Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG)

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.     

Polarized electronic spectra of Z-DNA single crystals

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.     

Properties of apyrase and inorganic pyrophosphatase inStreptomyces aureofaciens

Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified fromS. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates, diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain lengthn ≦ 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity, effect of inhibitors, pH optimum and effect of Mg²⁺ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphates of chain lengthn = 15, 40 and 60, were detected.     

Insulin antagonism of glucocorticoid induction of tyrosine aminotransferase in cultured foetal hepatocytes

Polyadenylated RNA from developing Artemia salina cysts was fractionated by centrifugation through a sucrose gradient containing methylmercuric hydroxide (CH3HgOH). Aliquots of each fraction were directly added to a rabbit reticulocyte lysate to program protein synthesis in vitro. The translation products were assayed for eukaryotic elongation factor Tu (eEF-Tu) by immunoprecipitation with an antibody raised in rabbits and purified by affinity chromatography. The immunoprecipitated radioactivity was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. Sequences coding for eEF-Tu sediment in the 20-S region of the gradient and form a major component of the poly(A)-containing RNA. The mRNA of the 20-S region, comprising about 10% of the poly(a)-containing RNA fractionated on the gradient, has been translated in vitro and 30% of the translation products represent immunoprecipitable eEF-Tu protein chains with an Mr of 50000.     

Mineral element content in Ulva lactuca L. with reference to eutrophication in Hong Kong coastal waters

The amounts of tissue nitrogen, phosphorus, potassium, sodium, calcium and iron were estimated in the green alga Ulva lactuca L. collected from 9 rural and 14 urban littoral sites in the waters around Hong Kong Island during 1978 and 1979. The mean levels of tissue nitrogen and phosphorus were respectively 65% and 87% more in urban sites than in rural ones. Very significant correlation (r = 0.920) was found between the logarithmic concentration of seawater inorganic nitrogen and that of tissue nitrogen. The same applied to soluble reactive phosphorus in seawater and tissue phosphorus (r = 0.886). The levels of potassium, sodium and calcium in the alga were relatively uniform. However considerable variation in the level of iron was detected. The potential use of Ulva as an indicator species for eutrophication is discussed.     

Radioimmunoassay for the detection and quantitation of bruceantin

A radioimmunoassay for a new anticancer drug, bruceantin, has been developed using [³H]acetylbruceantin and antibody induced by immunizing rabbits with succinylbruceantin-bovine serum albumin conjugates. [³H]Acetylbruceantin was synthesized by reacting bruceantin with [³H]acetyl anhydride. The assay is simple and reproducible. The standard curve was linear on a logit-log plot, and the lower limit of sensitivity of the assay was 1 ng/ml. Using this assay, drug levels were easily determined in tissues of experimental animals following bruceantin administration. The assay procedure does not require sample extraction for plasma, urine, and bile. Bruceantin in other tissues can be extracted quantitatively with ethanol before being measured by the radioimmunoassay.     

Regulation of proton leakage from broken chloroplasts by CF0.

Measurements of proton translocation in CF₁-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF₀ is still dependent on light intensity with a first-order rate constant equal to mR₀, where R₀ is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF₀ is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF₁.CF₀ complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.     

Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.     

A new plaque system for canine distemper: characteristics of the green strain of canine distemper virus.

A Vero cell adapted Green strain of canine distemper virus (CDV) was tested for its plaque-forming capacity in different cell lines. Plaque formation was observed in HEp-2, BS-C-1, and HeLa cells but not in Vero or dog kidney cells even though replication and cytopathology were observed in the latter cell types. In the cells in which the virus was capable of producing plaques, the plaques were observed within 24 h post infection and continued to increase in size with subsequent cellular destruction such that by 72 h postinfection the size of the plaques approached 0.5 mm. With the use of the plaquing technique, it was possible to demonstrate the thermal lability of the virus as well as the kinetics of adsorption. Thus, it was shown that the half-life of the virus was 125 min at 25 degrees C, 75 min at 35 degrees C, and 65 min at 37 degrees C. The rate of adsorption of CDV to HEp-2 cells was 17.2% in 30 min at 37 degrees C and continued slowly for 4 h before completion. Application of this rapid plaque-forming assay to plaque-reduction tests for CDV antibody and for CDV-infected cells by the infectious center assay are described.