Barbara G. Green

Expression, purification and characterization of recombinant human insulin-like growth factor I in yeast

Insulin-like growth factor I (IGF-I) is a 70 amino acid (aa) protein that is structurally similar and functionally related to insulin. We have inserted a synthetic gene coding for human IGF-I into a Saccharomyces cerevisiae expression vector utilizing the MF alpha 1 promoter and pre-pro leader peptide. This vector directs the expression and secretion of native, biologically active growth factor. Cleavage of the pre-pro alpha factor leader sequence in vivo results in the secretion of a 70-aa recombinant IGF-I molecule with the native N-terminal glycine residue. Human IGF-I purified from yeast culture supernatant is equipotent to serum-derived IGF-I in inhibiting [125I]IGF-I binding to type-I IGF receptors and crude human serum-binding proteins. Recombinant IGF-I is also equipotent to human IGF-I in the stimulation of DNA synthesis in rat aortic smooth-muscle cells. In contrast, yeast recombinant IGF-I is less potent than serum-derived IGF-I in binding to type-2 IGF receptors. The ability to produce native, biologically active IGF-I in yeast will allow the elucidation of binding domains through the expression and characterization of specific structural analogs.

Comparison of the Proteoglycanolytic Activities of Human Leukocyte Elastase and Human Cathepsin G in Vitro and in Vivo

In this study, we evaluated the in vitro and in vivo potency of human leukocyte elastase (HLE) and human cathepsin G (HCG) as proteoglycanases. In vitro evaluation was done using bovine nasal septum aggrecan and aggrecan/hyaluronan aggregate as substrates. Enzyme activity was assessed by the ability of the proteinases to abrogate the ability of aggrecan to aggregate with hyaluronan. In vivo activity of the proteinases was tested by injecting purified HLE and HCG intra-articularly into rabbit stifle joints and quantifying the levels of proteoglycan released into synovial fluids. On a molar basis, HCG was at least tenfold more potent than HLE as a proteoglycanase in vitro. Moreover, HCG was twofold more potent as a proteoglycanase in vivo. In contrast, HLE hydrolyzed elastin approximately 22-fold faster than HCG, but was only slightly more rapid than HCG when [3H]-transferrin was used as substrate. These data indicate that HCG is more potent than HLE as a proteoglycanase both in vitro and in vivo. Thus, HCG could be more important in the pathogenesis of rheumatoid arthritis than previously suspected.

PMN elastases: A comparison of the specificity of human isozymes and the enzyme from other species toward substrates and inhibitors

The human elastases isolated from polymorphonuclear neutrophils (PMN) and purulent sputum displayed identical kinetic constants toward substrates and inhibitors. The elastases from the two sources yield identical N-terminal sequences and were recognized by antiserum prepared against human sputum elastase (HSE) isozyme-4 (I-4). The data support the proposal put forth by Twumasi and Liener (1977, J. Biol. Chem. 252, 1917-1926) that the human elastase from sputum is of PMN origin. PMN elastases from other species displayed kinetic constants toward both substrates and inhibitors significantly different from the human enzyme. Therefore, extrapolation of inhibitor profiles from these elastases to the human source should be avoided. Four groups of isozymes were resolved from HSE by FPLC. Only the most basic isozyme (I-4) was obtained as a single species. The isozymes displayed identical macroscopic kinetic constants toward several substrates and two classes of inhibitors. The similar partition ratios observed with a cephalosporin-derived inhibitor suggest that the microscopic rate constants are also identical. The data support the proposal suggested by Baugh and Travis (1976, Biochemistry 15, 836-841) that HLE isozymes differ only in carbohydrate content. Whatever the source of human PMN elastase heterogeneity, it does not result in heterogeneous catalytic properties. In addition, a new protein was identified in elastase preparations derived from human sputum. This protein displayed homology to serine proteases and properties suggesting that it is identical to azurocidin.

Evidence that insulin and concanavalin-A can co-bind to solubilized insulin receptors without inhibiting each other

Abstract Concanavalin A (Con A) precipitates detergent-solubilized insulin receptors (free of or bound to [ 125 I ]insulin) prepared from fat cell membranes resulting in a loss of [ 125 I ]insulin binding capacity (or bound hormone) in the soluble fraction. The losses can be recovered by redissolving the precipitates with methyl-α-D-mannoside; the sugar also inhibits precipitation. [ 125 I ]insulin also binds to insoluble Con A-occupied receptors. At all concentrations of Con A tested, the initial amounts of free or hormone-bound receptors were completely accounted for by their distribution between soluble and insoluble states. We conclude that Con A and insulin can co-bind to independent sites on the insulin receptor without inhibiting each other and that previously reported decreases in insulin binding to solubilized insulin receptors were likely due to precipitation by Con A of the receptors.

Inhibition of nitric oxide synthase by benzoxazolones

Abstract A series of benzoxazolones has been synthesized using modifications of literature methods. The synthetic benzoxazolone analogs, along with commercially available analogs, were evaluated as inhibitors of nitric oxide synthases (NOS). Structure-activity relationships are also discussed.

Impaired insulin-like growth factor I-mediated stimulation of glucose incorporation into glycogen in vivo in the ob/ob mouse

SummaryThe ability of insulin to modulate glucose metabolism is impaired in insulin resistant ob/ob mice. It has been shown that insulin-like growth factor I stimulates the uptake and metabolism of glucose in muscle through the insulin-like growth factor receptor not the insulin receptor. Thus, we have compared the abilities of insulin-like growth factor I and insulin to stimulate the in vivo incorporation of [14C]-glucose into glycogen in the diaphragm of ob/ob mice and their lean littermates. The animals used in these studies were 12–14 weeks old and the serum insulin levels of the ob/ob mice were 16-fold higher than in their lean littermates. There were no differences in the serum levels of glucose or insulin-like growth factor I. Both insulin and insulin-like growth factor I stimulate the incorporation of [14C]-glucose into glycogen in lean mice. Significant stimulation occurs at doses as low as 1 μg/kg of either peptide. The effective doses of insulin and insulin-like growth factor I are quite similar, which indicates that the effect of insulin-like growth factor I is mediated by the insulin-like growth factor receptor and not the insulin receptor. In contrast, greater than 100 μg/kg of insulin-like growth factor I is required to stimulate [14C]-glucose incorporation into glycogen in the diaphragm of ob/ob mice. Thus, ob/ob mice are resistant to the action of both insulin and insulin-like growth factor I. In contrast to the decrease in the number of insulin receptors which occurs in ob/ob mice, there is no significant difference in the number of type 1 insulin-like growth factor receptors or in their affinity for insulin-like growth factor I in muscle membranes prepared from lean and ob/ob mice. In addition, the ability of insulin-like growth factor I to stimulate the catalysis of Val5-angiotensin II phosphorylation by the partially purified muscle type 1 insulin-like growth factor receptor is not decreased in ob/ob mice as compared with their lean littermates. These data indicate that the loss in sensitivity of the ob/ob mouse of both insulin and insulin-like growth factor I is most likely mediated by a post-receptor defect in metabolism and not by receptor down-regulation or desensitisation.

Characterization of Inducible Nitric-oxide Synthase by Cytochrome P-450 Substrates and Inhibitors INHIBITION BY CHLORZOXAZONE

Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3 ± 0.7 μM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.

Phosphorous acid analogs of L-680,833, a potent monocyclic β-lactam inhibitor of human leukocyte elastase

Abstract Analogs of the monocyclic β-lactam human leukocyte elastase (HLE) inhibitor L-680,833 in which the carboxyl group is replaced by phosphorous acid moieties were synthesized and found to be potent inhibitors of the enzyme ( k inact / K i in the range 217,000–1,326,000 M −1 s −1 ). Cellular activity was demonstrated by inhibition of the generation of the N-terminal cleavage product Aα-(1–21) from the Aα chain of fibrinogen.

PROTEOGLYCAN-DEGRADING ACTIVITY OF HUMAN STROMELYSIN-1 AND LEUKOCYTE ELASTASE IN RABBIT JOINTS. QUANTITATION OF PROTEOGLYCAN AND A STROMELYSIN-INDUCED HABR FRAGMENT OF AGGRECAN IN SYNOVIAL FLUID AND CARTILAGE

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.