Purification, characterization, and kinetic mechanism of S-adenosyl-L-methionine:macrocin O-methyltransferase from Streptomyces fradiae

S-Adenosyl-L-methionine:macrocin O-methyltransferase catalyzes conversion of macrocin to tylosin, the terminal and main rate-limiting step of tylosin biosynthesis in Streptomyces fradiae. The O-methyltransferase was stabilized in vitro and purified to electrophoretic homogeneity. The purified enzyme had a molecular weight of 65,000 and consisted of two identical subunits of 32,000 with an isoelectric point of 4.5. The enzyme required Mg2+, Mn2+, or Co2+ for maximal activity and was catalytically optimal at pH 7.5-8.0 and 31 degrees C. The O-methyltransferase catalyzed the conversion of macrocin to tylosin at a stoichiometric ratio of 1:1. The enzyme also mediated conversion of lactenocin----desmycosin. The corresponding Vmax/Km ratios for the two analogous conversions were similar, and both enzymic conversions were susceptible to extensive competitive and noncompetitive inhibitions by macrolide metabolites. Steady-state kinetic studies for initial velocity, substrate analogue, and product inhibitions have allowed formulation of Ordered Bi Bi as the reaction mechanism for macrocin O-methyltransferase.     

Morphometric characteristics of hepatocellular dysplasia

Morphometric study of liver biopsies from six entities (normal tissue, post-hepatitis cirrhosis, post-alcoholic cirrhosis, cancer-related cirrhosis, hepatocellular adenoma and hepatocellular adenocarcinoma) confirmed that this technique can be a valuable adjunct to histopathologic study in the examination of such specimens. As expected, measurements in cirrhotic nodules showed two populations of cells. The so-called "large dysplastic cells" had nuclear and cellular areas close to those of normal hepatocytes and should thus be considered to be hyperplastic elements, not precancerous elements. The smaller dysplastic cells had morphometric values close to those of the corresponding hepatocellular carcinomas, indicating that these cells are the truly precancerous ones. Therefore, while the study confirmed that hepatic cirrhosis is a precancerous lesion, it also showed that the term hepatocellular dysplasia must be restricted to the smaller type of cells found in such nodules.     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

Oscillations in glycolysis: multifactorial quantitative analysis in muscle extract

A multifactorial quantitative analysis of oscillations in glycolysis was conducted in the postmicrosomal supernatant of rat muscle homogenates incubated in the presence of yeast hexokinase. Oscillations in adenine nucleotides, D-fructose 1,6-bisphosphate, triose phosphates, L-glycerol 3-phosphate, ³HOH generation from D-[5-³H]glucose, NADH and L-lactate production were documented. The occurrence of such oscillations were found to depend mainly on the balance between the consumption of ATP associated with the phosphorylation of D-glucose, as catalyzed by both yeast and muscle hexokinase, and the net production of ATP resulting from the further catabolism of D-fructose 6-phosphate, as initiated by activation of phosphofructokinase. The oscillatory pattern was suppressed in the presence of D-fructose 2,6-bisphosphate. It is proposed that the quantitative information gathered in this study may set the scene for further studies in extracts of cells other than myocytes, e. g. hepatocytes and pancreatic islet cells, in which no oscillation of glycolysis was so far observed.     

Monofunctional DNA-platinum(II) adducts block frequently DNA polymerases.

The question of whether monofunctional DNA platinum(II) adducts block synthesis of DNA by purified DNA polymerases of different types and origin has been investigated by comparing the time dependence of synthesis arrest and of DNA adduct formation. Activated salmon testis DNA is used as a suitable substrate for DNA synthesis allowing to probe inhibition by platinum(II) monoadducts for the variety of inherent template-primers. Reaction amplitudes are related to defined mixtures of dichloro and chloroaqua platinum(II) complexes. It is found that (i) all investigated DNA polymerases seem arrested (100% efficiency) at bifunctional DNA adducts. (ii) human DNA polymerase beta bypasses most of the monofunctional lesions of the three platinum(II) complexes investigated. (iii) Klenow fragment is blocked by monoadducts with increasing efficiency in the order cis-diamminechloroaquaplatinum(II) (0%) less than meso-[1,2-bis(2,6- dichloro-4-hydroxyphenyl)ethylenediamine] chloroaquaplatinum(II) (50%) less than trans-diamminechloro-aquaplatinum(II) (75%). (iv) Escherichia coli DNA polymerase I, Thermus aquaticus DNA polymerase, Physarum polycephalum DNA polymerase alpha, and calf thymus DNA polymerase alpha appear to be arrested by monoadducts. According to these examples, blocking efficiencies depend on the cis/trans-stereogeometry of fixation of the carrier ligands at platinum(II) residues, on the size/chemical nature of the platin(II) carrier ligand and on the type/origin of DNA polymerase.     

Immunologic comparisons of Pneumocystis carinii strains obtained from rats, ferrets, and mice using convalescent sera from the same sources.

Pneumocystis carinii (Pc) infections were developed in animals immunosuppressed by dexamethasone treatment either from activation of latent infection (ferret) or trans-tracheal inoculation of Pc obtained from infected lungs of the homologous species (rat, mouse). Convalescent antisera were obtained by stopping dexamethasone treatment after 2-4 wk and allowing 5-8 wk for recovery. Parasites from infected lungs were purified by differential filtration, solubilized in loading buffer, subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis, and blotted to polyvinylidene fluoride sheets for Western analysis. Antisera from each animal species were reacted on Western blots of antigens from rat, ferret, and mouse. Each combination of antigen and antibody from the same species of animal showed reaction with 5 or more bands of Pc antigen. Convalescent mouse antibody did not react with rat or ferret antigens. Convalescent rat antibody reacted with a mouse antigen at about 66 kDa but not with ferret antigen, and convalescent ferret antibody showed minimal, probably non-specific reactions with both rat and mouse antigens. Variations in reactions indicate antigenic differences in Pc strains infecting these animals.     

Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Rotational correlation times of peptides determined by perturbed angular correlations of γ-rays

The technique of Perturbed Angular Correlations of -rays has been used to study the rotational correlation times in aqueous solution of the peptides: oxytocin, glycyltryptophan, cholecystokinin and the glycopeptide ristocetin. These peptides were labelled with excited ^111mCd through the covalent coupling of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to the primary amines-of the peptides. The experimental correlation times are in good accordance with calculations based on the molecular weight. This indicates that the ^111mCd-DTPA is rigidly bound to the molecules. In the case of ristocetin, the correlation time was measured at 2°C, 25°C and 38°C. These experiments show the expected linear dependence on the viscosity divided by temperature. The feasibility of determining rotational correlation times for peptides without lysines and with correlation times in the ns region is thus demonstrated. Also, the correlation time of ^111mCd-DTPA coupled to the lysines of bovine serum albumin was determined. The measured correlation time is about 5 times less than the calculated correlation time. This effect is assigned to local motion. In spite of this, experiments show that ^111mCd-DTPA-bovine-serum-albumin is significantly immobilised by aggregation with immunoglobulins. The nuclear quadrupole interactions, necessary for determining the correlations times, were determined for ^111mCd-DTPA-ristocetin and ^111mCd-DTPA-bovine-serum-albumin by adding sucrose to a concentration of 63% and cooling to 2°C. This showed a small but significant difference between the two molecules. We interpret this as due to different conformations, possibly different coordination numbers. Offprint requests to: E. Danielsen