The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

Human immunodeficiency virus protein gp120 interferes with β-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes

Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of³²P into a soluble acidic protein of 80,000M _r , which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to³H-myristic acid indicated that the acidic 80,000M _r protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M _r MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK _m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee.     

A facile method to prepare C-terminal fluorescently labelled peptides by an Fmoc strategy

Summary Spectrophotometric peptide probes, derivatized at the C-terminus, are conveniently prepared by means of an Fmoc solid-phase strategy. Using a resin such as Sasrin, the fully protected peptide can be cleaved from the resin with hydrazine, yielding the protected peptide-hydrazide which is subsequently oxidized to the azide. An amino-containing chromophore or fluorophore such as 5-[(2-aminoethyl)-amino]-naphthalene sulfonic acid (EDANS) can be coupled directly to this activated carboxyl group. This allows for specific placement of the fluorophore at the C-terminal carboxyl group in the presence of trifunctional amino acids.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-substrate adhesion and metastatic potential of cultured mesothelioma cells induced by asbestos

Cell-substrate adhesion was quantified for two cultured mesothelioma cell lines (epitheliomatus and sarcomatous) on glass, fibronectin and laminin substrates. Interference reflection microscopy (IRM) was used to image the adhesion patterns of cells and a grey level analysis was employed to quantify adhesion. Sarcomatous cells demonstrated marked adhesion to glass and fibronectin-coated substrates but not to laminin-coated substrate, with the greatest adhesion occurring on the fibronectin-coated surface. This adhesion was accompanied by cytoplasmic spreading. By contrast, epitheliomatous cells showed little tendency to adhere to any of the substrates and only showed significant spreading when in contact with the laminin substrate (P < 0.01). A bioassay was used to determine the metastatic potential of each of the cell lines. Via the intravenous route, the sarcomatous cells killed the host rats in 24.7 ± 1.5 (S.D.) days compared to 27.3 ± 0.9 (S.D.) days for the epitheliomatous cells (P < 0.01). After subcutaneous inoculation of tumour cells, the sarcomatous cells killed the host rats in 54.7 ± 0.7 (S.D.) days compared to 48.5 ± 0.5 (S.D.) days for the epitheliomatous cells (P < 0.01). We conclude that the results of the metastasis bioassays were consistent with the predicted behavior of these cell lines based on their ability to adhere to substrates in the in vitro adhesion assays.     

Effect of agitation speed on the synthesis of Mucor miehei acid protease

Different experiments using Mucor miehei CBS 370.65 were carried out to study the effect of agitation speed on the production of the mold acid protease. The experiments were conducted in shake flasks at a fixed substrate concentration of 58 g l⁻¹ of total carbohydrates and at shaker speeds from 80 to 380 rev min⁻¹. Enzyme production was found to be directly proportional to the shaker speeds, with the highest concentration of enzyme of 1,400 Soxhlet Rennet units (SU) ml⁻¹ obtained at 380 rev min⁻¹. The yield of product to substrate at 380 rev min⁻¹ was determined to be 27,081.0 SU g⁻¹ substrate and the productivity of the process was 221 SU g⁻¹ h⁻¹. Enzyme production was partially growth associated, and glucose supported both cell growth and enzyme production. Product formation and cell concentration were directly related to the rate of substrate consumption. The rate of product formation decreased when product started to accumulate, suggesting that the process was affected by feedback repression.     

On the ecology of Colurellidae (Rotifera)

A material consisting of 45 colurellid rotifers from diverse waters in south and central Sweden was analyzed to reveal their relationships to their substrate. Periphytic substrates were generally preferred. Most species are obviously mobile and more or less euryecious, but a few show a predilection for bog environments. Some colurellids are euryhaline, but when inhabiting fresh water they prefer rivers and other environments with a high oxygen concentration, probably because of problems with osmoregulation.