A fast and simple radiometric assay for adenosine deaminase using reversed-phase thin-layer chromatography

A new quantitative radiometric assay for adenosine deaminase is described. The reaction conditions are similar to those used in other radioassays and are shown to result in an activity which increases linearly with time and with enzyme concentration. An original feature of the technique resides in the use of reversed-phase thin-layer chromatography to separate adenosine from inosine. The separation is complete, fast, and reproducible. Both compounds can be recovered almost quantitatively from the plates. The assay is very simple and allows the determination of up to 36 samples in 3 h.     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

The decreasedin vitro chemotactic activity of polymorphonuclear leukocytes of newborns and infants

Using Boyden's technique, a statistically significant decrease in the chemotactic activity of polymorphonuclear (PMN) leukocytes was found during the early postnatal period, i.e. in the cord blood and in blood of newborns within the first 10-15 d of life after stimulation of cells with both zymosan-activated adult serum (ZAS) and with an abacterial filtrate of Escherichia coli broth culture (ECF). After this period, the responsiveness of leukocytes to both chemotactic agents increased and remained at the same level during the whole observation period, i.e. up to the age of 6 months. Nevertheless even then it did not reach fully the responsiveness of the leukocytes of mothers and pregnant women. Zymosan-activated serum was shown to be a more potent chemotactic stimulus to leukocytes of infants as compared to the E. coli filtrate.     

Origin of sclerotia-like cells in submerged Claviceps purpurea producing clavine alkaloids

Ultrathin sectioning of submerged mycelium of Claviceps purpurea Tul. producing clavine alkaloids revealed yeast-like budding resulting in asexual sporesblastospores. These deciduous spores were born by extended hyphal cells and retained the same ultrastructure of cell organelles. Both the extended hyphae and the blastospores resembled the cells of ergot sclerotial tissue. A surface culture of C. purpurea Tul. producing no alkaloids was used as a reference.     

Penicillinamidohydrolase inEscherichia coli

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) fromEscherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. SomeN-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial Tates of the enzyme hydrolysis of different substrates were found when using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space.N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.     

Some histochemical studies on the prostate, urethral and bulbourethral glands of the one-humped camel (Camelus dromedarius)

Synopsis The histochemical localization of carbohydrates, ribonucleoproteins (RNA), lipids, some hydrolytic enzymes, succinate and lactate dehydrogenase and acetylcholinesterase were investigated in the prostate, urethral and bulbourethral glands of the camel. These glands probably secrete carbohydrate-protein complexes. In the bulbourethral glands, they are sulphated mucopolysaccharides. RNA was seen in the cytoplasm of the prostate and urethral glands. Neutral lipids were cytoplasmic and present in moderate amounts in the prostate and urethral glands and in traces, in the bulbourethral gland. Acid phosphatase-containing granules were abundant in the prostate, moderate in the urethral glands and in traces in the bulbourethral glands. Alkaline phosphatase was observed in the apical cytoplasm of the prostate and bulbourethral glands and in the ducts of the urethral glands. ATPase and adenosine 5-monophosphatase were seen in the basal laminae and interstitial tissue. In the urethral glands, adenosine 5-monophosphatase was distributed diffusely in the cytoplasm. Succinate dehydrogenase was seen in the urethral and bulbourethral glands. Varying degrees of lactate dehydrogenase activity was observed in all the glands. Acetylcholinesterase was confined to neural elements. The pars disseminata and the urethral glands were considered as two distinct glandular zones along the pelvic urethra. The significance of these histochemical results is discussed.