Semisynthesis and purification of homogeneous plasmenylcholine molecular species.

Methods for the efficient use of limiting amounts of fatty acid probes in the synthesis of individual molecular species of plasmenylcholine have been developed. Plasmenylcholine molecular species were synthesized through acylation of homogeneous 1-O-(Z)-hexadec-1'-enyl-sn-glycero-3-phosphocholine utilizing fatty acid anhydrides generated in situ from combined pools of reactant and recycled fatty acids by repeated addition of small amounts (10 mol%) of N,N'-dicyclohexylcarbodiimide. The efficient generation of reactive anhydrides was accomplished through minimizing irreversible formation of N-acyl urea adducts by maintaining a persistent molar excess of fatty acid (with respect to carbodiimide) during the entire reaction time course. The synthesis of multiple different sn-2 labeled plasmenylcholine probes for utilization in fluorescence, ESR, or 2H NMR spectroscopy as well as isotopically labeled plasmenylcholines for metabolic studies has been achieved in good yield (40-50% of theoretical yield based on fatty acid) by these methods. Rapid and effective purification methods utilizing high-performance liquid chromatography were developed for both large- and small-scale purifications of individual reaction mixtures which collectively resulted in the isolation of homogeneous plasmenylcholine molecular species in high yield from limiting amounts of fatty acid probes.     

Sedimentation of homogeneous double-strand DNA molecules.

Sedimentation velocity studies have been carried out with isolated double-strand DNA fragments prepared by digestion of PM2 phage with the restriction endonuclease Hae III. The results show that DNA molecules shorter than about 200 base pairs behave almost exactly as rigid rods with a diameter of 27 A. The behavior of the larger fragments (up to 1735 base pairs) can be described very well by either the theory of Yamakawa and Fujii (Yamakawa, H., and Fujii, M. (1973), Macromolecules 6. 407) using the same diameter and a persistence length of 575 A, or the theory of Hearst and Stockmayer (Hearst, J.E., and Stockmayer, W.H. (1962), J. Chem. Phys. 37, 1425) using a diameter of 20 A and a persistence length of 525 A.     

A homogeneous isoenzyme of human liver acid phosphatase.

An isoenzyme of human liver acid phosphatase (orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2) has been purified 4560-fold to homogeneity. The purification procedure includes ammonium sulfate fractionation, acid treatment, ion exchange chromatography on O-(carboxymethyl)-cellulose and DEAE-cellulose, Sephacryl S-200 chromatography, and affinity chromatography on Concanavalin A-Sepharose 4B. The homogeneous enzyme is a glycoprotein having 4% carbohydrate by weight in the form of mannose and glucosamine. In polyacrylamide gel electrophoresis under varied conditions of pH and cross-linking, the purified enzyme displays a single protein band coincident with activity. The native enzyme has a molecular weight of 93,000 as determined by gel elution chromatography and consists of two equivalent polypeptide chains. The subunit weight is 50,000–52,000 by sodium dodecyl sulfate gel electrophoresis. l-(+)-Tartrate is a strong competitive inhibitor of the enzyme; K_i is 4.3 × 10^?7m at pH 4.8 in 50 mm sodium acetate/100 mm sodium chloride. K_i values for a number of other inhibitors are given. Although it catalyzes the hydrolysis of a variety of phosphomonoesters, this isoenzyme of human liver acid phosphatase does not hydrolyze adenosine 5′-diphosphate, adenosine 5′-triphosphate, pyrophosphate, or choline phosphate at a detectable rate. The values of V differ with different alcohol or phenol leaving groups. The pH dependence of K_m and V values for the hydrolysis of p-nitrophenyl phosphate have been determined together with the pH dependence of K_i for l-(+)-tartrate. The pH dependence of both K_m and V indicate the effect of substrate ionization (pK ~ 5.2) and the involvement of a group in the EScomplex having a pKa value of approximately 6–7 which is ascribed either to a phosphoryl-enzyme intermediate or to the ionization of substrate in the ES-complex. An irreversible modification of the enzyme and a rapid loss of enzymic activity occurs upon treatment of the enzyme with Woodward's reagent K. The enzyme is protected against inactivation by the presence of competitive inhibitors. These and other data suggest that at least one carboxylic acid group plays an important role in catalysis.     

Computerized topographical analysis of functionally homogeneous neuronal sets.

A computer-assisted analysis of the spatial distribution of neurons having homogeneous characteristics is described in this paper. The camera lucida drawings of sections of a brain nucleus and the points representing the neurons labeled on the basis of a specific behavior of discharge rates were digitized on a personal computer Amiga 2000 or IBM compatible. Our software provided: a) the computerized, stereotaxically oriented reconstruction of the stored sections and of the plotted neurons; b) the identification within each section of the mass center (MC) of the units sharing a given behavior and of the area where the density of such neurons was maximal (MDA). The routine was tested on the spatial distribution of neuronal responses to serotonin in the lateral vestibular nucleus.     

Rotation of sickle cells in homogeneous magnetic fields.

Deoxygenated sickle cells (HbS) have been shown to orient in homogeneous magnetic fields because of magnetic anisotropy of the hemes. The time of rotation is proportional to 1/H2 and is in agreement with theory. Structural information concerning the orientation of HbS molecules in the fibers is obtained from these experiments and is shown to agree substantially with existing models.     

Purification of homogeneous murine immunoglobulins with anti-fructofuranan specificity.

Homogenous IgAK immunoglobulins ABPC-47 new, EPC-109, UPC-61 (anti-2,1 fructofuranan specificity) and homogeneous IgAK ABPC-48 and IgGK UPC-10 (anti-2,6 fructofuranan specificity) were purified by affinity chromatography by using inulin- and grass levan-Sepharose columns, respectively.     

Seven-color, homogeneous detection of six PCR products.

We describe an extension of the fluorogenic PCR 5'-nuclease assay, or "Taq-Man" assay. Sequence-specific probes consisted of a novel nonfluorescent quencher, nitrothiazole blue (NTB), at the 3' terminus and six different reporter dyes at the 5' terminus. The six reporters were 6-FAM, dR110, dR6G, dTMR, dROX and JAZ dyes. The seventh color was from aluminum phthalocyanine tetrasulfonate and was utilized as a "passive reference" to calibrate concentration variations. Our test system was a set of three single-nucleotide polymorphisms (SNPs). Each SNP system consisted of two primers and two sequence-specific probes, each labeled with a different reporter dye and NTB. Following PCR, the reactions were diluted with water and measured in a microcuvette on a luminescence spectrometer in synchronous scanning mode. In this method, both the excitation and emission wavelengths were scanned, with a fixed wavelength difference (delta gamma) between excitation and emission wavelengths. The spectral overlap in the set was evaluated by calculation of the condition number of the 7 x 7 matrix (dye fluorescence vs. wavelength). The small value of the condition number (1.5) proved that the cross-talk between the dyes was minimal. SNP analyses of known, synthetic target sequences and genomic DNA were plotted both as normalized, subtracted spectra and as data points in three separate dot plots.     

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.     

Preparation and some properties of homogeneous Neurospora crassa assimilatory NADPH-nitrite reductase.

The Neurospora crassa assimilatory NADPH-nitrite reductase (NAD(P)H: nitrite oxidoreductase, EC 1.6.6.4), which catalyzes the NADPH-dependent formation of ammonia from nitrite, has been purified to homogeneity as judged by polyacrylamide gel electrophoresis. The specific activity of the purified enzyme is 26.9 mumol nitrite reduced/min per mg protein, which corresponds to a turnover number of 7800 min(-1). The enzyme also has associated NADH-nitrite reductase, NADPH-hydroxylamine reductase and NADH-hydroxylamine reductase activities. The stoichiometry of 3 mol NADPH oxidized per mol nitrite reduced and ammonia formed has been confirmed. The visible absorption spectrum of the nitrite reductase reveals maxima at 280,390 (Soret) and 580 (alpha) nm. The latter bands are indicative of the occurrence of siroheme as a prosthetic group. The A280nm/A390nm ratio of 7.0 and the Soret/alpha ratio of 3.8 are compatible with values reported for other purified siroheme-containing enzymes. These results are discussed in terms of the comparative biochemistry of various enzymes involved in nitrite, hydroxylamine and sulfite metabolism in Neurospora crassa and other organisms.     

Preparation of a homogeneous soluble D-beta-hydroxybutyrate apodehydrogenase from mitochondria.

D-beta-Hydroxybutyrate dehydrogenase of bovine heart mitochondria has been purified to apparent homogeneity. The membrane-bound enzyme is first released by phospholipase A digestion of the mitochondria. Lithium bromide, 0.4 M, is used to aid release, and dithiothreitol is required to stabilize the enzyme. The membranous material is removed by centrifugation, and the apoenzyme is recovered in the supernatant and precipitated with ammonium sulfate to 50 percent of saturation. The main purification (100-fold) is achieved by selective adsorption and elution on controlled pore glass beads. The purified enzyme has been purified approximately 250-fold from the mitochondria. The purified enzyme is homogeneous as shown by poly-acrylamide gel electrophoresis in sodium dodecyl sulfate or acid-urea systems; a sharp band is obtained which is equivalent to a subunit molecular weight of 31,500. The apoenzyme is devoid of lipid and is completely inactive as isolated. It can be reactivated by adding aqueous microdispersions of lecithin or phospholipids containing lecithin. The apoenzyme is stable, i.e. it has a half-life of about 450 hours at 0-2 degrees in 0.4 M lithium bromide, containing 5 mM dithiothreitol at pH 7, and is soluble at these conditions, existing mainly as a monomer and dimer in dilute solution. It has a tendency to associate into larger aggregates when the salt concentration is lowered. The enzyme does not have a distinctive amino acid composition as compared with other proteins or soluble dehydrogenases. The purified apodehydrogenase is well suited for study of specific protein-lipid interaction, as well as the molecular basis for the role of phospholipid in this lipid-requiring enzyme.     

Subunits of Panulirus japonicus hemocyanin. 2. Cooperativity of the homogeneous hexamers

Hexameric hemocyanin from a spiny lobster, Panulirus japonicus, comprises three major subunits (Ib, II and III) and one minor subunit (Ia), as reported in the preceding paper in this journal. It has previously been shown that the O2 equilibria of Panulirus hemocyanin can be described by a concerted model extended to three affinity states [Makino, N. (1986) Eur. J. Biochem. 154, 49-55]. In this study the equilibrium binding of O2 to the reassociated subunits (Ib, II and III) was examined at various pH in the presence or absence of Ca2+ in order to test the applicability of the three-state model to the homogeneous hexamers. The hexameric structure of the reassembled subunits was less stable than that of the native protein under the conditions examined. The model could be fitted to the O2-binding isotherms of the homohexamers composed of the subunits II or III, if the molecular dissociation of the protein was taken into account. It was postulated that the monomeric hemocyanin has the same ligand affinity as that of the hexamer in the intermediate-affinity state (S). The fitting of the model to the O2 binding of the subunit I was unsuccessful mainly because of the low cooperativity of the assembled subunits.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Purification and properties of a homogeneous beta polymerase.

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) FROM THE Novikoff hepatoma has been purified over 200 000-fold (based on the increase in specific activity), by ammonium sulfate fractionation and chromatography on DEAE-Sephadex, phosphocellulose, hydroxylapatite, and DNA-cellulose. The enzyme is remarkably stable through all stages of purification until DNA-cellulose chromatography when it must be kept in buffers containing 0.5 M NaCl and 1 mg/ml bovine serum albumin for stability. The enzyme appears to be homogeneous as evidenced by a single stainable band when subjected to electrophoresis in polyacrylamide gels of different porosity. The stainable band corresponds to the DNA polymerase as determined by slicing sister gels and assaying for enzyme activity. The specific activity of the homogeneous preparation is about 60 000 units/mg. The enzyme lacks detectable exonuclease or endonuclease activity. It has a molecular weight of 32 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. In sucrose gradients, the molecular weight is estimated at 31 000. The isoelectric point of the hydroxylapatite fraction enzyme is 8.5. The Novikoff beta-polymerase requires all four deoxyribonucleoside triphosphates, primer-template, and a divalent cation for maximal activity. The apparent Km for total deoxyribonucleoside triphosphate is 7-8 muM and for DNA 125 mug/ml. Activated DNA, rendered 7% acid soluble by DNase I, is the preferred primer-template, although a number of synthetic polynucleotides can by efficiently utilized, particularly in the presence of Mm2+ optimum is 7 mM; the Mn2+ optimum is 1 mM. The pH optimum is 8.4 in Tris-HCl or 9.2 in glycine buffer. The beta-polymerase is sstimulated about twofold by NaCl or KCl at an optimum of 50-100 MM, and the enzyme maintains considerable activity at high ionic strengths. The DNA polymerase is inhibited by ethanol, acetone, and a variety of known polymerase inhibitors. Glycols stimulate the enzyme as does spermine or spermidine. Unlike most beta-polymerases, the Novikoff enzyme is moderately sensitive to N-ethylmaleimide.     

Zero-sized effect of nano-particles and inverse homogeneous nucleation. Principles of freezing and antifreeze

It was found that freezing of water in terms of homogeneous nucleation of ice never occurs even in ultra-clean micro-sized water droplets under normal conditions. More surprisingly, at sufficiently low supercoolings, foreign nano-particles exert no effect on the nucleation barrier of ice; it is as if they physically "vanished." This effect, called hereafter the "zero-sized" effect of foreign particles (or nucleators), leads to the entry of a so-called inverse homogeneous-like nucleation domain, in which nucleation is effectively suppressed. The freezing temperature of water corresponds to the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation. The freezing temperature of water is mainly determined by (i) the surface roughness of nucleators at large supercoolings, (ii) the interaction and structural match between nucleating ice and the substrate, and (iii) the size of the effective surface of nucleators at low supercoolings. Our experiments showed that the temperature of -40 degrees C, commonly regarded as the temperature of homogeneous nucleation-mediated freezing, is actually the transition temperature from the inverse homogeneous-like nucleation regime to foreign particle-mediated heterogeneous nucleation in ultra-clean water. Taking advantage of inverse homogeneous-like nucleation, the interfacial tensions between water and ice in very pure water and antifreeze aqueous solutions were measured at a very high precision for the first time. The principles of freezing promotion and antifreeze and the selection for the biological ice nucleation and antifreeze proteins are obtained. The results provide completely new insights into freezing and antifreeze phenomena and bear generic implications for all crystallization systems.