Coenzyme B12-dependent diol dehydrase: Chemical modification with 2,3-butanedione and phenylglyoxal

The apoenzyme of diol dehydrase was inactivated by two arginine-specific reagents, 2,3-butanedione and phenylglyoxal, in borate buffer. In both cases, the inactivation followed pseudo-first-order kinetics. Kinetic data show that the incorporation of a single reagent molecule per active site of the enzyme is necessary for the complete inactivation. The modification with 2,3-butanedione was reversed by dilution of the reagent and borate concentrations (65% activity recovered). 1,2-Propanediol (substrate) partially protected the enzyme against inactivation. The holoenzyme was almost insensitive to 2,3-butanedione and phenylglyoxal, indicating that the essential arginine residue is prevented from the attack of these reagents either by direct blockage with the bound coenzyme or by an indirect conformational change caused by coenzyme binding. The inactivation of diol dehydrase by 2,3-butanedione did not result in dissociation of the enzyme into subunits. From these results, we concluded that the essential arginine residue is located at or in close proximity to the active site of diol dehydrase.     

Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Macromolecule synthesis of Escherichia coli BB at a lower or transient growth state.

Macromolecule synthesis in Escherichia coli BB at lower growth rates was investigated. The results indicate that a deviation in ribonucleic acid (RNA) content per cell at a lower growth rate from the exponential relationship to a specific growth rate is entirely attributable to the presence of nonviable cells, in which the RNA content is lower than in viable cells. Based on this fact, a mathematical expression of macromolecule contents versus specific growth rate was devised. Moreover, continuous changes in macromolecule content during unbalanced growth from late-logarithmic phase to stationary phase were measured. Although growth rates changed continuously, the data on deoxyribonucleic acid (DNA) or RNA content versus the specific growth rate calculated from the increments in cell number satisfactorily fitted the exponential lines obtained under balanced growth at a higher growth rate. However, no such relationship was observed in the plot of DNA or RNA content versus the specific growth rate calculated from the increments in optical density.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Summary Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, andToxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle’s minimum essential medium, or Medium 199 with Hanks’ salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses.Ae. aegypti RML-12,Ae. albopictus C6/36,Ae. pseudoscutellaris AP-61, andTx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as inAe. albopictus C6/36) to comparable or higher titers (as inAe. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes. Use of trade names and commercial sources is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.     

Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C.

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current-clamp conditions and an outward current under voltage-clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA-induced outward current was slightly inhibited by treatment with GDP-beta-S and was completely inhibited by treatment with GTP-gamma-S. The activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate, suppressed the GABAB receptor-mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor-mediated response.     

Augmentation of retinoic acid-induced granulocytic differentiation in HL-60 leukemia cells by serine/threonine protein phosphatase inhibitors.

To evaluate the involvement of protein phosphatases (PP) in differentiation of human myelogenous leukemia HL-60 cells, we made use of potent inhibitors of PP1 and PP2A, calyculin-A (CAL-A) and okadaic acid (OKA). CAL-A and OKA could augment all-trans retinoic acid (ATRA)-induced granulocytic differentiation, whereas the differentiation toward macrophage lineage by 12-o-tetradecanoylphorbol acetate (TPA) was unchanged in the presence of CAL-A. CAL-A augmented the phosphorylation of 18K, 23K and 30K proteins induced by ATRA. The PP1 and PP2A were identified and were present mainly in the cytosol of HL-60 cells. These results suggest that either PP1 or PP2A or both may be involved in regulating granulocytic differentiation of HL-60 cells.     

Competitive exclusion through reproductive interference

A simple differential equation model was developed to describe the competitive interaction that may occur between species through reproductive interference. The model has the form comparable to Volterra's competition equations, and the graphical analysis of the outcome of the two-species interaction based on its zero-growth isoclines proved that: (1) The possible outcome in this model, as in usual models of resource competition, is either stable coexistence of both species or gradual exclusion of one species by the other, depending critically upon the values of the activity overlapping coefficient c_ij; (2) but, for the same c_ij-values, competitive exclusion is much more ready to occur here than in resource competition; (3) and moreover, the final result of the competition is always dependent on the initial-condition due to its non-linear isoclines, i.e., even under the parameter condition that generally allows both species to coexist, an extreme bias in intial density to one species can readily cause subsequent complete exclusion of its counterparts. Thus, it may follow that the reproductive interference is likely to be working in nature as an efficient mechanism to bring about habitat partitioning in either time or space between some closely related species in insect communities, even though they inhabit heterogeneous habitats where resource competition rarely occurs so that they could otherwise attain steady coexistence.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae.

The presence of diol dehydratase and glycerol dehydratase was shown in several bacteria of Enterobacteriaceae grown anaerobically on 1,2-propanediol and on glycerol, respectively. Diol dehydratases of Enterobacteriaceae were immunologically similar, but distinct from that of Propionibacterium freudenreichii.