Inhibition of enzymes by metal ion-chelating reagents. Theory and new graphical methods of study

1. The mechanism of inhibition of enzymes by metal ion-chelating reagents is discussed and equations derived. 2. Two distinct mechanisms are postulated and graphical methods are given for differentiating between them. 3. Where the metal ion is actually removed from the enzyme to form a co-ordination complex in solution, a procedure is described for obtaining the stability constant for metal-enzyme interaction, the number of metal ions involved and the stoicheiometry of metal ion-ligand interaction.     

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.     

The mechanism of intestinal absorption of phosphatidylcholine in rats

1. The mechanism of absorption of phosphatidylcholine was studied in rats by injecting into the intestine phosphatidylcholine specifically labelled either in the fatty acid or in the glycerol moiety or with (32)P, when considerable amounts of 1-acyl-lysophosphatidylcholine were found in the intestinal lumen. 2-([(14)C]Acyl)phosphatidylcholine gave markedly more radioactive unesterified fatty acids in the lumen, compared with the 1-([(14)C]acyl) derivative. Some of the radioactivity from either the fatty acid or the glycerol moiety of the injected phosphatidylcholine appeared in the mucosal triacylglycerols. 2. Injection of (32)P-labelled phosphatidylcholine or (32)P-labelled lysophosphatidylcholine led to the appearance of radioactive glycerylphosphorylcholine, glycerophosphate and P(i) in the mucosa. 3. Rat mucosa was found to contain a highly active glycerylphosphorylcholine diesterase. 4. It was concluded that the dietary phosphatidylcholine is hydrolysed in the intestinal lumen by the pancreatic phospholipase A to 1-acylglycerylphosphorylcholine, which on entering the mucosal cell is partly reacylated to phosphatidylcholine, and the rest is further hydrolysed to glycerylphosphorylcholine, glycerophosphate, glycerol and P(i). The fatty acids and glycerophosphate are then reassembled to give triacylglycerols via the Kennedy (1961) pathway.     

Glucose transport in Entamoeba histolytica

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.     

Passive Electrical Properties of Microorganisms: V. Low-Frequency Dielectric Dispersion of Bacteria

Very high dielectric constants have been observed for bacteria at low frequencies. High dielectric constants such as these can be explained by a theory which has been developed for the low-frequency dielectric dispersion of porous charged particles and which has been tested successfully through measurements with ion exchange resins. The bacterial cell wall is electrically similar to an ion exchange resin. Observations show that the theory provides a quantitative explanation for the low-frequency dielectric dispersion of bacteria.     

The respiration of insects at reduced pressures II. The uptake of oxygen by Tenebroides mauritanicus

The respiration of larvae of Tenebroides mauritanicus exposed to reduced pressures in air was measured in a specially designed chamber by means of gas chromatography. Both O₂ uptake and CO₂ output were progressively reduced as the pressure was lowered from 200 to 35 mm Hg. There were differences in the amounts of gases exchanged when the insects were exposed under dry or moist conditions. When expired CO₂ was allowed to accumulate, in some experiments it had a marked stimulating effect on O₂ uptake between 60 and 200 mm. The respiratory quotient at the various pressures was calculated and the values found under different conditions of pressure and moisture are discussed.

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Zusammenfassung In der ersten Veröffentlichung dieser Reihe beschrieben Monro, Dumas und Buckland (1962) die Kohlendioxyd-Ausatmung der Larven von T. mauritanicus bei verringertem atmosphärischen Druck. Unter 65 mm nahm die CO₂-Ausatmung deutlich und fortschreitend ab. Um zu zeigen, daß dieser Verringerung der CO₂-Produktion tatsächlich eine Abnahme der Atmung entspricht, wurde ein Apparat entwickelt, mit dessen Hilfe O₂-Aufnahme und CO₂-Abgabe der Insekten gaschromatographisch gemessen werden konnten.Bei dreistündiger Exposition wurde unter den experimentellen Bedingungen gefunden, daß sich im Vergleich mit der Reaktion bei höherem Druck sowohl O₂-Aufnahme als auch CO₂-Abgabe bei 100, 60 und 30 mm fortschreitend verringert. Bei so niedrigem Druck war die Atmung während der Expositionszeit gleichmäßiger als bei höherem Druck, bei dem von Zeit zu Zeit deutliche Schwankungen auftraten. Bemerkenswert war ein deutlicher Unterschied in der Respirationsrate bei feuchtigkeitsgesättigter Luft und unter trockenen Bedingungen. Bei hoher Luftfeuchtigkeit war die O₂-Aufnahme zwischen 100 und 400 mm größer als bei Trockenheit und die CO₂-Abgabe war größer von 65 mm aufwärts. Daraus wird gefolgert, daß in diesem Druckbereich bei Trockenheit der Austausch beider Gase durch dauernden oder zeitweisen Verschluß der Atmungswege behindert wird.In einigen Versuchen wurde CO₂ durch Absorption aus der die Insekten umgebenden Atmosphäre entfernt. Abwesenheit von CO₂ verringerte die O₂-Aufnahme im Bereich von 60–200 mm besonders bei Feuchtigkeit. Bei Trockenheit war die O₂-Aufnahme bei hohem CO₂-Gehalt signifikant geringer. Hier mag wiederum Schließung der Atmungswege die stimulierende Wirkung des CO₂ vermindert haben.Berechnungen des Respirationsquotienten aus den verfügbaren Daten zeigen eine stetige Abnahme dieses Wertes mit abnehmendem Druck bei Feuchtigkeit, während der R.Q. in trockener Umgebung bei jedem Druck ziemlich konstant blieb. Es werden keine endgültigen Schlüsse aus diesen Berechnungen gezogen, aber es wird angenommen, daß die feuchte Umgebung einen freieren Gasaustausch durch die Atmungswege erlaubt und daher bessere Bedingungen zur Bestimmung des tatsächlichen R.Q.-Wertes bei vermindertem Druck bietet.

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In this paper pressures are given as absolute pressures expressed in mm Hg.     

Probes of DNA Structure and Interactions: Effects of Copper II on Ultraviolet-Induced Pyrimidine Dimer Formation

Cu(II) affects the yield of cyclobutyl dimers induced in DNA by 254 nm radiation. The effects are a function of r, the ratio of Cu(II) to DNA phosphate, and of the ultraviolet (UV) fluence; they seem to reflect two types of copper complexes with DNA. The first probably involves “exterior” binding to the bases of native DNA and increases [unk]TT formation (without affecting [unk]UT yield) by raising the energy levels of bases other than thymine. The second seems to occur only at high ratios (rs) and only after the structure has been opened locally by UV radiation; it involves “interior” binding of Cu(II) to the bases. This complex tends to decrease dimer yield by holding the bases apart and/or by lowering the energy levels of bases other than thymine. These results illustrate the potential use of DNA photoproducts and ligands to probe the structure and interactions of DNA in vitro and perhaps also in vivo.     

THE HUMAN CHROMOSOME : Electron Microscopic Observations on Chromatin Fiber Organization

Human lymphocytes were grown in short-term tissue culture and were arrested in metaphase with Colcemid. Their chromosomes were prepared by the Langmuir trough-critical point drying technique and were examined under the electron microscope. In addition, some chromosomes were digested with trypsin, Pronase, or DNase. The chromosomes consist entirely of tightly packed, 240 ± 50-A chromatin fibers. Trypsin and Pronase treatments induce relaxation of fiber packing and reveal certain underlying fiber arrangements. Furthermore, trypsin treatment demonstrates that the chromatin fiber has a 25–50 A trypsin-resistant core surrounded by a trypsin-sensitive sheath. DNase digestion suggests that this core contains DNA.     

FINE STRUCTURE OF PACINIAN CORPUSCLES IN THE MESENTERY OF THE CAT

Pacinian corpuscles in the mesentery of adult cats were fixed with either glutaraldehyde, osmium tetroxide or permanganate solutions by close intra-arterial injection through the mesenteric artery, and were processed, after electron staining and Epon embedding, for electron microscopy. Better resolution of the corpuscle's ultrastructure was obtained than available heretofore. The myelinated segment of the corpuscle contains blood vessels separated from the axon by collagen fibers and 3 to 4 layers of lamellae. No blood vessels are found in the central core, though access from the vessels is afforded by diffusion through the "cleft" of the inner core. Two cell types are discernible in the inner core hemilamellae; the "clear cells" in which pinocytotic vesicles and organelles abound and reflect the greater metabolic activity of these cells, in contrast to the "dark cells." The ultraterminal is ellipsoidal in form with projections into the "cleft" which give this portion an irregular appearance in section. The terminal and ultraterminal are packed with mitochondria, and "synaptic" vesicles are seen in the ultraterminal. The innermost laminae of the inner core cells are in close apposition to the terminal and break their regular pattern of hemilamellation to surround the small ultraterminal projections at the apical part of the corpuscle.     

Electron Microscopic Observations on the Ribonucleic Acid of Murine Leukemia Virus

The ribonucleic acid (RNA) of murine leukemia virus (MLV) Rauscher strain was observed by the aid of electron microscopy with the use of the protein monolayer technique. RNA was observed directly after release from virus particles or after isolation by sedimentation in sucrose density gradients. Molecules were found in an extended linear form. Many of the RNA filaments released by detergent treatment contained curled regions, suggesting the linear filaments were originally coiled within the virus particle. The relationship of the curled areas to the containment of the RNA within the virus particle is discussed, and a mechanism for the inclusion of RNA in the budding virion is proposed. Treatment of the extended MLV-RNA with dimethyl sulfoxide resulted in the collapse of the molecule forming a tangled complex. Treatment with urea or heating at 50 C in 3 mm NaCl also produced this effect. Also under the conditions in which MLV-RNA was linear, RNA from Rous sarcoma virus also was linear, but Newcastle disease virus RNA and ribosomal RNA of rat liver had collapsed structures. The results indicated that the RNA of MLV, and perhaps other RNA-containing tumor viruses, has a specific unique conformation dependent upon hydrogen bonds.