Ox liver glutamate dehydrogenase. The use of chemical modification to study the relationship between catalytic sites for different amino acid substrates and the question of kinetic non-equivalence of the subunits.

The effect of pyridoxal 5'-phosphate on the activity of ox liver glutamate dehydrogenase towards different amino acid substrates was investigated. Both alanine and glutamate activities decreased steadily in the presence of pyridoxal 5'-phosphate. The alanine/glutamate activity ratio increased as a function of inactivation by pyridoxal 5'-phosphate, indicating that glutamate activity is lost more rapidly than alanine activity. A mixture of NADH, GTP and 2-oxoglutarate completely protected the alanine and glutamate activities against inactivation by pyridoxal 5'-phosphate. The activity of glutamate dehydrogenase towards glutamate and leucine decreased steadily in a constant ratio in the presence of pyridoxal 5'-phosphate. The effect of leucine on the alanine and glutamate activities as a function of inactivation by pyridoxal 5'-phosphate was studied. The results are interpreted to suggest that the subunits of glutamate dehydrogenase hexamer are kinetically non-equivalent with regard to activity towards the two monocarboxylic amino acids as well as glutamate, and that all three substrates share the same active centre. However, leucine is also able to bind at a separate regulatory site.     

Respiratory behavior in the pond snail Lynmaea stagnalis

Summary Previously (Syed et al. 1991) we described the ventilatory behavior of the pond snail Lymnaea stagnalis and identified motor neurons that innervate various muscles involved in this behavior. In the present study we describe an interneuronal network that controls ventilatory behavior in Lymnaea. An identified interneuron, termed the input 3 interneuron (Ip.3.I), was found to be involved in the opening movement of the pneumostome (expiration), whereas another identified interneuron known as visceral dorsal 4 (V.D.4) caused its closure (inspiration). These cells have reciprocal inhibitory connections with each other, which accounts for their opposing effects on common follower motor neurons. In isolated brain preparations a third identified interneuron, right pedal dorsal 1 (R.Pe.D.1) initiated the respiratory cycle by the excitation of Ip.3.I. Whereas Ip.3.I in turn excited R.Pe.D.1, the connections between R.Pe.D.1 and V.D.4 were mutually inhibitory. Both Ip.3.I and V.D.4 were active during spontaneously occurring respiratory behavior as recorded from semi-intact preparations, and selective hyperpolarization of V.D.4 during such spontaneous activity disrupted the respiratory behavior. Regarding peripheral feedback, the mechanical stimulation of the pneumostome during its opening movements not only caused closure but also inhibited Ip.3.I in the middle of its discharge. Ip.3.I and V.D.4 were also found to be multifunctional, inhibiting both locomotor and whole body withdrawal neural networks. We conclude from these results that the rhythmic patterned activity underlying respiratory behavior in Lymnaea is generated centrally, and that the network described here therefore comprises a central pattern generator.     

Population structure and dynamics oi Juniperus excelsa in Balouchistan,Pakistan

Abstract. 60 monospecific stands of Juniperus excelsa were sampled at four locations in Balouchistan. Density, basal area and height of individuals were recorded. Soils were analysed for selected physical and chemical characteristics and the degree of disturbance due to logging and burning was noted. The density of juniper trees (> 6 cm dbh) ranged from 56 to 332 stems / ha (average 174 stems / ha). Higher densities were recorded for relatively undisturbed stands and on west facing slopes. Density of seedlings and saplings (< 6 cm dbh) was strongly correlated with tree density and tree basal area. Among the edaphic variables CaC0₃ was correlated with juniper density and basal area. Diameter distributions within stands were mostly skewed and unimodal with gaps appearing in large size classes. The male to female ratio was close to 1. Cross-sections of 16 trees were used to determine age and growth rate. Number of rings in trees with 20 to 30 cm dbh ranged from 95 to 221 (x = 160 ± 38). Diameter and age were not related. Mean annual diameter increment ranged from 6 to 16 yr / cm x = 10 ± 3 yr / cm). It is concluded that size class gaps and low seedling / sapling densities are the consequence of anthropogenic disturbance.     

Vascular pattern in the pancreas of the cat

Summary The pathways of microcirculation in the pancreas of the cat were investigated by scanning electron microscopy of Mercox preparations of the vascular bed. A portal system from islet to exocrine vessels as well as a direct arterial flow to the exocrine pancreas have been observed. Sphincters appear to exist in portal capillaries at the border between the endocrine and exocrine portions of the pancreas. Islets possess an independent venous drainage.A portion of these results was presented on the occasion of the 6th European Anatomical Congress, Hamburg 1981 (see Syed Ali 1981, for abstract).     

Effects of Metals on Streptomyces coelicolor Growth and Actinorhodin Production

Actinorhodin production by Streptomyces coelicolor was used as a model system to study the effects of metals on growth and polyketide synthesis in a streptomycete. Numerous metals were tested in cultures grown in liquid media. Mercury and cadmium were highly toxic, and copper, nickel, and lead were less so, but all tended to inhibit both growth and antibiotic synthesis to a similar extent. Unexpectedly, manganese, cobalt, zinc, and, to a lesser extent, chromium caused complex effects that in general resulted in some enhancement of growth yield but a reduction in antibiotic titers. These complex effects meant that cobalt, manganese, and zinc had lower 50% inhibitory concentrations for antibiotic yields compared with those for biomass. The physiologically active divalent cations calcium and magnesium were also tested. Calcium at high concentrations was particularly effective in reducing antibiotic titers and enhancing growth yields. By adding calcium at different phases of growth, it could be demonstrated that it was most effective in reducing the antibiotic yield when added during the early growth phase. Addition during the antibiotic-producing phase resulted in little reduction of final actinorhodin titers.     

Adhesion of an Amylolytic Arthrobacter sp. to Starch-Containing Plastic Films

Cells of the amylolytic bacterium KB-1 (thought to be an Arthrobacter sp.) adhered (~70%) to the surface of plastic films composed of starch-poly (methylacrylate) graft copolymer (starch-PMA), but did not adhere (<10%) to films composed of polymethylacrylate (PMA), polyethylene (PE), carboxymethyl cellulose, or a mixture of PE plus poly (ethylene-coacrylic acid) (EAA), starch plus PE, or starch plus PE and EAA. About 30% of the cells adhered to gelatinized insoluble starch. Dithiothreitol (5 mM), EDTA (5 mM), and soluble starch (1%, wt/vol) had little effect on the adhesion of KB-1 cells to starch-PMA films. However, glutaraldehyde-fixed cells, azide-treated cells, and heat-killed cells did not bind to starch-PMA plastic, suggesting that the observed adhesion required cell viability. Culture supernatant from 5-day-old KB-1 cultures contained a proteolytic enzyme that inhibited cell adhesion to starch-PMA plastics. Trypsin-treated KB-1 cells also lost their ability to bind to starch-PMA plastic. When washed free of trypsin and suspended in fresh medium, trypsin-treated bacteria were able to recover adhesion activity in the absence, but not in the presence, of the protein synthesis inhibitor chloramphenicol. These results suggested that adhesion of KB-1 to starch-PMA plastic may be mediated by a cell surface protein. Although KB-1 bacteria bound to starch-PMA plastic, they did not appear to degrade starch in these films. Evidence of starch degradation was observed for starch-PE-EAA plastics, where <10% of the bacteria was bound, suggesting that cell adhesion may not be a prerequisite for degradation of some starch-containing plastics.     

Orotate phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase from yeast: kinetic analysis with chromium (III) pyrophosphate

The reactions catalyzed by orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) from yeast differ in the kinetic mechanisms by which they are activated by divalent metal ions. Moreover, whereas OPRTase is activated specifically by Mg(II) or Mn(II), the reactions catalyzed by HGPRTase can utilize a wider range of divalent metal ions, including Mg(II), Mn(II), Co(II), and Zn(II). In this report we describe the results of a kinetic analysis of the effects of the addition of Cr(III) pyrophosphate (Cr-PPi) to the OPRTase and HGPRTase assay solutions, which delineates further the differences between these enzyme activations by metal ions. (1) Cr-PPi is an effective competitive inhibitor of the OPRTase catalysis, when the steady-state forward velocity of orotidine monophosphate (OMP) formation is examined over a range of phosphoribosyl alpha-pyrophosphate (PRibPP) concentrations, whereas pyrophosphate (PPi) has been reaffirmed to be a noncompetitive product inhibitor under the same conditions. (2) Cr-PPi itself serves as a substrate for the OPRTase-catalyzed reverse pyrophosphorolysis of OMP and does not inhibit the utilization of PPi as substrate during this reaction. (3) In contrast, Cr-PPi, at concentrations as high as 6 mM, has no effect on the HGPRTase-catalyzed formation of inosine monophosphate, whereas the inhibition exhibited by PPi during this reaction is noncompetitive but defined by two sets of lines in the double reciprocal plot of the initial velocity versus 1/PRibPP. (4) Cr-PPi is not a substrate for the HGPRTase-catalyzed pyrophosphorolysis of IMP under the conditions of these assay procedures.     

A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum.

1. On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction. The rate became approximately constant after declining to 4-5% of the initial value. Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate. The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH. 2. Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution. At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min. 3. Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy. 4. 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective. 5. The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation. 6. Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8. 7. The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates. 8. The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues.     

Polyprenol phosphates and mannosyl transferases in Phaseolus aureus

The transfer of mannose from GDP[¹⁴C]mannose to lipid and to insoluble polymer by a particulate preparation of Phaseolus aureus has been investigated. The evidence favours the lipid being a prenol phosphate mannose. Of a range of prenol phosphates tried, betulaprenol phosphate was the most effective exogenous acceptor of mannose. Most of the insoluble [¹⁴C]polymer formed was glycoprotein in nature although small quantities of ¹⁴C were associated with glucomannan and galactoglucomannan fractions. Time studies failed to reveal a typical precursor-product relationship between the lipid and polymer fractions but on incubation of [¹⁴C]mannolipid with the particulate fraction a small transfer (0·5–0·7%) of [¹⁴C] to polymer was detected. p-Hydroxymercuribenzoate inhibited (by 90%) the transfer of [¹⁴C] from GDP[¹⁴C]-mannoseto polymer and simultaneously increased (3-fold) the [¹⁴C] recovered in the lipid fraction. The effect was nullified by mercaptoethanol. Attempts to solubilize the transfer system were only partially successful. The formation of a chromatographically identical mannolipid was demonstrated in particulate fractions of Codium fragile and tomato roots.