Autoreduction and aggregation of fungal laccase in solution phase: possible correlation with a resting form of laccase

This paper reports results of a reexamination of some poorly understood peculiarities of laccases, an enzyme family which has been extensively studied in our laboratories as well as by others for some years. The issue that is reconsidered here is the previously proposed existence of "active" and "resting" forms of laccases. The presence of fungal laccases with partly reduced active sites is demonstrated. Of further interest is that an aggregated state in solution, not to our knowledge previously noted for laccase, has been found by using small-angle X-ray scattering as well as thorough analysis of the results of several biochemical experiments. Under some conditions, this aggregated state may correlate with the resting form of the laccases, although this resting form could have a broader significance. It was shown that Trametes ochracea laccase had some anomalous characteristics, which could be correlated with the high concentration of the "resting" enzyme. The mechanism of formation of resting laccase is suggested. Knowledge of the resting state is of importance for in vitro studies. Additionally, a suggestion about the possible regulatory role of this form in vivo is mentioned.     

The lumbosacral transition: morphologic variations and their functional significance

The articular facet of a superior articular process of the sacrum is directed backward, inward, and upward with marked variations. 4 angles characterize the orientation of this facet: a) The relative angle of tilt: i.e. the angle between the articular facet and the upper end-plate of the sacrum, measured in a sagittal plane. b) The absolute angle of tilt: i.e. the angle between the articular facet and the horizontal plane, measured in a sagittal plane. c) The tilted part-angle of opening: i.e. the angle between the articular facet and the sagittal plane, measured in a plane parallel to the upper end-plate of the sacrum. d) The horizontal part-angle of opening: i.e. the angle between the articular facet and the sagittal plane, measured in a horizontal plane. These 4 angles are determined by characteristic straights within the articular facet and certain reference planes (upper end-plate of the sacrum, horizontal plane, sagittal plane). Only 2 intersecting straights suffice for an adequate determination of a geometrical plane; therefore, if we know the relative angle of tilt and the tilted part-angle of opening, we are able to construct or to calculate the absolute angle of tilt as well as the horizontal part-angle of opening by using the range of inclination of the sacrum. The shape as well as the orientation of the articular facets at the superior articular processes of the sacrum do not depend on the inclination of the pelvis nor on the inclination of the sacrum nor on the range of the lumbosacral angle. Only the absolute angle of tilt shows a reference to the inclination of the sacrum because the relative angle of tilt shows a certain constancy. The orientation of the articular facets is slightly influenced by static moments, but considerably determined by dynamical requirements. At spines with irregular numbers of praesacral vertebrae, the orientation of the lumbosacral articular facets do not differ from the orientation of these facets at spines with the regular number of 24 praesacral vertebrae. This, however, does not prove right at spines, that have a lumbosacral "transitional vertebra". Such lumbosacral transitional vertebrae detract much from the stability of the lumbosacral region of the spine.     

Anions induce conformational changes and influence the activity and photoaffinity-labelling by 8-azido-ATP of isolated cytochrome c oxidase.

The biphasic effect of anions on the activity of isolated bovine heart cytochrome c oxidase is paralleled by changes in the visible oxidized spectra, indicating the different conformational changes in the enzyme induced by bromide, chloride, sulphate, phosphate, ADP and ATP. Photoaffinity-labelling of most subunits of the isolated enzyme by low concentrations of 8-azido-[gamma-32P]ATP is strongly increased by ATP, ADP and unlabelled 8-azido-ATP in an unspecific manner. With the reconstituted enzyme less subunits are labelled and this labelling is only little affected by nucleotides. The data suggest a highly dynamic structure for isolated bovine heart cytochrome c oxidase.     

A new type of prosome-like particle, composed of small cytoplasmic RNA and multimers of a 21-kDa protein, inhibits protein synthesis in vitro

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.     

Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit

The type II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase from bovine heart, consisting of a dimeric regulatory subunit and two catalytic subunits, was converted to a heterodimer by limited tryptic digestion. Loss of the tetrameric structure was accompanied by proteolysis of the regulatory subunit to a form with an apparent molecular weight of 45 000 vs. 52 000 for the native subunit. The proteolyzed subunit behaved as a monomer, in contrast to the dimeric native subunit. Amino acid sequence analysis established that proteolysis removed 45 residues at the N-terminus, indicating that these 45 residues constitute the dimerizing domain of this protein. The kinetic properties of this heterodimer were indistinguishable from those of the native tetramer: half-maximal kinase activation occurred at 48 nM cAMP with a Hill coefficient of 1.45, the regulatory subunit bound 1.5 equiv of cAMP with half-maximal binding occurring at 33 nM, and kinetics for dissociation of bound cAMP were biphasic, indicating the presence of two different binding sites. These observations suggest that residues 1-45 function only in the formation of dimers and that dimerization has little influence on other functional properties of the regulatory subunit. More extensive proteolysis cleaved the monomeric fragment at Lys-311. The fragments resulting from this second cleavage did not dissociate, and the complex inhibited the catalytic subunit in a cAMP-dependent manner.     

Basolateral K channels in an insect epithelium. Channel density, conductance, and block by barium

K channels in the basolateral membrane of insect hindgut were studied using current fluctuation analysis and microelectrodes. Locust recta were mounted in Ussing-type chambers containing Cl-free saline and cyclic AMP (cAMP). A transepithelial K current was induced by raising serosal [K] under short-circuit conditions. Adding Ba to the mucosal (luminal) side under these conditions had no effect; however, serosal Ba reversibly inhibited the short-circuit current (Isc), increased transepithelial resistance (Rt), and added a Lorentzian component to power density spectra of the Isc. A nonlinear relationship between corner frequency and serosal [Ba] was observed, which suggests that the rate constant for Ba association with basolateral channels increased as [Ba] was elevated. Microelectrode experiments revealed that the basolateral membrane hyperpolarized when Ba was added: this change in membrane potential could explain the nonlinearity of the 2 pi fc vs. [Ba] relationship if external Ba sensed about three-quarters of the basolateral membrane field. Conventional microelectrodes were used to determine the correspondence between transepithelially measured current noise and basolateral membrane conductance fluctuations, and ion-sensitive microelectrodes were used to measure intracellular K activity (acK). From the relationship between the net electrochemical potential for K across the basolateral membrane and the single channel current calculated from noise analysis, we estimate that the conductance of basolateral K channels is approximately 60 pS, and that there are approximately 180 million channels per square centimeter of tissue area.     

Metabolic changes during substrate shifts in continuous cultures of the yeast Candida boidinii variant 60

Summary Substrate shift experiments in chemostat cultures with either methanol or glucose as carbon source were performed with the yeast Candida boidinii variant 60. At low dilution rates of 0.064 h^–1 the culture may be easily shifted from methanol to glucose medium and back again to methanol. From these experiments it can be seen that glucose does not give rise to any catabolite inhibition of alcohol oxidase. Alcohol oxidase and formaldehyde dehydrogenase seem to be regulated by a repression-derepression mechanism, as small basal activities of both these enzymes can still be measured during growth on glucose. On the other hand, formate dehydrogenase activity is completely absent in the presence of glucose. This kind of regulation seems to favor the smooth switch from growth on glucose to methanol metabolism.With methanol or glucose, growth yields (Y_S) of 0.3 and 0.35, respectively may be obtained, and oxygen consumption (Q_{O 2}) is much higher in methanol cultures than in glucose-grown cells. Accordingly, the RQ values during growth on methanol decrease to about 0.5. Based on the yield coefficient of 0.3, it is possible to calculate that 38% of the methanol consumed must be incorporated into biomass, whereas 62% of the methanol is oxidized to CO₂. The corresponding RQ of 0.56 could not be experimentally ascertained.The activities of three mitochondrial enzymes were found to be higher in methanol-grown cells than in cells from glucose cultures. The low activites of enzymes for the phosphogluconate route in methanol-grown cells indicates that a cyclic oxidation of formaldehyde via hexose phosphate to CO₂ cannot be of great importance for methanol metabolism.List of Symbols D 1/h Dilution rate - 1/h Specific growth rate - Q_{CO 2} mmol/g·h Specific CO₂ production rate - Q_{O 2} mmol/g·h Specific O₂ comsumption rate - Q_S g/g·h Specific substrate consumption rate - RQ ./. Respiratory quotient (Q_{CO 2}/Q_{O 2}) - S_O g/l Substrate concentration in the feeding medium - $#x0073;$#x0304 g/l Substrate concentration in the fermentor - $#x0078;$#x0304 g/l Biomass in the fermentor - Y_{O 2} g/mmol O₂ Biomass yield on oxygen - Y_S g/g Biomass yield on carbon source     

STUDIES ON THE BIOCHEMISTRY AND FINE STRUCTURE OF SILICA SHELL FORMATION IN DIATOMS. II. THE STRUCTURE OF THE CELL WALL OF NAVICULA PELLICULOSA (BRÉB.) HILSE

The cell wall of the freshwater diatom Navicula pelliculosa (Bréb.) Hilse is composed of the silica shell and an organic skin which surrounds it. Isolated skins can be prepared by first removing the contents of the cell by mechanical shaking, followed by a posttreatment of these isolated cell walls with HF vapor to remove the silica shell. T h e skins can also be seen in sections, particularly well after the silica shell has been removed B y H. F; vapor. The origin and morphological composition of the shin in N. pelliculosa are not yet completely ascertaincd. As parts of the cell wn11, both the silica shell and the skin are extracellularly located. The growth of the silica shell, however, occurs intracellularly inside a vesicle delimited by a triple-layered membrane, the silicalemma. This membrane or secondary excreted organic material or both in various proportions may compose the skin.     

Synthesis and characterization of iodopropyl derivatives of adenosine 3′,5′-cyclic-monophosphate: Potential affinity labels of cAMP binding sites in enzymes

The syntheses of two potential cAMP affinity lables, 1,N ⁶-(3-iodopropyleno)adenosine 3′,5′-cyclic-monophosphate and 2′-O-(2-iodo-3-hydroxypropyl) adenosine 3′,5′-cyclic-monophosphate, by a two-step chemical procedure are described. TheN ⁶- and 2′-O-allyl intermediates were prepared selectively by alkylation of cAMP in organic and alkaline aqueous solutions, respectively. Treatment of theN ⁶-allyl derivative withN-iodosuccinimide resulted in iodine addition to the double bond and cyclization to theN ¹ position of the purine ring. The iodohydrin analog was synthesized by reaction of 2′-O-allyl-cAMP with potassium iodide and thallium trichloride in acetate buffered solution. The products were isolated by column chromatography and characterized by thin-layer chromatography, elemental analysis, and ultraviolet,¹³C, and¹H NMR spectroscopy. The cAMP analogs were found to react with lysine and cysteine. Both cAMP derivatives were tested for their reaction with the low-K _m cAMP phosphodiesterase of human platelets. The ribose-substituted analog functioned as a competitive inhibitor (K _I =0.72 μM) and caused a time-dependent irreversible inactivation of the phosphodiesterase. In contrast, the purine-substituted derivative acted neither as a reversible competitive inhibitor nor as an irreversible inactivator of the enzyme. These results indicate the specificity of these potential cAMP analogs in their interaction with the phosphodiesterase.