The active site of the P99 beta-lactamase from Enterobacter cloacae.

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.     

Amino acid sequence around the thiol and reactive acyl groups of human complement component C4.

Activation of the fourth component of complement (C4) by C1s results in the generation of a reactive acyl group, able to react with putrescine, and in the release of a free thiol group that cannot be detected in the native haemolytically active molecule. Both the reactive acyl group and the free thiol group have been shown to reside in C4d, a fragment of the alpha'-chain of C4b derived from digestion of the molecule with the control proteins C3b inactivator and C4-binding protein. Peptides derived from CNBr digestion of [1,4-14C]putrescine-labelled and iodo(2-14C]acetic acid-labelled C4d have been obtained and used to establish a continuous sequence of 88 residues from the N-terminus of the molecule. The thiol and reactive acyl groups are contained in an octapeptide that shows near identity with the equivalent sequences reported for alpha 2-macroglobulin and C3. Other adjacent short sections also show homology of sequence between the three proteins, and it is highly likely that they contribute to the overall structure that gives a unique reactivity to the thiol ester bond postulated to exist in the native forms of the three proteins.     

Dynein ATPase is inhibited selectively in vitro by erythro-9-[3-2-(hydroxynonyl)]adenine

Dynein (ATP phosphohydrolase, EC 3.6.1.3) extracted from sea urchin sperm tails was inhibited by erythro-9-[3-2-(hydrosynonyl)]adenine in a dosedependent fashion; at the 50% inhibitory concentration, 0.23 mM, twelve other ATP-metabolizing enzymes were notsignificantly affected. Actomyosin and myosin ATPase activities were enhanced 1.5- to 2-fold by millimolar concentrations of erythro-9-[3-2-(hydroxynonyl)]adenine. Enzyme kinetic analysis supported a model of linear mixed-type inhibition, which suggests that the binding site for erythro-9-[3-2-(hydroxynonyl)]adenine on dynein is remote from the ATPase active site. As a selective inhibitor $\text{in}\text{vitro}$, erythro-9-[3-2-(hydroxynonyl)]adenine appears to offer a biochemical criterion for identifying dynein isozymes in tissue extracts.     

Characterization of a prolyl endopeptidase from Flavobacterium meningosepticum. Complete sequence and localization of the active-site serine.

A prolyl endopeptidase was purified from Flavobacterium meningosepticum. It was digested with trypsin. Two oligonucleotides, based on tryptic peptide sequences and used in PCR experiments, amplified a 300-base pair (bp) fragment. A 2.4-kilobase EcoRI fragment that hybridized to the 300-bp probe was cloned in lambda ZAP and sequenced from both strands. It contains a reading frame of 2115 bp, encoding the complete protein sequence of 705 amino acids. Ion-spray mass spectrometry experiments demonstrated the presence of an NH2-terminal signal peptide: the periplasmic mature protease is 685 residues in length for a molecular mass of 76784 Da. The prolyl endopeptidase showed no general sequence homology with known protein sequences except with that of porcine brain prolyl endopeptidase. In order to identify the active-site serine, the prolyl endopeptidase was labeled with [3H]diisopropyl fluorophosphate. One labeled peptide was purified and sequenced. The active-site serine was located in position 536 within the sequence GRSNGG. This sequence is different from the active-site sequence of the trypsin (GDSGGP) and subtilisin (GTSMAS) families.     

The mechanism of leukotriene B4 export from human polymorphonuclear leukocytes

Recently, we characterized the export of leukotriene (LT) C4 from human eosinophils as a carrier-mediated process (Lam, B. K., Owen, W. F., Jr., Austen, K. F., and Soberman, R. J. (1989) J. Biol. Chem. 264, 12885-12889). To determine whether a similar mechanism regulates the release of leukotriene B4 (LTB4), human polymorphonuclear leukocytes (PMN) were preloaded with LTB4 by incubation with 25 microM leukotriene A4 (LTA4) at 0 degrees C for 60 min. PMN converted LTA4 to LTB4 in a time-dependent manner as determined by resolution of products by reverse-phase high performance liquid chromatography and quantitation by integrated optical density. When PMN preloaded with LTB4 were resuspended in buffer at 37 degrees C for 0-90 s, there occurred a time-dependent release of LTB4 but little formation or release of 20-hydroxy-LTB4 and 20-carboxy-LTB4. When PMN were preloaded with increasing amounts of intracellular LTB4 by incubation with 3.1-50.0 microM LTA4 and were then resuspended in buffer at 37 degrees C for 20 s, there occurred a concentration-dependent and saturable release of LTB4 with a Km of 798 pmol/10(7) cells and a Vmax of 383 pmol/10(7) cells/20 s. The release of LTB4 was temperature-sensitive with a Q10 of 3.0 and an energy of activation of 19.9 kcal/mol. The rate of LTB4 release at 37 degrees C is about 50 times the rate of 20-carboxy-LTB4 release. PMN preloaded with LTB4 and resuspended at 0 degree C for 1-60 min in the presence of 30 microM LTA5 progressively converted LTA5 to LTB5. The rate of LTB4 release at 0 degree C was inhibited over the entire time period, peaking at about 50% at 30 min. These results indicate that the release of LTB4 from PMN is a carrier-mediated process that is distinct from its biosynthesis.     

Microbiological aspects of aerobic thermophilic treatment of swine waste.

A thermophilic strain (D2) identified as a Bacillus sp. was isolated from an aerobic digestor of swine waste after several months of operation at 55 degrees C. Aerobic thermophilic batch treatment of swine waste inoculated with strain D2 was studied in a 4-liter fixed-bed reactor. Stabilization of the waste was achieved in less than 30 h when the original chemical oxygen demand (COD) was between 15 and 20 g/liter or in less than 48 h when the COD was around 35 g/liter. When the COD was higher than 30 g/liter, the pH of the waste reached 9.2 to 9.5 during the treatment, and periodic adjustment of the pH to 8.5 was necessary to maintain the activity of the biofilm. In this reactor, ammoniacal nitrogen was completely eliminated by desorption in less than 72 h of incubation. The different packing materials used resulted in similar rates of degradation of organic matter. The thermophilic treatment was also efficient in the 75-liter digestor, and stabilization was achieved in approximately 50 h. A bank of 22 thermophilic bacterial strains originating from different environments and adapted to the thermophilic treatment of swine waste was established. This thermophilic treatment allows, in one step, rapid stabilization of the waste, elimination of the bad smell, and complete elimination of ammonia nitrogen by stripping.     

Completion of the amino acid sequences of the A and B chains of subcomponent C1q of the first component of human complement.

The sequences of amino acid residues 109--224 of the A chain, and residues 109--22 of the B chain, of human subcomponent C1q are given. These results, along with previously published sequence data on the N-terminal, collagen-like, regions of the A and B chains [Reid (1979) Biochem. J. 179, 367--371] yield the complete amino acid sequences of the A and B chains of subcomponent C1q. The asparagine residue at position A-124 has been identified as the major site of asparagine-linked carbohydrate in subcomponent C1q. When the sequences of the C-terminal, 135-residue-long, 'globular' regions of A and B chains are compared they show 40% homology. The degree of homology over certain stretches of 15--20 residues, within the C-terminal regions, rises up to values of 73%, indicating the presence of strongly conserved structures. Structure prediction studies indicate that both the A and B chain C-terminal regions may adopt a predominantly beta-type structure with apparently little alpha-helical structure.     

Inheritance of allozymes in Atlantic herring (Clupea harengus harengus)

Progeny from single pair crosses of Atlantic herring were examined to determine the heritability of genetic variation at seven polymorphic allozyme loci. Mendelian inheritance of codominant autosomal alleles was established for IDH-2, LDH-1, LDH-2, ME-2, PGM-1, and PGI-2. This demonstration of Mendelian inheritance is essential for accurate interpretation of allozymic variation among natural populations of this pelagic species.     

Prostaglandin biosynthesis and stimulation of cyclic AMP in primary monolayer cultures of epithelial cells from mouse mammary gland

Cyclic AMP levels in primary monolayer cultures of epithelial cells prepared from mid-pregnant mice are stimulated by prostaglandin E₁ and E₂. Prostaglandin F_1α and F_2α have only a slight effect upon cyclic AMP levels. In the absence of phosphodiesterase inhibitors the rise in cyclic AMP produced by PGE₁ is only transient and the levels return to normal within 30 minutes. High concentrations (16 mM) of theophylline are needed to prevent this decline, suggesting that the phosphodiesterase activity of epithelial cells in culture is high. However, theophylline alone produced only a small increase in basal cyclic AMP levels even over a 2-hour period indicating that basal cyclic AMP is turned over more slowly than cyclic AMP produced in response to stimulation with PGE₁.Both PGE and PGF synthesis were monitored using radioimmunoassay procedures previously reported. The observed levels were found to decrease as cell density increased and were sensitive to the addition of agents such as collagen and naproxen.