Quantitative Isolation of Undegraded Polysomes from Aged Pea Tissue in the Absence of Contaminants and Artefacts

Undegraded polysomes were isolated successfully from aged peaepicotyls by grinding frozen tissue in at least 10 volumes ofbuffer A (0.2 M Tris-HCl, pH 8.5; 0.2 M sucrose; 60 mM KCl;30 mM MgCl₂), taking care to prevent the tissue from thawingprior to homogenization. Supposedly pure polysomes, derivedfrom the membrane-bound polysome fraction, were apparently contaminatedwith membranes, and contained polysomes clumped together vianascent chains. Problems with contaminants and artefacts werepartially alleviated by the use of polyoxyethylene tridecylether as a detergent replacing Triton X-100; further alleviatedby the use of large volumes of detergent-containing buffer toresuspend the membrane-bound polysome; and almost completelyeliminated by brief treatment of resuspended polysomes withprotease K. Optimal conditions for isolating polysomes fromaged tissue are given. ¹ Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received April 24, 1985; Accepted September 2, 1985)     

Mitochondrial aldehyde dehydrogenase from higher plants

Aldehyde dehydrogenase has been purified to homogeneity from mitochondria of potato tubers and pea epicotyls. Although the enzyme had a high affinity for glycolaldehyde it also had a high affinity for a number of other aliphatic and arylaldehydes. It is proposed that the codification glycolaldehyde dehydrogenase (EC 1.2.1.22) should be abandoned in favour of mitochondrial aldehyde dehydrogenase (EC 1.2.1.3). The purified enzyme showed esterase activity and had properties similar to those reported for the mammalian mitochondrial aldehyde dehydrogenase. Although the natural substrate(s) for the enzyme is not known, the kinetic properties of the enzyme are consistent with it playing a role in the oxidation of acetaldehyde, glycolaldehyde and indoleacetaldehyde.     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

The mechanism of guanosine triphosphate depletion in the liver after a fructose load. The role of fructokinase.

A Sephadex G-25 filtrate of a 100 000g supernatant of rat liver homogenate was shown to be able to phosphorylate fructose, with GTP as the phosphate donor. Attempts to separate ATP- and GTP-dependent fructokinase activities failed, indicating that there is a single enzyme able to use both nucleotides. With a partially purified enzyme, Km values for fructose of 0.83 and 0.56 mM were found with ATP and GTP as substrates respectively. Km values of 1.53 and 1.43 mM were found for GTP and ATP respectively. Both ADP and GDP inhibited the GTP- and ATP-dependent fructokinase activity. We conclude that the depletion of hepatic GTP caused by intravenous administration of fructose to mice and rats can be explained simply by the utilization of the nucleotide by fructokinase.     

Analysis of the inducible MEL1 gene of Saccharomyces carlsbergensis and its secreted product, alpha-galactosidase (melibiase)

We have determined both the nucleotide sequence of the MEL1 gene of Saccharomyces carlsbergensis and the N-terminal amino acid (aa) sequence of its extracellular gene product, alpha-galactosidase (melibiase) (alpha-Gal). The predicted translation product of MEL1 is a pre-alpha-Gal protein containing an 18 aa N-terminal signal sequence for secretion. The purified enzyme is a dimer consisting of two 50-kDal polypeptides, each of which is glycosylated with no more than eight side chains. The 5'-flank of the MEL1 gene contains a region (UASm) having certain areas of sequence homology to similar sites found upstream of the structural genes GAL1, GAL7 and GAL10, which are also regulated by the action of the products of genes GAL4 and GAL80. There are three TATA boxes between UASm and the initiation codon of pre-alpha-Gal, as well as a typical yeast cleavage/polyadenylation sequence in the 3'-flank of the gene.     

Efficacy of high-frequency ventilation in presence of extensive ventilation-perfusion mismatch

Ten anesthetized normal dogs were each given two methacholine inhalational challenges to produce large amounts of low ventilation-perfusion (VA/Q) regions but little shunt. After one challenge, high-frequency ventilation (HFV) was applied, whereas after the other conventional mechanical ventilation (MV) was used, the order being randomized. Levels of both ventilatory modes were selected prior to challenge so as to result in similar and normal mean airway pressures and arterial PCO2 levels during control conditions. Gas exchange was assessed by both respiratory and multiple inert-gas transfer. Comparing the effect of HFV and MV, no statistically significant differences were found for lung resistance, pulmonary hemodynamic indices, arterial and mixed venous PO2, expired-arterial PO2 differences, or inert-gas data expressed as retention-excretion differences. The only variables that were different were mean airway pressure (2 cm higher during HFV, P less than 0.04) and arterial PCO2 (10 Torr higher during HFV, P less than 0.002). These results suggest that in this canine model of lung disease characterized by large amounts of low VA/Q regions, HFV is no more effective in delivering fresh gas to such regions than is MV.     

Correlation of pulmonary ACE activity and capillary surface area during postnatal development

Although a considerable amount of information is available regarding the remodeling and growth of the pulmonary arterial circulation, relatively little is known regarding postnatal development of the pulmonary microcirculation. We hypothesized that the maximal velocity (Vmax) of pulmonary angiotensin-converting enzyme (ACE) activity, measured from indicator-dilution outflow curves using a synthetic substrate, 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), is directly related to the capillary endothelial cell surface area in the lungs of developing lambs. Accordingly we measured apparent kinetics of pulmonary ACE activity in 22 anesthetized ventilated lambs (2-171 days old) and compared our functional assessment to simultaneous in vivo determinations of CO diffusing capacity (DLCO) and postmortem structural assessment of alveolar septal dimensions using stereology and electron microscopy. There was a progressive increase in Vmax of ACE in this age group, with little change in apparent affinity for BPAP. Similar functional manifestation of growth was noted by an age-dependent increase in DLCO. Neither Vmax nor DLCO was significantly affected by an increase in left atrial pressure to 19 Torr (via inflation of a balloon in the left atrium), suggesting little recruitment of vessels under conditions of the present protocol. A close correlation was observed when either Vmax for ACE activity or DLCO was plotted vs. capillary endothelial cell surface area. Double logarithmic transformation of capillary endothelial cell surface area, Vmax-ACE and DLCO vs. lung volume revealed power functions with slopes all greater than that predicted from isotropic growth, suggesting selective differential postnatal development of the endothelium of the alveolar septum in lambs from 2-171 days of age.