Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

A simple kinetic test to distinguish exo-glucosidase and beta-glucosidase activities

Unusual kinetic behaviour was observed in assaying spectrophotometrically for exo-glucanase activity in a beta-glucosidase isolated from A. faecalis using p-nitrophenyl beta-cellobioside as substrate. At high substrate concentrations no phenol was released whereas at low concentrations a rapid release of phenol was detected and this increased in rate with extent of hydrolysis. These results are consistent with a model involving tight binding of the substrate to the enzyme and an initial exo-glucosidase-catalysed hydrolysis to produce glucose and p-nitrophenyl glucoside. Subsequent hydrolysis of the nitrophenyl glucoside results in phenol release, but only after sufficient concentrations have accumulated to compete with the cellobioside. This theory was confirmed by product analysis and by measuring the affinity of the substrate for the enzyme by its inhibition of p-nitrophenyl glucoside hydrolysis. Observation of such kinetic behaviour allows distinction between beta-glucosidase and exo-glucosidase activities.     

The effect of pH on the selection of carbaryl-degrading bacteria from garden soil

At an alkaline pH and in an aqueous solution carbaryl hydrolyses to form 1-naphthol, methylamine and carbon dioxide, but it is much more stable at an acid pH. Soil perfusion column experiments indicated that the rate of carbaryl degradation at pH 6.0 to 7.0 was limited by the rate of chemical hydrolysis. Bacterial communities of at least 12 and 14 members were selected in continuous cultures using carbaryl as the sole carbon and nitrogen source at pH 6.0. These communities were supported by the slow formation of hydrolysis products and a carbaryl-degrading bacterium was not selected after greater than 2000 h. A bacterial community of at least eight members was selected using equimolar 1-naphthol and methylamine as its sole carbon and nitrogen source. In contrast, after a lag of between 10 and 50 days, soil perfusion column and continuous culture enrichments at pH 5.2 and 5.0, respectively, led to the selection of a Pseudomonas sp. which could utilize carbaryl as its sole carbon and nitrogen source.     

Elucidation of the nucleotide sequence of chicken calcitonin mRNA: direct evidence for the expression of a lower vertebrate calcitonin-like gene in man and rat.

Calcitonin shows considerable divergence in amino acid sequence between lower vertebrates and higher vertebrates. Immunoreactive salmon-like calcitonin molecules are present in the thyroid of man and rat. Elucidation of the almost complete sequence of chicken calcitonin mRNA revealed that the calcitonin precursor in chickens had the same organisation as in higher vertebrates (man and rat) but showed considerable differences in amino acid sequence. cDNA probes specific for chicken calcitonin mRNA hybridized to poly(A)-rich RNA extracted from a case of medullary carcinoma of the thyroid and from murine thyroid. These results suggest the expression in man and rat of a gene coding for an avian calcitonin-like precursor.     

Abnormal high density lipoproteins from patients with liver disease regulate cholesterol metabolism in cultured human skin fibroblasts

Apolipoprotein B (apoB) of plasma low density lipoproteins (LDL) binds to high affinity receptors on many cell types. A minor subclass of high density lipoproteins (HDL), termed HDL1, which contains apoE but lacks apoB, binds to the same receptor. Bound lipoproteins are engulfed, degraded, and regulate intracellular cholesterol metabolism and receptor activity. The HDL of many patients with liver disease is rich in apoE. We tested the hypothesis that such patient HDL would reduce LDL binding and would themselves regulate cellular cholesterol metabolism. Normal HDL had little effect on binding, uptake, and degradation of 125I-labeled LDL by cultured human skin fibroblasts. Patient HDL (d 1.063-1.21 g/ml) inhibited these processes, and in 15 of the 25 samples studied there was more than 50% inhibition at 125I-labeled LDL and HDL protein concentrations of 10 micrograms/ml and 25 micrograms/ml, respectively. There was a significant negative correlation between the percentage of 125I-labeled LDL bound and the apoE content of the competing HDL (r = -0.54, P less than 0.01). Patient 125I-labeled HDL was also taken up and degraded by the fibroblasts, apparently through the LDL-receptor pathway, stimulated cellular cholesterol esterification, increased cell cholesteryl ester content, and suppressed cholesterol synthesis and receptor activity. We conclude that LDL catabolism by the receptor-mediated pathway may be impaired in liver disease and that patient HDL may deliver cholesterol to cells.     

Bacillus cereus 569/H penicillinase serine-44 acylation by diazotized 6-aminopenicillanic acid

Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

Effects of flooding on leaf litter decomposition in microcosms

Summary The effects of hydroperiod on decomposition rates of senescent Acer rubrum leaves were tested in microcosms in a controlled laboratory environment. Microcosm treatments included continuously flooded, continuously unflooded, and fluctuating hydroperiods. All flooding treatments promoted decomposition but variations in hydroperiod had no significant effects. A leaching experiment indicated the higher decay rates under flooded conditions were primarily due to high leaching losses from soaking. Unlike nutrient dynamics in the field, where net accumulation occurs, nitrogen and phosphorus in the litter in the microcosms exhibited net losses. The major external inputs which provide a source of nitrogen and phosphorus for immobilization in the field were lacking in the microcosms. Calcium, magnesium, and potassium exhibited net losses except for calcium in the unflooded microcosms. The microcosm results demonstrated the importance of external inputs to litter nutrient relations.     

The regulation of glycolysis and electron transport in roots

The respiration of roots and isolated root mitochondria was investigated in Phaseolus vulgaris L., Spinacea oleracea L.; Triticum aestivum L., and Zea mays L. Although the respiration of both intact roots and isolated mitochondria displayed resistance to cyanide and sensitivity to SHAM, the percentage resistance and inhibition in roots was not the same as that in the mitochondria, with the exception of wheat. Adding FCCP to roots stimulated oxygen uptake and equalized the effects of SHAM and cyanide on roots and mitochondria. In spinach and maize roots, FCCP stimulated both the cytochrome and alternative pathways, while in bean roots, only the alternative pathway was stimulated. FCCP had little effect on wheat root respiration rates. Potential in vivo rates of oxygen uptake were estimated by expressing rates obtained with isolated mitochondria on a fumarase activity basis, and fumarase activity on a root weight basis. In wheat roots the potential rate was approximately equal to the measured in vivo rate; in the other species the potential rates were substantially greater than measured rates, but approximately equal to uncoupled in vivo rates. Key glycolytic intermediates in roots were measured, and it was found that the phosphofructokinase and pyruvate kinase reactions were displaced far from equilibrium, the degree of displacement being approximately equal in roots with little, and roots with substantial, alternative path engagement. Thus, although glycolysis is controlled, the regulation of this pathway appears to be quite flexible. The results are discussed in terms of possible regulatory mechanisms.