Production of citric acid from n-paraffins bySaccharomycopsis lipolytica: Kinetics and balance of the fermentation

Summary The excretion of citric and isocitric acids was studied in a stirred fermentor with control of various fermentation parameters. Growth being nitrogen-limited, excretion was found to start at the end of the growth phase, constant production rates being then obtained for the two acids for about 90 h. A linear relationship between these production rates and cell density is observed, thus allowing a definition of specific production rates. Variation of these rates with temperature, aeration, pH and medium iron content were studied. The main effects observed are those of pH, which shows a clear optimum at pH 5, and of iron content, the lower values of which promote citric acid excretion. During the excretion phase rate measurements for all reactants (n-paraffins-oxygen) and products (carbon dioxide-citric and isocitric acids) show that good carbon and oxygen balance are obtained. Comparison with a similar fermentation using glucose is also presented and discussed.Abbreviations v.v.m. volume of air per volume of medium per minute - st.p.m. strokes per minute This work is a part of a Doctorat de Spécialité thesis submitted by R. Marchal to the University of Nancy 24-7-1975     

Kinetic properties of a magnesium ion- and calcium ion-stimulated adenosine triphosphatase from the outer-membrane fraction of rat spleen mitochondria

1. Isolated outer membranes from rat spleen mitochondria can be stored in liquid N(2) for several weeks without significant loss of ATPase (adenosine triphosphatase) activity. 2. The ATPase reaction has a broad pH optimum centering on neutral pH, with little significant activity above pH9.0 or below pH5.5. 3. A sigmoidal response of the ATPase activity to temperature is observed between 0 and 55 degrees C, with complete inactivation at 60 degrees C. The Arrhenius plot shows that the activation energy above the transition temperature (22 degrees C) (E(a)=144kJ/mol) is one-third of that calculated for below the transition temperature (E'(a)=408kJ/mol). 4. The outer-membrane ATPase (K(m) for MgATP=50mum) is inactive unless Mg(2+) is added, whereas the inner-membrane ATPase (K(m) for ATP=11mum) is active without added Mg(2+) unless the mitochondria have been depleted of all endogenous Mg(2+) (by using ionophore A23187). 5. The substrate for the outer-membrane ATPase is a bivalent metal ion-nucleoside triphosphate complex in which Mg(2+) (K(m)=50mum) can be replaced effectively by Ca(2+) (K(m)=6.7mum) or Mn(2+), and ATP by ITP. Cu(2+), Co(2+), Sr(2+), Ba(2+), Ni(2+), Cd(2+) and Zn(2+) support very little ATP hydrolysis. 6. Univalent metal ions (Na(+), K(+), Rb(+), Cs(+) and NH(4) (+), but not Li(+)) stimulate the MgATPase activity (<10%) at low concentrations (50mm), but, except for K(+), are slightly inhibitory (20-30%) at higher concentrations (500mm). 7. The Mg(2+)-stimulated ATPase activity is significantly inhibited by Cu(2+) (K(i)=90mum), Ni(2+) (K(i)=510mum), Zn(2+) (K(i)=680mum) and Co(2+) (K(i)=1020mum), but not by Mg(2+), Ca(2+), Ba(2+) or Sr(2+). 8. The outer-membrane ATPase is insensitive to the inhibitors oligomycin, NN'-dicyclohexylcarbodiimide, NaN(3), ouabain and thiol-specific reagents. A significant inhibition is observed at high concentrations of AgNO(3) (0.5mm) and NaF (10mm). 9. The activity towards MgATP is competitively inhibited by the product MgADP (K(i)=0.7mm) but not by the second product P(i) or by 5'-AMP.     

Characterization of the extracellular haemoglobins of Artemia salina.

The following factors were measured for extracellular haemoglobins of Artemia salina: a minimal molecular weight of globin chain per haem group (based on the iron and haem contents), the absorption coefficients, the absorption spectra of various derivatives and the amino acid compositions. These were compared with those of the haemoglobins of other invertebrates. Three Artemia haemoglobins (I, II and III) had similar molecular structures, constructed from two-globin subunits of 122000-130000mol.wt. Since the minimal mol.wt. was determined to be 18000, this suggests that one globin subunit was bound by seven haem groups, and hence one haemoglobin molecule (240000-260000mol.wt.) should contain 14 haem groups. A successful identification of this high-molecular-weight subunit required first the denaturation of haemoglobin in 1% sodium dodecyl sulphate before sodium dodecyl sulphate gel electrophoresis. Denaturation by prolonged incubation (12-36 h) at room temperature in the presence of 0.1% sodium dodecyl sulphate [Bowen, Moise, Waring & Poon (1976) Comp. Biochem. Physiol. B55, 99-103] was accompanied by extensive proteolysis, resulting in low recovery of the stainable protein and heterogeneous gel patterns. Regardless of which electrophoretic system was used, the high-molecular-weight subunit was always present provided that 1% sodium dodecyl sulphate was present during denaturation. These results contrast with those obtained by Bowen et al. (1976). However, preferential cleavage of the globin subunit (alpha) seemed to occur in vitro when standard conditions were used, producing two specific fragments having mol.wts. of 80000 (beta) and 50000 (gamma).     

Regulation of the central metabolism in relation to citric acid production in Saccharomycopsis lipolytica

The mechanism of the massive extracellular production of citric and isocitric acids by Saccharomycopsis lipolytica grown on n-paraffins has been studied. When growth stops, because of nitrogen limitation, the intracellular concentration of ATP sharply rises whereas that of AMP and ADP decreases to a low level. At the same time production of acids begins. The activity of the NAD-dependent isocitrate dehydrogenase which requires AMP for activity becomes very low and prevents the oxidative function of the citric acid cycle whereas isocitrate lyase is not inhibited. As citrate synthase inhibition by ATP appears to be insufficient to stop n-paraffin degradation, citric and isocitric acids accumulation can take place. Massive excretion of these acids, however, probably still involves other physiological changes brought about by nitrogen limitation, possibly some permeabilization of the cell to these acids.This work is a part of a Doctorat de Spécialité Thesis submitted by R. Marchal to the University of Nancy (1975)     

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E₁ (10⁻⁸ − 10⁻⁷M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE₁, tetracaine and PMSF. The inhibitory effect of PGE₁ is strongly potentiated by theophylline (1.0 mM).Addition of the Ca²⁺ ionophore A23187 to platelet suspensions overcomes PGE₁ and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O₂ consumption. Tetracaine and dibucaine, however, block the responses to A23187.Formation of MDA and RCS (a mixture of PG endoperoxides and TXA₂) due to stimulation by thrombin and collagen depends upon activation of Ca²⁺-dependent phospholipase A₂ (PLA₂) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE₁, antagonizes the mobilization of the Ca²⁺ which is normally required for PLA₂ activity. Thrombin-stimulated platelets exhibit enhanced ⁴⁵Ca uptake which probably reflects exchange of extracellular Ca²⁺ with an increased available pool of exchangeable intracellular Ca²⁺. PGE₁ strongly suppresses this ⁴⁵Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca²⁺ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca²⁺ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O₂ uptake are nearly normal indicating that activation of PLA₂ can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca²⁺ to activate PLA₂. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA₂ activity.     

Biophysical characterization of Artemia salina (L.) extracellular haemoglobins.

Sedimentation coefficients (s0 20,w) of 11.57 +/- 0.10 S and 11.52 +/- 0.09 S were assigned for Artemia salina (L.) extracellular haemoglobins II and III respectively. These values are not significantly different. The molecular weights, M0w and M0z, of the native haemoglobins as determined by the high-speed sedimentation-equilibrium method were for haemoglobin II 239 400 +/- 7200 and 240 400 +/- 2600 respectively, and for haemoglobin III 216 300 +/- 6500 and 219 300 +/- 4500 respectively. The observed increase of Mapp. with concentration suggested that association was occurring over the concentration range investigated. Exposure of haemoglobin II to either 6 M-guanidinium chloride or to low pH (pH 4) resulted in dissociation to units of approximately half the size of the native protein, with molecular weights approx. 115 000. Electron-microscopic observations indicated a molecular structure composed of two stacked lobed discs. These results strongly support the dimeric model for Artemia haemoglobins proposed by Moens & Kondo [(1978) Eur. J. Biochem. 82, 65-72].     

Immunohistochemical localisation of substances reactive to antisera against α-and β-endorphin and met-enkephalin in the brain of Rana temporaria L.

Summary In the brain of Rana temporaria, two distinct systems reactive with - and -endorphin antisera, respectively, and with a met-enkephalin antiserum, have been detected immunohistochemically.Neurons reacting with - and -endorphin antisera are located (1) in the preoptic nucleus, and (2) in the pars ventralis of the tuber cinereum. Immunoreactive nerve fibres of both groups of perikarya end in the infundibular floor near the capillaries and the preoptico-hypophysial tract. Control reactions have shown that the immunoreactivity is suppressed by the corresponding antigens, but also by -LPH. In view of these results the immunoreactive systems examined correspond to an /-endorphin system or a lipotropinergic system.Neurons reacting with the met-enkephalin antiserum are located in the paraventricular organ. Intense immunofluorescence was observed in the infundibular floor. Controls show that the labelling by met-enkephalin antiserum is exclusively suppressed by met-enkephalin.In the pituitary gland, on the other hand, - and -endorphin antisera reveal: 1) the MSH/ACTH-like cells of the pars intermedia and 2) the ACTH-like cells of the pars distalis.Supported by the D.G.R.S.T., Contrat no 77.7.0648     

lamB mutations in E. coli K12: Growth of λ host range mutants and effect of nonsense suppressors

Summary Over sixty EMS induced mutations affecting gene lamB, presumably the structural gene for the receptor in Escherichia coli K12, were examined for growth of host range mutants and effect of nonsense suppressors. By the first criterion the mutations could be grouped in three classes. Bacteria with class I mutations allow growth of mutants with extended host range (noted h) of the type already described (Appleyard, Mac Gregor and Baird, 1956). Bacteria with class II mutations allow growth of h mutants with still more extended host range (noted hh ^*). No host range mutants of could be found which would grow on bacteria with class III mutations. Using nonsense suppressors it was found that class I and II consist of missense mutations, while class III consists of nonsense mutations. Exceptions are likely to exist (especially in class III) but were not found among the mutations tested. These observations are briefly discussed in terms of outer membrane protein integration and of phage receptor interaction.     

Karyotype analysis ofAscaris lumbricoides var.suum

Twelve synaptonemal complexes are present in both oocyte and spermatocyte pachytene nuclei ofAscaris lumbricoides var.suum, as determined by 3-D reconstruction of the nuclear contents from electron microscopy of serial sections and therefore, n=12 in the strain ofAscaris described here. In the female the heterochromatic end of each synaptonemal complex is attached to the nuclear envelope and the other end is free in the nucleoplasm. In the male neither end ot the synaptonemal complex is attached, but there is a heterochromatic knob at one end of each complex. Five additional large heterochromatic masses are present in the spermatocyte nucleus and these may be the sex chromosomes described by earlier workers.