfate-gel electrophoresis. The NH₂-terminal sequences of these subunits determined by Edman degradation were as follows: α subunit, Thr-Val-Asn-Ile-Ser-His-Thr-Ala-Gln-Ile-Gln-Gln-Phe-Phe-Gln-Gln-(X)-(X)-Gly -Phe-Gly; β subunit, Thr-Val-Lys-Ile-Ser-His-Thr-Ala-Asp-Ile-Gln-Ala-Phe-Phe-Asn-Gln-Val-(X)-Gly-Leu-Asx. The COOH-terminal amino acid residues were determined to be alanine for the α subunit and glycine for the β subunit by three different methods: carboxypeptidase digestion, tritium labeling, and hydrazinolysis. These results indicate that the enzyme consists of two nonidentical subunits, α and β.     

Preparation of extracts from Juniper leaves for electrophoresis

The action of phenolase and subsequent reaction; in plant tissues with a high phenol to protein ratio causes a reduction in enzyme activity during extraction. To overcome the action of phenolases in Juniper leaves, an extraction buffer was developed which contained sodium tetraborate, sodium ascorbate, sodium meta-bisulfite, sodium diethyldithiocarbamate, and germanium dioxide. This extraction buffer was used in combination with an extraction procedure using liquid nitrogen, polyvinylpyrrolidone, n-butanol, and diethyl ether. Modifications in the buffer or extraction procedure resulted in a reduction in both the quality and number of isoenzymes obtained. Differences obtained in isoesterase banding by changing the extraction procedures should caution the systematic botanist that extraction methods may have critical effects in isoenzyme studies.     

Syntheses of p-aminophenyl α-d-talopyranoside and 1-thio α-d-talopyranoside as analogs of glycosidase substrates

p-Nitrophenyl and p-aminophenyl α-d-talopyranoside and 1-thio-α-d-talopyranosides were prepared for studies on specificity of glycosidases. Reaction of α-d-talopyranose pentaacetate with p-nitrophenol gave exclusively p-nitrophenyl 2,3,4,6-tetra-O-acetyl-α-d-talopyranoside (2) in 63% yield. A similar reaction with p-nitrobenzenethiol afforded the 1-thio analog (3) of 2 in 41.8% yield; the p-nitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-talopyranoside (6) was also obtained in low yield (6.7%). The two α-d-talosides 2 and 3 were catalytically deacetylated in near-quantitative yields by methanolic sodium methoxide. The p-nitrophenyl α-d-talopyranoside (4) and 1-thio-α-d-talopyranoside (5) were reduced with palladium on barium sulfate catalyst to the corresponding p-aminophenyl talosides. The acetylated p-nitrophenyl d-talosides 2, 3, and 6 were determined, from their 250-MHz n.m.r. spectra, to exist in the ⁴C₁ (d) conformation in chloroform solution.     

Operation of the glucose-fatty acid cycle after glycogenolysis induced by treatment with nicotinic acid.

The intramembranous segment of glycophorin A has been localized to a 35-amino acid peptide. This has been isolated by a new procedure in which acid-insoluble peptides of a tryptic digest of detergent-purified glycophorin A are fractionated by countercurrent distribution. Amino acid sequence analyses, using both manual and automatic Edman degradation techniques, indicate that this peptide has a unique sequence in contrast to earlier work (J. P. Segrest, I. Kahane, R. L. Jackson, and V. T. Marchesi, 1973, Biochem. Biophys. Res. Commun., 49, 964–969). Ambiguities at three positions have been resolved, and sequencing errors at two additional positions have been corrected. One segment of this peptide has an uninterrupted stretch of 22 uncharged amino acids, and it is likely that this is the part which spans the lipid bilayer of the membrane. The complete 35-residue peptide has an apparent molecular weight in the 6000–8000 range, when analyzed on sodium dodecyl sulfate gels, suggesting that it forms dimers under these conditions. This result is consistent with our earlier proposal that intact glycophorin A molecules exist as dimers in sodium dodecyl sulfate which are stabilized by noncovalent associations between hydrophobic segments of their polypeptide chains.