CHEMICAL PACEMAKERS : III. ACTIVATION ENERGIES OF SOME RATE-LIMITING COMPONENTS OF RESPIRATORY SYSTEMS

1. In a previous paper it was found that 11,200 calories is obtained for the energy of activation in the oxidation of succinate to fumarate in the presence of crude beef heart extract when succino-dehydrogenase was made the limiting factor. 16,000 calories was obtained with this preparation when cytochrome-cytochrome oxidase was made the limiting factor. In the present paper activation energies of the components of this enzyme system are further studied. 2. Oxidation of p-phenylenediamine catalyzed by the extract and known not to involve the dehydrogenase component yields Arrhenius equation plots indicating a pacemaker reaction with a µ of 9,500 calories. 3. An activation energy of 17,500 calories is obtained for the oxidation of succinate to fumarate in the presence of the beef heart extract partially poisoned by pyrophosphate. Evidence is presented that this value corresponds to a link in the respiratory chain other than that of succino-dehydrogenase or cytochrome c-cytochrome oxidase. 4. Addition of a suitable amount of cresyl blue to a beef heart extract reaction mixture, completely inhibited by cyanide, restores the oxidation of succinate to normal in the presence of pure oxygen. In this system, in which the dye is substituted for the oxidase, when the enzyme extract (dehydrogenase) is made the limiting factor, a µ of 18,500 calories is obtained; when cresyl blue is made the limiting factor, the µ value is 22,000 calories. 5. Results of these experiments indicate that energies of activation are associated not with the enzyme as such, but with the particular reaction steps involving them as catalysts.     

TEMPERATURE CHARACTERISTICS FOR HEART BEAT FREQUENCY IN MICE

An apparatus is described which permits the simultaneous recording of body temperature and heart beat frequency in young mice. When heart beat frequency is related to body temperature the values of the temperature characteristic for the inbred albino strain used are 22,400– calories over the range 15 to 20°+, 16,000± calories from 20+ to 27°, and 11,000± calories from 27 to 35°+.     

TEMPERATURE CHARACTERISTICS FOR THE GROWTH OF THE SPORANGIOPHORES OF PHYCOMYCES

1. With constant temperature and light intensity, the rate of elongation of a sporangiophore of Phycomyces is constant for many hours. 2. With constant light intensity, values of µ, the "critical thermal increment," have been computed for the elongation of different sporangiophores. These values group themselves closely about the modes 11,000, 16,000, 20,000, 26,000, and 33,000 calories. Sporangiophores from the same culture need not have similar increments. 3. The significance of these results for studies of growth is discussed.     

TEMPERATURE CHARACTERISTICS FOR METABOLISM OF CHLORELLA : I. THE RATE OF O2 UTILIZATION OF CHLORELLA PYRENOIDOSA WITH ADDED DEXTROSE

The temperature characteristic for the rate of O₂ consumption by Chlorella pyrenoidosa suspended in Knop solution containing 1 per cent glucose was studied between 1° and 27°C. with the Warburg technic. The value of µ was found to be about 19,000 ±1,000 cal. There is some indication of a critical temperature at 20°C., with shift to a lower µ above this temperature. The effect of sudden changes in temperature on the rate of respiration and the variation of the latter with time at constant temperatures are discussed. It is concluded that the "normal" respiration (in absence of external glucose) does not appear in the determination of this temperature characteristic.     

TEMPERATURE AND FORWARD MOVEMENT OF PARAMECIUM

1. The rate of forward movement in Paramecium as affected by changes in temperature can be described accurately in terms of the Arrhenius equation. See PDF for Equation 2. For the range from 6–15°, µ = 16,000; from 16–40°, µ = 8,000. These values fall within the limits characteristic for chemical processes. 3. On the principle of velocity control by the slowest rate, it is assumed that in Paramecium at temperatures above normal, control passes from one underlying reaction to another. 4. The views expressed by Rice, the recent results of Crozier, and certain µ values given by Arrhenius all suggest that µ = 16,000 may represent an oxidation, and µ = 8,000 either a modified oxidation or an hydrolysis. 5. For the system of controls, the catenary series O → A → E with the lower µ value attached to the precursor reaction is adequate. We may also assume a cyclical system analogous to Meyerhof''s conception of carbohydrate metabolism in muscle. In this case it is necessary to assign µ = 16,000 to the oxidation of A and E and µ = 8,000 to the synthesis E → O. This model also accounts for the fact that the data might be interpreted as involving, apparently, a depletion of A at the higher temperature.     

Respiratory Chain of Colorless Algae II. Cyanophyta

Whole cell difference spectra of the blue-green algae, Saprospira grandis, Leucothrix mucor, and Vitreoscilla sp. have one, or at the most 2, broad α-bands near 560 mμ. At −190° these bands split to give 4 peaks in the α-region for b and c-type cytochromes, but no α-band for a-type cytochromes is visible. The NADH oxidase activity of these organisms was shown to be associated with particulate fractions of cell homogenates. The response of this activity to inhibitors differed from the responses of the NADH oxidase activities of particulate preparations from the green algae and higher plants to the same inhibitors, but is more typical of certain bacteria. No cytochrome oxidase activity was present in these preparations. The respiration of Saprospira and Vitreoscilla can be light-reversibly inhibited by CO, and all 3 organisms have a CO-binding pigment whose CO complex absorbs near 570, 535, and 417 mμ. The action spectrum for the light reversal of CO-inhibited Vitreoscilla respiration shows maxima at 568, 534, and 416 mμ. The results suggest that the terminal oxidase in these blue-greens is an o-type cytochrome.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

CRITICAL THERMAL INCREMENTS FOR RHYTHMIC RESPIRATORY MOVEMENTS OF INSECTS

The rhythm of abdominal respiratory movements in various insects, aquatic and terrestrial, is shown to possess critical increments 11,500± or 16,500± calories (Libellula, Dixippus, Anax). These are characteristic of processes involved in respiration, and definitely differ from the increment 12,200 calories which is found in a number of instances of (non-respiratory) rhythmic neuromuscular activities of insects and other arthropods. With grasshoppers (Melanoplus), normal or freshly decapitated, the critical increment is 7,900, again a value encountered in connection with some phenomena of gaseous exchange and agreeing well with the value obtained for CO₂ output in Melanoplus. It is shown that by decapitation the temperature characteristic for abdominal rhythm, in Melanoplus, is changed to 16,500, then to 11,300—depending upon the time since decapitation; intermediate values do not appear. The frequency of the respiratory movements seems to be controlled by a metabolically distinct group of neurones. The bearing of these results upon the theory of functional analysis by means of temperature characteristics is discussed, and it is pointed out that a definite standpoint becomes available from which to attempt the specific control of vital processes.     

TEMPERATURE CHARACTERISTIC FOR PRODUCTION OF CO2 BY PHASEOLUS SEEDLINGS

The temperature characteristic for respiratory production of CO₂ by young seedlings of Phaseolus aureus (Roxb.) is µ = 16,500 calories, 12–21°C., even when the analyses depend upon the use of many seedlings crowded in a small respiration chamber, provided reasonable precautions are taken to avoid injury and to permit proper thermal adaptation. There is evidence of a definite critical temperature at 20–21°. These findings agree quantitatively with those obtained with other similar seedlings, and contradict the results reported by Kurbatov and Leonov (1930); the reasons for this are analysed.     

Studies on the origin of bacterial viruses. V. The effect of temperature on the terramycin-resistant and phage-producing cells of Bacillus megatherium cultures

The growth rates, the mutation frequency rate constants of the terramycin-resistant cells, the burst size of the phage-producing cells, and the ratio of phage to cells all have a temperature coefficient of about 2 from 20 to 35° (µ = 9 x 10³ calories), with a maximum at 40°. The mutation frequency rate constant (or time rate constant) of the phage-producing cells increases from 20 to 45° with a temperature coefficient of about 3 (µ = 2 to 3 x 10⁴ cal.). The change in the values for the growth rate, mutation rate, and cell volume occurs in less than 1 hour, after the temperature is changed. The value for the burst size of phage-producing cells changes for 3 to 4 hours. Prolonged growth of megatherium 899 at 48 to 50° results in the production of C + S phage, in place of T. Returning the culture to 25° results in the production of small T phage.     

Influence of growth temperature on respiratory characteristics of mitochondria from callus-forming potato tuber discs

The uninhibited respiration of mitochondria, isolated from potato tuber discs (Solanum tuberosum L. cv. Bintje) incubated on a callus-inducing medium at 28°C, is higher than that of mitochondria from tissue incubated at 8°C. This respiration is composed of a CN-sensitive and a CN-resistant part. The capacity of the CN-resistant alternative oxidase pathway is larger in mitochondria from 28°C tissue than in mitochondria from 8°C tissue (35% and 8% of uninhibited respiration, respectively). The alternative pathway is operative both in mitochondria from 28°C tissue and 8°C tissue.

The observed difference in uninhibited respiration, is not only caused by lower values of respiration via the alternative pathway in mitochondria from 8°C tissue, but also by lower values of respiration via the cytochrome pathway.

A positive correlation has been demonstrated between the incubation temperature (ranging from 4-37°C) and the relative capacity of respiration via alternative pathway in the mitochondria. Induction of alternative pathway is not directly correlated with growth (in terms of increase in fresh weight) of the potato tuber discs.

     

TEMPERATURE CHARACTERISTICS FOR THE METABOLISM OF CHLORELLA : III. THE CATALYTIC DECOMPOSITION OF HYDROGEN PEROXIDE BY CHLORELLA PYRENOIDOSA

The decomposition of hydrogen peroxide by intact Chlorella cells follows a first order course at very low temperatures, but at higher temperatures gives falling first order constants. Between 0.6° and 20°C. the value of µ is 10,500 calories.     

ON BIOLOGICAL OXIDATIONS AS FUNCTION OF TEMPERATURE

1. The critical thermal increments are calculated for respiratory processes (O₂ consumption, CO₂ production) in various plants and animals. They are characteristically found to be of two, possibly three, types: µ = 11,500, and 16,100 or 16,700. The first is commonly encountered above 15°, the second below that temperature, but these relations may be reversed. (The value of µ may be significantly changed in inanition.) 2. For reduction of methylene blue by bacteria, through removal of H from succinic acid, µ = 16,700. This process (Quastel and Whetham, 1924) at constant temperature is a function of the hydroxyl ion concentration. The suggestive identity is pointed out of the critical increment for this reduction phenomenon with that deduced for biological respirations in which a dehydrogenation mechanism is supposed to be of widespread occurrence, and in connection with which Fe very likely has a catalytic rôle. The action of OH'' is believed to be revealed in the value µ = 11,500, frequently obtained in connection with respiration. 3. A somewhat lower µ (16,140) is associated with the oxidation of Fe'''', and may be compared with (1) that of respiration in sea urchin eggs, for which (Warburg) iron is catalyst, and (2) that for some simple reactions in which Fe is known to serve as catalyst; it is not found for oxidative reactions in which Fe is not involved. 4. The bearing of these findings is discussed in relation to the theory of functional analysis of concurrent catalyzed reactions in protoplasm. It is shown that for a number of activities in which the effects of respiration may safely be assumed, the values of the critical increments are consistent with those determined for processes of respiration. 5. The further development of these views may lead to an extremely important method of identifying controlling reactions in undisturbed living matter.     

PULSATION FREQUENCY OF THE ADVISCERAL AND ABVISCERAL HEART BEATS OF CIONA INTESTINALIS IN RELATION TO TEMPERATURE

The frequency of pulsation of the heart of Ciona intestinalis increases with temperature in both advisceral and abvisceral direction, according to the Arrhenius equation. The increase in pulsation is the same in both directions. The following µ values were obtained: 8,000–, 12,000+, 16,000, in several combinations, with critical temperatures at 10°, 15°, and 20°C. The values found are comparable with earlier findings for activity of the heart in different animals. This quantitative correspondence suggests anew the conception that temperature characteristics may be employed for recognition of controlling processes. The fact that the µ''s and the critical temperature are the same for advisceral and abvisceral beats, indicates that the general metabolic condition of the two ends of the heart is the same in any one individual.     

TEMPERATURE CHARACTERISTICS FOR THE PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS AND ZEA MAYS

The rates of production of CO₂ by germinating seeds of Lupinus albus and Zea mays were studied between temperatures 12.5° and 25°C. with the HCl-Ba(OH)₂ titration method. The temperature characteristics found are different from those previously obtained for the oxygen consumption of the same seeds germinated in the same manner. For Lupinus, the temperature characteristics above and below the critical temperature of 20° are 16,100 ± and 24,000 ± calories respectively. For Zea, no evidence of a critical temperature was found in this region, and the temperature characteristic is 20,750 ± calories throughout the range of temperature tested. The possible interpretations of the difference in the values of temperature characteristics for oxygen consumption and for production of CO₂ are noted.     

ACTION POTENTIALS IN THE NERVOUS SYSTEM OF THE CRAYFISH : IV. INFLUENCE OF TEMPERATURE ON NERVE IMPULSES ARISING "SPONTANEOUSLY" IN ABDOMINAL GANGLIA

Increase in temperature elicits an increase in the number of nerve impulses arising spontaneously from deafferented crayfish ganglia. This alteration in gross frequency gives an apparent temperature characteristic of 27,000 calories. Changes in the number of active fibers and in the frequency of discharge of individual units account for the alterations in the gross frequency. The change in number of active units gives a n value of 17,500 calories. Individual fibers fall into two groups with respect to the effect of temperature on their frequency of discharge. One of these groups exhibits a µ value of 14,500 calories and the other yields a µ value of 7,000 calories.     

The Effect of Growth and Measurement Temperature on the Activity of the Alternative Respiratory Pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19°C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28°C but was unchanged in soybean cotyledons grown at 14°C compared with plants grown at 28°C. When the short-term response of tissue respiration was measured over the temperature range of 35°C to 9°C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19°C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.     

Persistence of PCR-Detectable Bacteroides distasonis from Human Feces in River Water

To evaluate the persistence of PCR-detectable Bacteroides distasonis in surface water, whole human feces were dispersed into water from the Ohio River and incubated in flasks in the laboratory or in diffusion chambers in situ. Duplicate samples were taken daily, and material that pelleted at 16,000 × g was assayed by PCR. Persistence of PCR-detectable DNA from this anaerobe depended upon temperature and predation, two of the factors shown by others to influence the survival of aerobic bacteria detected by culture. B. distasonis was detected by PCR for at least 2 weeks at 4°C but for only 4 to 5 days at 14°C, 1 to 2 days at 24°C, and 1 day at 30°C. In filtered water or in the presence of cycloheximide, a eukaryotic inhibitor, persistence at 24°C was extended by at least a week.     

Studies in biochemical genetics in Drosophila

The metabolism of the imaginal discs of wild type, miniature, vestigial, and four-jointed varieties of Drosophila was investigated using the Cartesian diver ultramicrorespirometer. Wild type and vestigial wing disc respiration is inhibited by cyanide and azide and thus is mediated by an iron or copper porphyrin system, presumable cytochrome-cytochrome oxidase. Respiration is also inhibited by certain hydroxynaphthoquinones, believed to inactivate some enzyme between cytochromes b and c. The respiration of the vestigial and miniature wing discs is increased to normal by the addition of ascorbic acid and to a lesser extent by p-phenylenediamine and hydroquinone, hence the cytochrome oxidase and cytochrome c systems of vestigial and miniature wing discs are normal and the effects of these genes are on enzymes below cytochrome c in the respiratory chain. The respiratory enzymes of the developing imaginal discs of insects are similar to those of a wide variety of cells from bacteria to mammals. The correlation of these biochemical findings with embryological studies of the discs is discussed.     

TEMPERATURE CHARACTERISTIC FOR THE ANAEROBIC PRODUCTION OF CO2 BY GERMINATING SEEDS OF LUPINUS ALBUS

The rate of anaerobic production of CO₂ by germinating seeds of Lupinus albus was studied as a function of temperature between 7.5° and 18°C. The mean value for the temperature characteristic was found to be 21,500± calories, which is slightly lower than that for the same process under aerobic conditions (23,500± calories). The values for the individual µ''s in the two cases overlap considerably. The possible identity of the processes underlying the production of CO₂ aerobically and anaerobically is discussed.