Biochemical characterization of arylsulfatase-C isozymes in human fibroblasts

Arylsulfatase-C and sterol sulfatase were thought to be identical enzymes whose X-linked locus escapes inactivation. However, recent evidence shows that they are not identical but that arylsulfatase-C in human fibroblasts exists in two isozymic forms, designated as slow and fast. We now report that the two forms are enzymatically different. When assayed with an artificial fluorogenic substrate, the slow form showed a pH optimum of 8.00 and a Km of 228 microM. In contrast, the fast form showed a pH optimum of 7.67 and a Km of 86.7 microM with substrate inhibition occurring above 0.33 mM. The heat stability of the fast form was slightly below that of the slow form. Polyclonal antibodies raised against the slow form did not cross-react with the fast form. Hence, the two isozymic forms of arylsulfatase-C are enzymatically and structurally different and the slow form is associated with sterol sulfatase activity.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

Postcanine dental form in the mustelidae and viverridae (Carnivora: Mammalia)

This study investigates whether the gross morphology of mustelid and viverrid postcanine dentitions corresponds with differences in diet. For each species, the predominant foods ingested are used to form predictions of dental form and measurements of the carnassial and molar teeth determine the extent of shearing and crushing surfaces on the postcanine teeth. Principal components analysis distinguishes species according to morphological differences in the dentition and these differences are compared with predictions of dental form based on diet. Dietarily specialized species are more likely to be correspondingly specialized in the dentition and species with varied food sources are more likely to possess dental characteristics that are generalized in function. Consumers of foods with high fracture resistance, such as vertebrate tissue and hard-surfaced invertebrates, possess specialized shearing or crushing postcanine teeth. On the other hand, species that consume foods of lesser fracture resistance, such as fruit and soft invertebrates, differ greatly in dental form and are more generalized in dental function. A few species possess postcanine dentitions that do not correspond with diet; the absence of dental-dietary correlation in these species suggests that other factors, such as phylogeny, are important in determining dental form.     

Human adenosine deaminase. Distribution and properties.

Adenosine deaminase exists in multiple molecular forms in human tissue. One form of the enzyme appears to be "particulate". Three forms of the enzyme are soluble and interconvertible with apparent molecular weights of approximately 36,000, 114,000, and 298,000 (designated small, intermediate, and large, respectively). The small form of adenosine deaminase is convertible to the large form only in the presence of a protein, which has an apparent molecular weight of 200,000 and has no adenosine deaminase activity. This conversion of the small form of the enzyme to the large form occurs at 4 degrees, exhibits a pH optimum of 5.0 to 8.0, and is associated with a loss of conversion activity. The small form of the enzyme predominates in tissue preparations exhibiting the higher enzyme-specific activities and no detectable conversion activity. The large form of adenosine deaminase predominates in tissue extracts exhibiting the lower enzyme specific activities and abundant conversion activity. The small form of adenosine deaminase shows several electrophoretic variants by isoelectric focusing. The electrophoretic heterogeneity observed with the large form of the enzyme is similar to that observed with the small form, with the exception that several additional electrophoretic variants are uniformly identified. No organ specificity is demonstrable for the different electrophoretic forms. The kinetic characteristics of the three soluble molecular species of adenosine deaminase are identical except for pH optimum, which is 5.5 for the intermediate species and 7.0 to 7.4 for the large and small forms.     

Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, yeast RAS proteins are potent activators of adenylate cyclase. In the present work we measured the activity of adenylate cyclase in membranes from Saccharomyces cerevisiae which overexpress this enzyme. The response of the enzyme to added RAS2 proteins bound with various guanine nucleotides and their analogs suggests that RAS2 proteins are active in their GTP-bound form and are virtually inactive in their GDP-bound form. Also, active RAS2 protein is not inhibited by inactive RAS2, suggesting that the inactive form does not compete with the active form in binding to its effector.     

Stabilization of glucosaminephosphate synthase from rat liver by hexose 6-phosphates. Properties and interconversion of two molecular forms.

Glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19) prepared from rat liver by extraction in the presence of glucose 6-phosphate (Glc-6-P) followed by precipitation with (NH4)2SO4 is susceptible to digestion by trypsin. This enzyme, designated form A, can be converted to tryptic-insusceptible form B upon incubation with Glc-6-P or fructose 6-phosphate (Fru-6-P) at 37 degrees C. The two forms also differ in the degree of activation by dithiothreitol, the degree of inhibition by methyl-glyoxal and the behavior on DEAE-Sephadex and Sephadex G-200 column chromatography. During purification with DEAE-Sephadex followed by hydroxyapatite, form B is converted to form A if Fru-6-P is absent and form A to form B if Fru-6-P is present. The two forms are therefore intercovertible. Under the conditions of purification, form B is more stable than form A, since the purity and yield of the final product are greater with form B than with form A. These findings suggest that the two forms of glucosaminephosphate synthase differ conformationally and that the equilibrium position depends on the concentration of Fru-6-P. Glc-6-P is effective only when it gives rise to Fru-6-P by mediation of glucose-phosphate isomerase.     

Huddling and the control of water loss by the slug Limax pseudoflavus Evans

Limax pseudoflavus Evans form closely packed huddles within their day-time resting sites. Huddles will form in two populations of this species are mixed. Huddles will also form if L. pseudoflavus and L. flavus are mixed, but individuals tend to select conspecific neighbours within the huddles. Huddles form only after considerable milling around of the component slugs and form more frequently and involve more contact in dry compared with humid conditions. Moving slugs increase their evaporation rate by 65% compared with still animals of the same weight. Two slugs making close contact reduce their evaporation rate by 34% compared with unhuddled slugs. It is argued that huddles are non-social aggregations whose prime function is the conservation of water.     

A protein-farnesyl transferase inhibitor interferes with the serum-induced conversion of Candida albicans from a cellular yeast form to a filamentous form

A commercially available, cell permeable, protein-farnesyl transferase inhibitor interfered with the serum-induced morphological change in Candida albicans from a cellular yeast form to a filamentous form. The inhibitor has a negligible effect on the growth of C. albicans cells in the cellular yeast form, at the levels used to interfere with the morphological change. Conversion of C. albicans from the yeast form to filamentous form is associated with pathogenicity and hence protein-farnesyl transferase inhibitors are potentially of therapeutic value against C. albicans infection.     

Structural differences in the catalytic subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides.

There are significant differences in the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase isolated from Rhodopseudomonas sphaeroides. Two-dimensional peptide mapping of carboxymethylated large subunits clearly indicates that there are differences in the primary structure of the two proteins. These results are supported by limited proteolysis with three different proteases and by subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data, in conjunction with immunological studies and investigations on the regulation of the two enzymes, support the conclusion that the large subunits of form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase may be different gene products.     

Dynamics of fd coat protein in lipid bilayers

The dynamics of backbone and side-chain sites of the membrane-bound form of fd coat protein are described with solid-state 2H and 15N NMR experiments. The samples were isotopically labeled coat protein in phospholipid bilayers in excess water. The protein itself is immobile and does not undergo rapid rotation within the bilayer. Like the structural form of the protein, the membrane-bound form has four mobile residues at the N-terminus. The membrane-bound form differs from the structural form in having several mobile residues at the C-terminus. Many of the side chains of residues with immobile backbone sites undergo large amplitude jump motions. The dynamics are generally similar in both the structural and membrane-bound forms of the protein.     

Crystal structures of heptakis(2,6-di-O-ethyl)cyclomaltoheptaose

Heptakis(2,6-di-O-ethyl)-beta-cyclodextrin (DE-beta-CD) was crystallized in two forms from hexane and 95% aqueous methanol, respectively: A form I crystal with the space group P2(1)2(1)2(1) and a form II crystal with the space group P3(1). In both crystals, DE-beta-CD molecules are in a round shape with intramolecular O-3-H...O-2 hydrogen bonds. In the form I crystal, the DE-beta-CD molecules are arranged along the twofold screw axis to form a helically extended polymeric chain by including the 6-O-ethyl groups of the adjacent molecule. One hexane molecule with twofold disorder is located in the intermolecular channel along the a-axis. In contrast, the DE-beta-CD molecules in the form II crystal form a helical arrangement along the threefold screw axis. One methanol and one water molecule are included on the O-6 side of the molecular cavity. The water molecule links the methanol molecule and two ethoxy groups of the adjacent DE-beta-CD molecule with hydrogen bonds. The result suggests the important role of solvent in the formation of helical arrangement of DE-beta-CD molecules.     

Pyruvate kinase isozymes of Mucor racemosus: control of synthesis by glucose.

A variety of cultural conditions were examined to determine the relationship between pyruvate kinase isozyme patterns and morphology in Mucor racemosus. The results indicate that M. racemosus has two isozymes of pyruvate kinase, form A and form B, which are clearly separable on ion-exchange columns (diethylaminoethyl-cellulose). Addition of glucose to cultures growing on amino acids in air resulted in the induction of form A and the termination of form B synthesis. Cycloheximide added at the same time as glucose blocked the formation of form A but did not interfere with the termination of form B synthesis. Removal of glucose resulted in termination of form A synthesis and the induction of form B. Cycloheximide blocked the induction of form B and did not interfere with the termination of form A synthesis. The data show that the isozyme type is not directly related to morphology, but depends only on the presence or absence of glucose.     

The skull evolution of oviraptorosaurian dinosaurs: the role of niche partitioning in diversification

Oviraptorosaurs are bird‐like theropod dinosaurs that thrived in the final pre‐extinction ecosystems during the latest Cretaceous, and the beaked, toothless skulls of derived species are regarded as some of the most peculiar among dinosaurs. Their aberrant morphologies are hypothesized to have been caused by rapid evolution triggered by an ecological/biological driver, but little is known about how their skull shapes and functional abilities diversified. Here, we use quantitative techniques to study oviraptorosaur skull form and mandibular function. We demonstrate that the snout is particularly variable, that mandibular form and upper/lower beak form are significantly correlated with phylogeny, and that there is a strong and significant correlation between mandibular function and mandible/lower beak shape, suggesting a form–function association. The form–function relationship and phylogenetic signals, along with a moderate allometric signal in lower beak form, indicate that similar mechanisms governed beak shape in oviraptorosaurs and extant birds. The two derived oviraptorosaur clades, oviraptorids and caenagnathids, are significantly separated in morphospace and functional space, indicating that they partitioned niches. Oviraptorids coexisting in the same ecosystem are also widely spread in morphological and functional space, suggesting that they finely partitioned feeding niches, whereas caenagnathids exhibit extreme disparity in beak size. The diversity of skull form and function was likely key to the diversification and evolutionary success of oviraptorosaurs in the latest Cretaceous.     

Inactive proenzyme to tissue-type plasminogen activator from human melanoma cells, identified after affinity purification with a monoclonal antibody

The human 66 000 mol. wt. plasminogen activator (HPA66; tissue-type plasminogen activator) has been purified from melanoma cells by a one-step affinity method with a monoclonal antibody. HPA66 purified in this way consists mainly of a one-polypeptide chain form with small amounts (15%) of a form containing two polypeptide chains held together by one or more disulphide bridges. The one-chain form was converted to the two-chain form by catalytic amounts of plasmin. During the conversion, the enzyme activity of HPA66, as measured by an [125I]plasminogen conversion assay and with a chromogenic substrate, increased linearly with the percentage of the two-chain form. A linear regression analysis showed that all enzyme activity could be accounted for by the two-chain form, while the one-chain form had no measurable enzyme activity (detection limit approximately 5% of the activity of the two-chain form). Together with previous findings of inactive proenzymes to murine and human approximately 50 000 mol. wt. (urokinase-type) plasminogen activators, these findings indicate that plasminogen activators are generally formed from inactive one-chain proenzymes which are converted to active two-chain enzymes by limited proteolysis, thus demonstrating a third step in a cascade reaction leading to extracellular proteolysis.     

Lovastatin and simvastatin are modulators of the proteasome

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.     

Locating Interrupted Hydrogen Bonding in the Secondary Structure of PM2 Circular DNA by Comparative Denaturation Mapping

Previous studies with HCHO have revealed a reaction with superhelical DNA that strongly suggests that this DNA consists of small regions of interrupted secondary structure. To map these sites in PM2 DNA, the following set of experiments was performed using electron microscopy. (i) A denaturation map of nicked form II was obtained using Inman's alkaline-HCHO conditions. (ii) The superhelical form I was reacted with HCHO at 30 C until equilibrium was achieved at the interrupted sites (3.6% reactivity). The excess HCHO was removed rapidly and X-ray treatment was employed to nick these prereacted molecules. These form II molecules containing HCHO (form II HCHO) were also subjected to denaturation mapping. It would be expected that the HCHO-unpaired regions would serve as induction sites for the propagation of melting. Hence, depending on the location of the induction sites; we would anticipate either the creation of new regions of melting or a normal denaturation map shifted to lower pH values. Comparison of the development of progressive denaturation of form II and form II HCHO reveals that the latter is the case. The denaturation maps of form II are highly organized patterns of adenine-thymine (AT)-rich regions, with a total of five regions at extreme pH conditions. There are six highly organized regions for form II HCHO, i.e., smaller adjacent loops, at low denaturation conditions where no denaturation is seen for form II. These coalesce into the pattern for form II containing four of five A-T-rich regions observed for form II. Hence we conclude that the regions of altered hydrogen bonding in superhelical PM2 DNA are four to six in number and they map in the A-T-rich regions of the DNA.     

Changes of pyruvate dehydrogenase in brain during alloxan diabetes

Alloxan diabetes causes a decrease in the active form of pyruvate dehydrogenase in rat brain. The effect is severe in the cerebellum and brain stem compared to cerebral hemispheres. The changes observed in the total form are not as significant as those found in the active (dephosphorylated) form. The effects are reversed after administration of insulin to diabetic animals. The severity of diabetes was also found to affect the activity of pyruvate dehydrogenase with inverse correlation. There was a gradual increase in the proportion of active (dephosphorylated) form with increase of time after the onset of diabetes.     

Simple simulations of DNA condensation

Molecular dynamics simulations of a simple, bead-spring model of semiflexible polyelectrolytes such as DNA are performed. All charges are explicitly treated. Starting from extended, noncondensed conformations, condensed structures form in the simulations with tetravalent or trivalent counterions. No condensates form or are stable for divalent counterions. The mechanism by which condensates form is described. Briefly, condensation occurs because electrostatic interactions dominate entropy, and the favored coulombic structure is a charge-ordered state. Condensation is a generic phenomenon and occurs for a variety of polyelectrolyte parameters. Toroids and rods are the condensate structures. Toroids form preferentially when the molecular stiffness is sufficiently strong.     

Rat adipose tissue glycogen synthase. Evidence for multiple discrete kinetic species and their interconversion.

Rat adipose tissue glycogen synthase has been kinetically characterized. The classical D form has an apparent Km for UDP-glucose of 0.7 mM and 0.4 mM in the absence and presence of glucose 6-phosphate, respectively. The apparent Ka for glucose 6-phosphate is 0.6 mM. The effect of glucose 6-phosphate on the D form is to enhance the Vmax 7-fold. The I form is also affected by glucose 6-phosphate (Ka, 0.025 mM) but the Vmax is increased only by 20%; apparent Km values for UDP-glucose are 0.4 mM and 0.045 mM in the absence and presence of glucose 6-phosphate, respectively. In addition, two new kinetically distinguishable forms have been observed. The first, designated glycogen synthase Q, arises from an Mg2+ATP-dependent deactivation of the I form. The apparent Km values of glycogen synthase Q for UDP-glucose are identical with those of the I form; however, the apparent Ka for glucose 6-phosphate (0.2 mM) is 8-fold higher than that for the I form and one-third that for the D form. Preparations from fasted or diabetic rats contain a form of glycogen synthase, designated glycogen synthase X, that has a much lower affinity for glucose 6-phosphate than the D form (apparent Ka, 3 mM); the apparent Km values for UDP-glucose are similar to those of the D form (0.7 mM and 0.3 mM in the absence and presence of glucose 6-phosphate, respectively). In preparations from fasted rats a stepwise Mg2+-dependent conversion was demonstrated of synthase X to D to Q to I; this sequential conversion was reversed on incubation with Mg2+ATP. In preparations from fed rats, synthase Q could be generated either by limited activation (from the D form) or, after conversion to the I form, by deactivation with Mg2+ATP. However, even prolonged incubation with Mg2+ATP failed to generate the D (or X) form.