Coenzyme binding by 3-hydroxybutyrate dehydrogenase, a lipid-requiring enzyme: lecithin acts as an allosteric modulator to enhance the affinity for coenzyme

The role of phospholipid in the binding of coenzyme, NAD(H), to 3-hydroxybutyrate dehydrogenase, a lipid-requiring membrane enzyme, has been studied with the ultrafiltration binding method, which we optimized to quantitate weak ligand binding (KD in the range 10-100 microM). 3-Hydroxybutyrate dehydrogenase has a specific requirement of phosphatidylcholine (PC) for optimal function and is a tetramer quantitated both for the apodehydrogenase, which is devoid of phospholipid, and for the enzyme reconstituted into phospholipid vesicles in either the presence or absence of PC. We find that (i) the stoichiometry for NADH and NAD binding is 0.5 mol/mol of enzyme monomer (2 mol/mol of tetramer); (ii) the dissociation constant for NADH binding is essentially the same for the enzyme reconstituted into the mixture of mitochondrial phospholipids (MPL) (KD = 15 +/- 3 microM) or into dioleoyl-PC (KD = 12 +/- 3 microM); (iii) the binding of NAD+ to the enzyme-MPL complex is more than an order of magnitude weaker than NADH binding (KD approximately 200 microM versus 15 microM) but can be enhanced by formation of a ternary complex with either 2-methylmalonate (apparent KD = 1.1 +/- 0.2 microM) or sulfite to form the NAD-SO3- adduct (KD = 0.5 +/- 0.1 microM); (iv) the binding stoichiometry for NADH is the same (0.5 mol/mol) for binary (NADH alone) and ternary complexes (NADH plus monomethyl malonate); (v) binding of NAD+ and NADH together totals 0.5 mol of NAD(H)/mol of enzyme monomer, i.e., two nucleotide binding sites per enzyme tetramer; and (vi) the binding of nucleotide to the enzyme reconstituted with phospholipid devoid of PC is weak, being detected only for the NAD+ plus 2-methylmalonate ternary complex (apparent KD approximately 50 microM or approximately 50-fold weaker binding than that for the same complex in the presence of PC). The binding of NADH by equilibrium dialysis or of spin-labeled analogues of NAD+ by EPR spectroscopy gave complementary results, indicating that the ultrafiltration studies approximated equilibrium conditions. In addition to specific binding of NAD(H) to 3-hydroxybutyrate dehydrogenase, we find significant binding of NAD(H) to phospholipid vesicles. An important new finding is that the nucleotide binding site is present in 3-hydroxybutyrate dehydrogenase in the absence of activating phospholipid since (a) NAD+, as the ternary complex with 2-methylmalonate, binds to the enzyme reconstituted with phospholipid devoid of PC and (b) the apodehydrogenase, devoid of phospholipid, binds NADH or NAD-SO3- weakly (half-maximal binding at approximately 75 microM NAD-SO3- and somewhat weaker binding for NADH).(ABSTRACT TRUNCATED AT 400 WORDS)     

Indication for deletion of two introns in theoxi-3 gene of a respiratory-competentSaccharomyces cerevisiae strain

Physical mapping of the mitochondrial DNA of the wild-typeSaccharomyces cerevisiae strainRXII revealed that most of the restriction sites as well as the location of the apocytochromeb gene were identical in comparison with the known maps of the mitochondrial genome in otherSaccharomyces cerevisiae strains. In the middle of theSalI linearized map of theRXII mitochondrial DNA, a deletion was detected which resulted in the loss of twoEcoRI and oneBamHI restriction sites. The corresponding region, however, exists in most other laboratory strains ofSaccharomyces mapped so far. This region overlaps the introns aI2 and aI3 surrounding exon A3 sequences of the subunit 1 of the cytochrome oxidase gene. The nucleotide sequence of the subunit 1 gene showed that theBamHI site was located close to the aI3-A4 intron-exon junction and the distalEcoRI site close to the aI2-A2 boundary. I therefore conclude that these two introns are deleted in the mitochondrial genome of strainRXII. The exon A3 must have been conserved since this strain was respiratory competent. This result, while being a good example of the morphological diversity of a genome with the same function, may contribute to an understanding of the role of introns in the mitochondrial split genes in yeast.     

Effect of breath-hold time on DLCO(SB) in patients with airway obstruction

The single-breath diffusing capacity of the lung for CO [DLCO(SB)] is considered a measure of the conductance of CO across the alveolar-capillary membrane and its binding with hemoglobin. Although incomplete mixing of inspired gas with alveolar gas could theoretically influence overall diffusion, conventional calculations of DLCO(SB) spuriously overestimate DLCO(SB) during short breath-holding periods when incomplete mixing of gas within the lung might have the greatest effect. Using the three-equation method to calculate DLCO(SB) which analytically accounts for changes in breath-hold time, we found that DLCO(SB) did not change with breath-hold time in control subjects but increased with increasing breath-hold time in both patients with asthma and patients with emphysema. The increase in DLCO(SB) with increasing breath-hold time correlated with the phase III slope of the single-breath N2 washout curve. We suggest that in patients with ventilation maldistribution, DLCO(SB) may be decreased for the shorter breath-hold maneuvers because overall diffusion is limited by the reduced transport of CO from the inspired gas through the alveolar gas prior to alveolar-capillary gas exchange.     

Respiratory adaptations to dead space loading during maximal incremental exercise

We examined the effects of dead space (VD) loading on breathing pattern during maximal incremental exercise in eight normal subjects. Addition of external VD was associated with a significant increase in tidal volume (VT) and decrease in respiratory frequency (f) at moderate and high levels of ventilation (VI); at a VI of 120 l/min, VT and f with added VD were 3.31 +/- 0.33 liters and 36.7 +/- 6.7 breaths/min, respectively, compared with 2.90 +/- 0.29 liters and 41.8 +/- 7.3 breaths/min without added VD. Because breathing pattern does not change with CO2 inhalation during heavy exercise (Gallagher et al. J. Appl. Physiol. 63: 238-244, 1987), the breathing pattern response to added VD is probably a consequence of alteration in the PCO2 time profile, possibly sensed by the carotid body and/or airway-pulmonary chemoreceptors. The increase in VT during heavy exercise with VD loading indicates that the tachypneic breathing pattern of heavy exercise is not due to mechanical limitation of maximum ventilatory capacity at high levels of VT.     

Endonuclease-free, protoplast-forming enzyme preparation and its application in fungal transformation.

An endonuclease-free, protoplast-forming enzyme complex was prepared from the "snail enzyme." The purified preparation has high protoplast-forming activity comparable to the crude enzyme complex without destroying circular plasmid DNA. Furthermore, a higher transformation rate was achieved by the application of the endonuclease-free enzyme complex in both yeast and filamentous fungal vector-host systems.     

Insensitivity of maximum expiratory flow to bronchodilation in normal dogs

We examined the effects of the inhaled parasympatholytic agent atropine and the sympathomimetic agent salbutamol on partitioned frictional pressure (Pfr) losses to the site of flow limitation (choke point, CP) in dogs to see how changes brought about by these agents would affect maximum expiratory flow (Vmax) and response to breathing 80% He-20% O2 (delta Vmax) in terms of wave-speed theory of flow limitation. In open-chest dogs, a Pitot-static tube was advanced down the right lower lobe to locate CP, to determine CP lateral and end-on pressures (PE), and to partition the airway into peripheral (alveoli to sublobar) and central (sublobar to CP) segments. Measurements were obtained at approximately 50% vital capacity. After inhalation, CP locations were unchanged with both bronchodilating agents. After atropine inhalation, Pfr central was decreased by one-half compared with base line. Despite the decrease in Pfr central, however, Vmax failed to increase after atropine because of altered bronchial area pressure (BAP) behavior at the CP site. After salbutamol inhalation, Pfr peripheral was reduced by about one-half compared with base line. However, Vmax failed to increase, because this reduction was too small to significantly increase the CP pressure head (i.e., PE). delta Vmax was also insensitive to these agents. Our results show mechanisms by which small changes in Pfr, as well as the complex interaction of changes in Pfr and BAP, may limit the use of Vmax in detecting bronchodilation at different airway sites.     

DNA-based immunization with chimeric vectors for the induction of immune responses against the hepatitis C virus nucleocapsid.

Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.     

Changes in Viability, Cell Composition, and Enzyme Levels During Starvation of Continuously Cultured (Ammonia-Limited) Selenomonas ruminantium

Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h⁻¹. Washed cell suspensions were subjected to long-term nutrient starvation at 39°C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss.     

Long-term nutrient starvation of continuously cultured (glucose-limited) Selenomonas ruminantium.

Selenomonas ruminantium, a strictly anaerobic ruminal bacterium, was grown at various dilution rates (D = 0.05, 0.25, and 0.35 h-1) under glucose-limited continuous culture conditions. Suspensions of washed cells prepared anaerobically in mineral buffer were subjected to nutrient starvation (24 to 36 h; 39 degrees C; N2 atmosphere). Regardless of growth rate, viability declined logarithmically, and within about 2.5 h, about 50% of the populations were nonviable. After 24 h of starvation, the numbers of viable cells appeared to be inversely related to growth rate, the highest levels occurring with the slowest grown population. Cell dry weight, carbohydrate, protein, ribonucleic acid (RNA), and deoxyribonucleic acid declined logarithmically during starvation, and the decline rates of each were generally greater with cells grown at higher D values. Both cellular carbohydrate and RNA declined substantially during the first 12 h of starvation. Most of the cellular RNA that disappeared was found in the suspending buffer as low-molecular-weight, orcinol-positive materials. During growth, S. ruminantium made a variety of fermentation acids from glucose, but during starvation, acetate was the only acid made from catabolism of cellular material. Addition of glucose or vitamins to starving cell suspensions did not decrease loss of viability, whereas a starvation in the spent culture medium resulted in a slight decrease in the rate of viability loss. Overall, the data indicate that S. ruminantium strain D has very little survival capacity under the conditions tested compared with other bacterial species that have been studied.     

Expiratory flow limitation in dogs with regional changes in lung mechanical properties

We examined maximum expiratory flow (Vmax) in two canine preparations in which regional changes in lung mechanical properties were produced. In one experiment serial bronchial obstructions were made to determine whether flow-limiting sites (choke points, CP) would occur in series. With the right lung tied off, constrictions were placed at the left lower lobar bronchus (LLL) and left main-stem bronchus. On deflation from total lung capacity, the obstructed LLL and nonobstructed left upper lobe (LUL) emptied into the obstructed left main-stem bronchus. Although a CP common to both lobes was identified at the main-stem obstruction, which limited total Vmax, we questioned whether there was also a CP at the lobar obstruction that fixed LLL flow. In that case the rate of LLL emptying would not be dependent on the presence of the common (i.e., central) CP and thus the flow contribution of the LUL. We found that when the LUL was removed, the LLL increased its rate of emptying. Thus a lobar CP did not fix LLL flow and CP did not occur in series. In a second experiment emphysema was produced in the left lung to reduce lung recoil, whereas the right lung was normal. CP were identified at approximately lobar bronchi of each lung, and the lungs were emptied at different rates. A CP common to both lungs was not identified. Our results indicate that in localized lung disease, if flows from the different regions are high enough, then wave speed is reached in proximal airways, and a CP occurs centrally rather than peripherally. On the other hand, if flows are low, then wave speed is reached peripherally and a CP common to all lung regions does not occur.