Biomedical subjects

J B Russell

Publications and source records attributed to J B Russell.

At least 55 records · Page 3Linked to original sources

PubMed1997

NAD-independent lactate and butyryl-CoA dehydrogenases of Clostridium acetobutylicum P262.

Clostridium acetobutylicum P262 cells that were growing on lactate and acetate had an NAD-independent lactate dehydrogenase(iLDH) activity of 200 nmol mg protein-1 min-1. Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold purification. Gel filtration indicated that the iLDH had a molecular weight of approximately 55 kDa, but two bands were always observed. Phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity. The semi-purified iLDH had a Vmax of 13,000 nmol mg protein-1 min-1 and a Km value of 3.5 mM for D-lactate. The Vmax and Km values for L-lactate were 300 nmol mg protein-1 min-1 and 0.7 mM. The iLDH had a pH optimum of 7.5, was not activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) or dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl viologen (BV). The iLDH did not have strong absorbance between 500 and 300 nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually no fluorescence at 450 nm. The crude extracts also had MTT-linked butyryl-CoA dehydrogenase activity (60 nmol mg protein-1 min-1). The NAD-independent butyryl-CoA dehydrogenase eluted from DEAE-cellulose as two fractions. The yellow fraction was extremely unstable, but the green fraction could be stored for short periods of time at 5 degrees C. The green-colored butyryl-CoA dehydrogenase had strong absorption at 450 nm, and gel filtration indicated that it had a molecular weight of 90 kDa. The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT, DCPIP, or MV, but not BV. Because the NAD-independent lactate and butyryl-CoA dehydrogenase could both be linked to low potential carriers, these two enzymes may function as oxidation-reduction system in vivo.

Butyryl-CoA Dehydrogenase↗

PubMed1997

The effect of nisin and monensin on ruminal fermentations In vitro.

When mixed ruminal bacteria and alfalfa were incubated in vitro, monensin and nisin both inhibited methane production so long as the concentrations were greater than 1 microM. Monensin- and nisin-dependent methane depressions caused a decrease in the acetate to propionate ratio (4.5 to 3.0). Total volatile fatty acid production was decreased by both monensin and nisin addition at concentrations greater than 2 microM. Starch-digesting ruminal bacteria were initially inhibited by monensin and nisin, but this effect disappeared after two to four transfers. Nisin always inhibited cellulolytic bacteria, but the nisin-dependent inhibition of cellulose digestion was no greater than the inhibition caused by monensin. Monensin and nisin also inhibited amino acid degradation, and nisin was more effective than monensin in controlling the growth of Clostridium aminophilum, an obligate amino acid-fermenting ruminal bacterium that can tolerate low concentrations of monensin. Because nisin was as potent as monensin, bacteriocins such as nisin may have potential as feed additives.

Acetates↗

PubMed1997

A Prevotella ruminicola B(1)4 operon encoding extracellular polysaccharide hydrolases.

When Escherichia coli XL1-Blue MRA (P2) was infected with lambda DNA containing Prevotella ruminicola B(1)4 chromosomal DNA, only a few plaques produced beta-1,4-endoglucanase activity, and all of these had mannanase activity. Positive phage contained a 17-kb SacI DNA fragment that gave six bands after EcoRI digestion. The EcoRI fragments were ligated into pBluescript and sequenced. The order of the fragments was verified by PCR and by restriction mapping. The DNA sequence contained 6 open reading frames (ORFs). The 4th and 5th ORFs encoded two related beta-1,4-endoglucanases. E. coli clones carrying ORF5 and ORF6 had beta-1,4-endoglucanase and mannanase activities, while a clone carrying only ORF6 hydrolyzed mannan but not carboxymethylcellulose. The 6th ORF had three regions of homology to mannanase A from Pseudomonas fluorescens. Based on these results, ORF6 encoded the mannanase gene. The 3rd ORF had 10 regions of homology with cellulose-binding protein A from Clostridium cellulovorans. The 1st and 2nd ORFs had no significant homology to genes or amino acid sequences in GeneBank or SwissProt. All of the ORFs except 1 encoded a potential signal peptide sequence. The upstream region of ORF1 contained four direct repeats and four inverted repeat elements, but no apparent sigma70 sequence-like promoter was present. The segment of DNA containing the 6 ORFs was preceded and followed by potential transcription termination signals suggesting a single transcriptional unit.

Amino Acid Sequence↗

PubMed1997

The ability of 2-deoxyglucose to promote the lysis of Streptococcus bovis JB1 via a mechanism involving cell wall stability.

The non-metabolizable glucose analog, 2-deoxyglucose (2-DG), decreased the growth rate and optical density of Streptococcus bovis JB1 20%, but it had an even greater effect on stationary phase cultures. Control cultures receiving only glucose (2 mg/ml) lysed very slowly (<5% decline in optical density in 48 h), but cultures that had been grown with glucose and 2-DG (2 mg/ml each) lysed much faster (>85% decline in optical density in 48 h). Cultures that were treated with inhibitors that decreased intracellular ATP (sodium fluoride, nigericin, and valinomycin or tetrachlorosalicylanilide) or membrane potential (sodium fluoride, nigericin, and valinomycin, tetrachlorosalicylanilide, or phenylmethylsulfonyl fluoride) did not promote lysis. 2-DG had its greatest effect when it was added at inoculation. If 2-DG was added at later times, less lysis was observed, and cells that were given 2-DG just prior to stationary phase were unaffected. Cells that were grown with glucose and 2-DG were more susceptible to cell wall-degrading enzymes (lysozyme and mutanolysin) than cells that had been grown only with glucose, but sublethal doses of penicillin during growth did not promote lysis after the cells had reached stationary phase. The idea that 2-DG might be affecting autolytic activity was supported by the observation that cultures washed and resuspended in fresh medium with or without 2-DG lysed at a slower rate than cultures that were not centrifuged or were resuspended in the culture supernatant.

Adenosine Triphosphate↗

PubMed1997

Unstimulated immature oocyte retrieval: early versus midfollicular endometrial priming.

OBJECTIVE: To assess the ability to retrieve transvaginally unstimulated immature oocytes from assisted reproductive technology (ART) patients and to determine their competency to mature, fertilize, and implant with early versus midfollicular exogenous estrogen endometrial priming. DESIGN: Prospective randomized study. SETTING: In vitro fertilization unit, private reproductive endocrinology and infertility practice. PATIENT(S): Fourteen patients, all had failed at least one IVF cycle. INTERVENTION(S): Early follicular endometrial priming was initiated on cycle day 3 with 2 mg 17 beta-E2 twice per day (group A). Midfollicular endometrial priming was initiated with 1 to 2 mg/d of 17 beta-E2 between cycle days 5 and 7 and gradually increased by 1 to 2 mg/d until the oocyte retrieval (group B). The oocytes were allowed to mature in 0.075 IU FSH or hMG, 0.5 IU of hCG, 1 microgram of 17 beta-E2 in Eagle's or Tissue Culture Media 199, fertilized, and transferred 72 hours later. MAIN OUTCOME MEASURE(S): Maturation, fertilization, and pregnancy rate. RESULT(S): Group A patients had 83 oocytes retrieved (11.8 +/- 6.1) versus 78 oocytes (11.1 +/- 2.7) from group B. The maturation rate in group A was 39.7% (34/83) versus 61.5% (48/78) in group B. The fertilization rate was 75.7% (25/34) in group A versus 75.0% (36/48) in group B. The cleavage arrest rate was significantly higher, 36.0% (9/25) in group A versus 8.3% (3/ 36) in group B. The number of embryos transferred was 1.8 embryos per retrieval in group A versus 4.0 embryos per retrieval in group B. One pregnancy was established in a patient with tubal disease in group B who delivered at 36 weeks gestation. CONCLUSION(S): Unstimulated immature oocyte retrieval can be performed successfully in ART patients. Midfollicular endometrial priming was able to achieve successful maturation (60%), fertilization (75%), and cleavage (92%), with the delivery of a successful pregnancy.

Adult↗

PubMed1997

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85.

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).

Journal Article↗

PubMed1997

Influence of monensin on Holstein steers fed high-concentrate diets containing soybean meal or urea.

We conducted two growth trials to evaluate the effects of monensin on amino acid sparing. When Holstein steers were fed a 90% concentrate diet supplemented with soybean meal (13.5% CP), the DMI, ADG, and efficiencies of feed and nitrogen utilization were greater than with urea (P < .10). Monensin improved ADG with both nitrogen supplements (P < .01), but the positive effects of monensin on efficiencies of feed (P = .12) and nitrogen (P = .26) utilization were greater for soybean meal than for urea. Increasing amounts of monensin (0, 11, or 22 mg/kg of DM) caused a linear increase in DMI with urea. Diets with soybean had greater intakes than diets with urea (P < .01); the greatest intake was of a soybean diet with monensin at 11 mg/kg of DM. Holstein steers fed soybean meal at 13.5% CP had lower DMI and greater efficiencies of feed and nitrogen utilization than steers fed 16.7% CP (P < .10). Crude protein level had no effect on ADG (P > .10). Monensin always increased the efficiencies of feed and nitrogen utilization (P < .05), but these trends were greater for diets with 16.7 than for those with 13.5% CP. Overall, monensin decreased DMI (P < .01), but this effect was greater for 16.7% than for 13.5% CP. Because the positive effects of monensin on diet NEg (P = .16) and efficiency of nitrogen utilization (P = .26) were greater for soybean meal than for urea, it seemed that monensin was sparing amino acids.

Animals↗

PubMed1997

Effect of forage quality and monensin on the ruminal fermentation of fistulated cows fed continuously at a constant intake.

A ruminal fermentation study was used to investigate the relationship between forage quality and monensin-dependent amino acid-sparing. Two fistulated cows were fed three concentrations of chopped (2.5 to 5.0 cm in length) timothy and alfalfa hays (100:0, 50:50 and 0:100) and two levels of monensin (0 and 350 mg.cow-1.d-1). The diets were offered 12 times per day (9 kg of DM/d), and the rumen reached a steady-state, reducing animal and day variation. Alfalfa hay had 1.4 times more CP and 1.4 times less NDF than timothy hay, and the substitution of timothy with alfalfa increased (P < .05) ruminal ammonia. Monensin had no effect on total ruminal ammonia when timothy hay was present in the diet, and it increased total ruminal ammonia with the 100% alfalfa diet. Effects on ruminal ammonia were, however, confounded by monensin-dependent decreases in ruminal pH (P < .05). Dissociated ammonia, the species most likely to be adsorbed from the rumen, declined when monensin was added to the 100% timothy diet (P < .05). Monensin had no effect on dissociated ammonia if alfalfa was 50% or 100% of the forage, but it counteracted alfalfa-dependent decreases in bacterial protein (P < .05). The idea that monensin could spare amino acids was supported by the observation that monensin decreased (P < .001) the specific activity of deanimation and increased bacterial protein at all combinations of alfalfa and timothy. Increases in bacterial protein could be explained by monensin-dependent increases in total VFA.

Ammonia↗

PubMed1997

The endogenous polysaccharide utilization rate of mixed ruminal bacteria and the effect of energy starvation on ruminal fermentation rates.

When mixed ruminal bacteria were starved in vitro for 24 h, cellular ATP decreased, but there was little change in cell protein. Starved ruminal bacteria derived most of their ATP from cellular polysaccharide. Because polysaccharide declined at a first-order rate of 23%/h, it was possible to estimate the endogenous polysaccharide utilization rate at various stages of starvation by multiplying the amount of utilizable polysaccharide remaining at each time point by 0.23. The bacteria initially had a rate of soluble carbohydrate fermentation that was > 717 micrograms of hexose equivalent/mg of protein per h. Starvation had little impact on the rate of soluble carbohydrate fermentation until 8 to 12 h, and the endogenous polysaccharide utilization rate was < 10 micrograms of hexose/mg of protein per h. The bacteria digested ball-milled cellulose at a rate of 24 micrograms of hexose/mg of protein per h for 8 to 12 h. Even bacteria that had been starved for 24 h fermented cellulose at a rate of 16 micrograms of hexose/mg of protein per h. The rate of methane production was initially 70 nmol of methane/mg of protein per min. Short periods of starvation (< 12 h) had little impact on methane production, but longer times caused an almost complete inhibition of methanogenesis. The rate of amino acid deamination was initially 31 nmol of ammonia/mg of protein per min, and the critical phase of starvation was again 8 to 12 h. Ruminal bacteria that were harvested at 24 h after feeding had 10-fold less polysaccharide than did bacteria that were harvested at 2 h after feeding, but this polysaccharide supported high rates of soluble carbohydrate and cellulose fermentation, deamination, and methane production.

Adenosine Triphosphate↗

PubMed1996

The acetate kinase of Clostridum acetobutylicum strain P262.

Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

Acetate Kinase↗

PubMed1996

Inducer expulsion is not a determinant of diauxic growth in Streptococcus bovis.

When Streptococcus bovis JB1 was repeatedly transferred in a medium that contained the non-metabolizable glucose analog, 2-deoxyglucose, it lost its phosphotransferase system (PTS) for glucose but was still able to take up glucose via a facilitated diffusion mechanism. The wild type (JB1) had an inducible enzyme II lactose, but the mutant (JB1(2DG)) had a constitutive lactose PTS. JB1(2DG) was no longer able to exclude lactose when it was provided with glucose, but it retained its ability to expel a non-metabolizable lactose analog. Because JB1(2DG) could utilize glucose and lactose simultaneously and grow in a non-diauxic fashion, it appeared that inducer expulsion was not an important catabolite regulatory mechanism. Based on these results, inducer expulsion may be an artifact of non-metabolizable sugars.

Biological Transport, Active↗

PubMed1996

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis.

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)-1(min)-1. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (Ks) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a Ks of 0.20 mM, but the Ks of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport.

Bacteriological Techniques↗

PubMed1996

Use of a modified Bacteroides-Prevotella shuttle vector to transfer a reconstructed beta-1,4-D-endoglucanase gene into Bacteroides uniformis and Prevotella ruminicola B(1)4.

A carboxymethyl cellulase (CMCase) gene from Prevotella ruminicola B(1)4 was reconstructed by adding a cellulose binding domain from a Thermomonospora fusca cellulase and was conjugally transferred from Escherichia coli to Bacteroides uniformis 0061 by using a chloramphenicol and tetracycline resistance shuttle vector (pTC-COW). pTC-COW was specifically constructed to facilitate conjugal transfer of vectors from B. uniformis donors to P. ruminicola recipients. B. uniformis transconjugants containing CMCase constructs cloned into pTC-COW expressed Cmr, but they did not produce the reconstructed CMCase until a xylanase promoter from P. ruminicola 23 was added upstream of the CMCase (pTC-XRCMC). The xylanase promoter allowed the B. uniformis transconjugants to produce large amounts of the reconstructed CMCase, which was present on the outside surface of the cells. Although the reconstructed CMCase alone did not allow B. uniformis to grow on acid-swollen cellulose, rapid growth was observed when two exocellulases were added to the culture supernatant. Under these conditions, the reconstructed CMCase permitted faster growth than the wild-type CMCase. The frequency of transfer of pTC-XRCMC from B. uniformis to P. ruminicola B(1)4 was increased 100-fold when strictly anaerobic conditions, nitrocelluose filters (cell immobilization), and more stringent selections were employed. Although the P. ruminicola B(1)4 (pTC-XRCMC) transconjugates expressed Tcr and had DNA that hybridized with a probe to the shuttle vector, these transconjugants did not produce detectable levels of the reconstructed CMCase even when xylan was the carbon source. On the basis of these results, it appears that not all of the promoters recognized by B. uniformis and P. ruminicola 23 are functional in P. ruminicola B(1)4. However, the results with B. uniformis suggest that the introduction of a P. ruminicola B(1)4 promoter should allow expression of the reconstructed CMCase in P. ruminicola B(1)4.

Anaerobiosis↗

PubMed1996

Use of potassium depletion to assess adaptation of ruminal bacteria to ionophores.

When mixed ruminal bacteria from cattle fed timothy hay were suspended in a medium containing a low concentration of potassium, monensin and lasalocid catalyzed a rapid depletion of potassium from cells. The ionophore-mediated potassium depletion was concentration dependent, and it was possible to describe the relationship with saturation constants. Mixed ruminal bacteria never lost more than 50% of their potassium (Kmax = 46%), and the concentrations of monensin and lasalocid needed to cause half-maximal potassium depletion (Kd) were 178 and 141 nM, respectively. When cattle were fed 350 mg of monensin per day, the ratio of ruminal acetate to propionate decreased from 4.2 to 2.9, and the Kd of monensin was eightfold greater than the value for mixed ruminal bacteria from control animals. Monensin supplementation also caused a twofold increase in the Kd of lasalocid. Lasalocid supplementation (350 mg per day) had no effect on the ruminal acetate-to-propionate ratio, but it caused a twofold increase in the Kd values of monensin and lasalocid. Increases in Kd occurred almost immediately after ionophore was added to the ration, and the Kd values returned to their prefeeding values within 14 days of withdrawal. Ionophore supplementation had no effect on the Kmax values, and approximately 50% of the population was always highly ionophore resistant. Because the Kd values of even adapted ruminal bacteria were low (< 1.5 microM), it appears that a large proportion of the ruminal ionophore is bound nonselectively to feed particles or ionophore-resistant bacteria.

Adaptation, Physiological↗

PubMed1996

The Intracellular pH of Clostridium paradoxum, an Anaerobic, Alkaliphilic, and Thermophilic Bacterium.

When the extracellular pH was increased from 7.6 to 9.8, Clostridium paradoxum, a novel alkalithermophile, increased its pH gradient across the cell membrane ((Delta)pH, pH(infin) - pH(infout)) by as much as 1.3 U. At higher pH values (>10.0), the (Delta)pH and membrane potential ((Delta)(psi)) eventually declined, and the intracellular pH increased significantly. Growth ceased when the extracellular pH was greater than 10.2 and the intracellular pH increased to above 9.8. The membrane potential increased to 110 (plusmn) 8.6 mV at pH 9.1, but the total proton motive force ((Delta)p) declined from about 65 mV at pH 7.6 to 25 mV at pH 9.8. Between the extracellular pH of 8.0 and 10.3, the intracellular ATP concentration was around 1 mM and decreased at lower and higher pH values concomitantly with a decrease in growth rate.

Journal Article↗

PubMed1996

An rRNA approach for assessing the role of obligate amino acid-fermenting bacteria in ruminal amino acid deamination.

Ruminal amino acid degradation is a nutritionally wasteful process that produces excess ruminal ammonia. Monensin inhibited the growth of monensin-sensitive, obligate amino acid-fermenting bacteria and decreased the ruminal ammonia concentrations of cattle. 16S rRNA probes indicated that monensin inhibited the growth of Peptostreptococcus anaerobius and Clostridium sticklandii in the rumen. Clostridium aminophilum was monensin sensitive in vitro, but C. aminophilum persisted in the rumen after monensin was added to the diet. An in vitro culture system was developed to assess the competition of C. aminophilum, P. anaerobius, and C. sticklandii with predominant ruminal bacteria (PRB). PRB were isolated from a 10(8) dilution of ruminal fluid and maintained as a mixed population with a mixture of carbohydrates. PRB did not hybridize with the probes to C. aminophilum, P. anaerobius, or C. sticklandii. PRB deaminated Trypticase in continuous culture, but the addition of C. aminophilum, P. anaerobius, and C. sticklandii caused a more-than-twofold increase in the steady-state concentration of ammonia. C. aminophilum, P. anaerobius, and C. sticklandii accounted for less than 5% of the total 16S rRNA and microbial protein. Monensin eliminated P. anaerobius and C. sticklandii from continuous cultures, but it could not inhibit C. aminophilum. The monensin resistance of C. aminophilum was a growth rate-dependent, inoculum size-independent phenomenon that could not be maintained in batch culture. On the basis of these results, we concluded that the feed additive monensin cannot entirely counteract the wasteful amino acid deamination of obligate amino acid-fermenting ruminal bacteria.

Amino Acids↗

PubMed1996

The effect of growth and starvation on the lysis of the ruminal cellulolytic bacterium Fibrobacter succinogenes.

Growing cultures of Fibrobacter succinogenes assimilated more ammonia than could be accounted for by cellular protein, RNA, or DNA and released large amounts of nonammonia nitrogen. The difference between net and true growth was most dramatic at low dilution rates, but mathematical derivations indicated that the lysis rate was a growth rate-independent function. The lysis rate was sevenfold greater than the true maintenance rate (0.07 h-1 versus 0.01 h-1). Because slowly growing cells had as much proton motive force and ATP as fast-growing cells, lysis was not a starvation response per se. Stationary-phase cells had a lysis rate that was 10-fold less than that of growing cells. Rapidly growing cells were not susceptible to phenylmethylsulfonyl fluoride, but phenylmethylsulfonyl fluoride increased the lysis rate of the cultures when they reached the stationary phase. This latter result indicated that autolysins of stationary-phase cells were being inactivated by a serine proteinase. When growing cells were treated with the glycolytic inhibitor iodoacetate, the proteinase-dependent transition to the stationary phase was circumvented, and the rate of lysis could be increased by as much as 50-fold.

Ammonia↗

PubMed1996

A Role for Fructose 1,6-Diphosphate in the ATPase-Mediated Energy-Spilling Reaction of Streptococcus bovis.

The amount of ATP produced by Streptococcus bovis was larger than the amount that could be attributed to growth and maintenance, and even glucose-limited continuous cultures used ATP inefficiently (spilled ATP). Rapid-dilution-rate cultures always spilled more ATP than those growing at slow dilution rates, but rates of ATP spilling could also be enhanced by amino acid deprivation (with only ammonia as a nitrogen source). Energy spilling and intracellular ATP were not correlated, but energy spilling was always greatest when the rate of lactate production was high. The relationship between lactate production and energy spilling was supported by the observation that amino acid deprivation increased lactate production and ATP spilling. The lactate production rate of nongrowing (energy-spilling) S. bovis cells was fructose 1,6-diphosphate (FDP) dependent, and previous work showed that the lactate dehydrogenase of S. bovis was activated by FDP (M. J. Wolin, Science 146:775-777, 1964). The role of FDP in energy spilling was supported by the observation that the membrane-bound ATPase of S. bovis could be stimulated by FDP. FDP decreased the K(infm) for ATP by as much as fivefold. Other glycolytic intermediates could not stimulate the ATPase of washed membrane preparations, and FDP had no effect on soluble ATPase activity.

Journal Article↗