Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.     

Optimization of L-malic acid production by Aspergillus flavus in a stirred fermentor

Effects of various nutritional and environmental factors on the accumulation of organic acids (mainly L-malic acid) by the filamentous fungus Aspergillus flavus were studied in a 16-L stirred fermentor. Improvement of the molar yield (moles acid produced per moles glucose consumed) of L-malic acid was obtained mainly by increasing the agitation rate (to 350 rpm) and the Fe(z+) ion concentration (to 12 mg/L) and by lowering the nitrogen (to 271 mg/L) and phosphate concentrations (to 1.5 mM) in the medium. These changes resulted in molar yields for L-malic acid and total C(4) acids (L-malic, succinic, and fumaric acids) of 128 and 155%, respectively. The high molar yields obtained (above 100%) are additional evidence for the operation of part of the reductive branch of the tricarboxylic acid cycle in L-malic acid accumulation by A. flavus. The fermentation conditions developed using the above mentioned factors and 9% CaCO(3) in the medium resulted in a high concentration (113 g/L L-malic acid from 120 g/L glucose utilized) and a high overall productivity (0.59 g/L h) of L-malic acid. These changes in acid accumulation coincide with increases in the activities of NAD(+)-malate dehydrogenase, fumarase, and citrate synthase.     

Properties of recombinant cells capable of growing on serine without NhaB Na+/H+ antiporter in Escherichia coli.

Escherichia coli HIT-1 has a mutation in the Na+/H+ antiporter gene, nhaB (P. Thelen, T. Tsuchiya, and E. B. Goldberg, J. Bacteriol. 173:6553-6557, 1991). This strain is not able to utilize serine as a carbon source (T. Ishikawa, H. Hama, M. Tsuda, and T. Tsuchiya, J. Biol. Chem. 262:7443-7446, 1987), because an active NhaB is required to maintain the electrochemical potential of Na+, which drives serine transport via the Na+/serine carrier, the major transport system for serine. We isolated recombinant cells from a cross between strains HIT-1 and Hfr, and these cells were able to grow on serine even though the NhaB Na+/H+ antiporter of the recombinant cells was still defective. We found that the activity of the H+/serine cotransport system, one of the minor serine transport systems in E. coli, was elevated in the recombinant cells. H+/serine cotransport activity was induced by leucine in the recombinant cells more strongly than in strain HIT-1. A kinetic analysis showed that the Vmax, but not the Km, of the transport system was much higher in the recombinant cells than in strain HIT-1 cells.     

Quantification and excretion profiles of pteridines in primate urine.

Biopterin, 6-hydroxymethyl-pterin, isoxanthopterin, neopterin and, pterin were quantified in stress-free collected spontaneous morning urine samples from Callithrix jacchus, Saguinus fuscicollis, Saguinus labiatus, Saimiri sciureus, Presbytis entellus, Cercopithecus albogularis, Cercocebus torquatus, Macaca fascicularis, Hylobates concolor, Pongo pygmaeus, and Gorilla gorilla. In most species, biopterin was the most frequent urinary pteridine followed by neopterin. Sex differences in biopterin and neopterin excretion were observed in Gorilla gorilla and Pongo pygmaeus. Pterin and isoxanthopterin were only present in minor concentrations. 6-hydroxymethyl-pterin was barely detectable and not present in the urine of Saguinus labiatus, Saimiri sciureus, and both male Gorilla gorilla and Pongo pygmaeus.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Chromosomal deletion 4p15.32----p14 in a Treacher Collins syndrome patient: exclusion of the disease locus from and mapping of anonymous DNA sequences to this region.

Treacher Collins syndrome is an autosomal dominant condition of bilateral craniofacial abnormalities of structures derived from the first and second branchial arches. A patient with severe manifestations of Treacher Collins syndrome and a de novo chromosomal deletion in region 4p15.32----p14 was identified. Anonymous DNA sequences of loci D4S18, D4S19, D4S20, D4S22, and D4S23 were mapped to the deleted region. DNA probes previously mapped to loci on chromosome 4p (D4S10, D4S15, D4S16, D4S26, D4S35, D4S95, D4S144, RAF1P1, QDPR, and HOX7) were not deleted in this patient. Linkage analysis between the D4S18, D4S23, and QDPR loci and Treacher Collins syndrome in eight families excluded the Treacher Collins syndrome locus from the region of the deletion.     

Calcium in bacteria: a solution to which problem?

Calcium and calcium-binding proteins including those resembling calmodulin are implicated in numerous diverse processes in bacteria. These processes include chemotaxis, sporulation, virulence, the transport of sugars and proteins, phosphorylation, heat shock, the initiation of DNAS replication, septation, nucleoid structure, nuclease activity and recombination, the stability of the envelope, and phospholipids synthesis and configuration. That such varied processes should have a common factor, calcium, suggests major underlying principles of calcium metabolism metabolism which have yet to be discovered.