A recombinant group 1 house dust mite allergen, rDer f 1, with biological activities similar to those of the native allergen.

Serum IgE directed against Der f 1, a protease found in the feces of Dermatophagoides farinae, correlates well with allergic sensitization to house dust mite in humans and is a risk factor for developing asthma. Native Der f 1 (nDer f 1) is produced as a pre-pro form and processed to an approximately 25-kDa mature form. We have expressed recombinant forms of Der f 1 (rDer f 1) in Pichia pastoris using AOX1-promoter expression vectors. Fusion of either the pro-enzyme form or the mature form to the Saccharomyces cerevisiae alpha factor pre-pro sequence resulted in secretion of the mature form of the protein from P. pastoris. The secreted protein was heterogeneously glycosylated at a single N-glycosylation site and had an apparent molecular mass of 35-50 kDa. Both the alpha factor signal peptide and the pro-enzyme region were efficiently processed during secretion. A version of the pro-enzyme with a mutated consensus N-linked glycosylation site was secreted from P. pastoris as a mature, unglycosylated, approximately 25-kDa protein. The IgE binding activity of this unglycosylated rDer f 1 was similar to that of glycosylated forms produced by P. pastoris and to nDer f 1 obtained from mites. Thus, oligosaccharides are not required for secretion from P. pastoris or for IgE binding in vitro. Recombinant and native versions of Der f 1 displayed protease activity on casein zymogram gels. The availability of a highly purified recombinant Der f 1 will facilitate experimental and clinical studies of mite allergy.

Identification, cloning, and characterization of a major cat flea salivary allergen (Cte f 1).

An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA), this protein detected IgE in 100 and 80% of experimental and clinical flea allergic dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization screening. This cDNA is 658 bp in length, and contains an open reading frame of 528 bp. The open reading frame encodes a protein of 176 amino acids, consisting of an 18 amino acid signal sequence and a 158 amino acid mature protein. The calculated molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively. The protein, named Cte f 1, is the first novel major allergen described for canine flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity. Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE binding activity equal to the native antigen, was isolated from the soluble fraction of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed and secreted a fully processed, correctly refolded and fully active form of rCte f 1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight intact disulfide bonds were present, matching the number observed in the native allergen. The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals, produced in these three expression systems, matched that of the native allergen. Competition ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte f 1 could be blocked by the different forms of rCte f 1.

Canine interleukin-13: molecular cloning of full-length cDNA and expression of biologically active recombinant protein.

Interleukin-13 (IL-13) regulates immune responses mediated by type 2 T helper lymphocytes (Th2) in the human and mouse. To study the function of this cytokine in the dog, we have isolated a cDNA that encodes the full-length canine IL-13 (CaIL-13) precursor polypeptide of 131 amino acids. CaIL-13 shares significant homology with the IL-13 amino acid sequences of cattle (54.1%), mouse (39.6%), and rat (36.6%) but shares the highest identity with human IL-13 (HuIL-13) (61.8%). The predicted CaIL-13 mature polypeptide of 111 residues was expressed in bacteria, and recombinant CaIL-13 (rCaIL-13) was isolated from inclusion bodies and refolded. rCaIL-13 stimulated the proliferation of TF-1 cells, which are derived from human erythroleukemia cells and respond to IL-13 as well as to a number of other human and murine cytokines. CaIL-13 mRNA was readily detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells from lymph nodes and peripheral blood. The gene sequence and biologically active recombinant protein for CaIL-13 will be useful reagents to determine the role of IL-13 in the regulation of canine immune responses.

Refolding and characterization of recombinant human soluble CTLA-4 expressed in Escherichia coli.

CD28 and CTLA-4 are homologous cell surface proteins expressed by T cells. CD28 is constitutively expressed by most T cells, whereas CTLA-4 is expressed by activated T cells. Both proteins are ligands for the costimulatory molecules CD80 and CD86 expressed by activated B cells, macrophages, and dendritic cells. A fusion protein comprising the CTLA-4 extracellular domain joined to a human immunoglobulin heavy chain constant region (CTLA4Ig) binds CD80 and CD-86 with high affinity and inhibits CD80/CD86-dependent immune responses in vitro and in vivo. Attempts at producing the CTLA-4 extracellular domain as an unfused protein have met with limited success. Here we describe the expression and purification of the CTLA-4 extracellular domain as a nonfused protein in Escherichia coli. The 12.5-kDa CTLA-4 extracellular domain was insoluble when expressed in E. coli and required denaturation, reduction, and refolding steps to become soluble and assume its proper conformation. The protein refolded into a mixture of monomers, disulfide-linked dimers, and higher order disulfide-linked aggregates. sCTLA-4 dimers were the predominant refold form when air was used as the oxidizing agent during the refold procedure. Purified sCTLA-4 dimers were 10- to 50-fold more potent than sCTLA-4 monomers at inhibiting T cell activation using a CD80-dependent in vitro bioassay.

Correlation of MRI and arthroscopic diagnosis of knee pathology in children and adolescents.

The accuracy of magnetic resonance imaging (MRI) in diagnosing knee pathology in the pediatric and adolescent population is not well established. The purpose of this study was to correlate the findings of MRI and knee arthroscopy in children and adolescents. One hundred and eight consecutive knee arthroscopies performed in patients ages 4-17 years between 1992 and 1996 were retrospectively reviewed. Fifty-three of these patients underwent preoperative MRI. Age-related comparisons were then made between MRIs and observed intraoperative meniscal and anterior cruciate ligament pathology. The pediatric group (ages 4-14 years) was demonstrated to have an appreciable decrease in sensitivity, specificity, positive predictive value, and accuracy for essentially all categories of pathologic changes. Conversely, negative predictive values for the pediatric group exceeded those of the adolescent group (ages 15-17 years) in each category. The ability of MRI to predict intraarticular knee pathology among adolescents is comparable to that in adults, whereas it is much less accurate in the pediatric population.

Polyethylene glycol conjugated insulin-like growth factor binding protein-1 (IGFBP-1) inhibits growth of breast cancer in athymic mice.

Insulin-like growth factor (IGF) binding protein-1 (BP-1) inhibits IGF-mediated proliferation of some breast cancer cell lines in vitro. Here we examined whether recombinant human wild-type IGFBP-1 (WT-BP-1) and IGFBP-1 conjugated with polyethylene glycol (PEG-BP-1) could inhibit breast cancer growth. Three breast cancer cell lines were used: MCF-7, MDA-MB-231 and MDA-MB-435A (ascites model). The cells were grown in agar with or without the BP-1 conjugates to investigate their effect on colony formation. Both WT-BP-1 and PEG-BP-1 inhibited anchorage-independent growth (AIG) of MCF-7 and MDA-MB-435A cells. AIG of MDA-MB-231 cells was not inhibited by PEG-BP-1, whereas WT-BP-1 significantly stimulated colony number. We also tested both forms of BP-1 in xenograft tumour models. Two solid breast tumour models were studied using MCF-7 and MDA-MB-231 cell lines, and one ascites model using the MDA-MB-435A cell line. PEG-BP-1 inhibited malignant ascites formation in the MDA-MB-435A model. Conversely, PEG-BP-1 did not significantly inhibit MCF-7 xenograft growth. However, the MDA-MB-231 tumour growth curves were significantly different by a constant amount, suggesting that PEG-BP-1 treatment inhibited early tumour growth of this cell line. In contrast, WT-BP-1 was ineffective in the MDA-MB-231 tumours. These data show that anti-IGF strategies can be used to inhibit breast cancer cell growth. Since PEG-BP-1 inhibited the in vivo, but not in vitro, growth of MDA-MB-231, we speculate that PEG-BP-1 may block host IGF functions required for optimal tumorigenesis. Because PEG-BP-1 has a prolonged serum half-life compared to WT-BP-1, we conclude that improvements in BP-1 pharmacological properties enhanced its antitumour effects in vivo.

Seasonal changes in haemostatic factors in young and elderly subjects.

Morbidity and mortality from cardiovascular disease are more common in colder seasons, especially in elderly people. Previous studies have shown higher fibrinogen levels in old people in the winter months. The present studies of haemostatic factors in relation to age and season have shown that fibrinogen, tissue plasminogen activator (tPA), protein S and protein C levels are higher in old (aged 75 years and over) than young (aged 25-30 years) subjects while antiplasmin levels are lower in old people. Antiplasmin and protein C levels are lower in winter in both young and old while plasminogen activator inhibitor (PAI) is higher, and tPA higher in old people only. This study illustrates the complex interrelationships of the haemostatic system and may suggest that in 'successful' elderly people the fibrinolytic system may alter to maintain the delicate balance between thrombogenic and fibrinolytic activity. Nevertheless, the results presented here suggest that both old age and cold weather may increase the risk of atherothrombotic disease.

Recombinant human insulin-like growth factor (IGF)-binding protein-1 inhibits somatic growth stimulated by IGF-I and growth hormone in hypophysectomized rats.

We have examined the effects of exogenously administered recombinant human insulin-like growth factor-binding protein-1 (rhIGFBP-1) alone and in combination with recombinant human insulin-like growth factor-I (rhIGF-I) or human GH on weight gain and tibial epiphysis enlargement in hypophysectomized rats. rhIGF-I, given twice daily by sc injection, increased both growth parameters in a dose-dependent manner. Coadministration of increasing amounts of rhIGFBP-1 with a constant amount of rhIGF-I (80 micrograms/injection, given twice daily) resulted in a dose-dependent inhibition of the growth-promoting effects of rhIGF-I. A rhIGFBP-1 dose of 9.8 micrograms/injection (an IGFBP-1/IGF-I molar ratio of 0.04:1) caused no significant effect on rhIGF-I-stimulated growth parameters, whereas a rhIGFBP-1 dose of 1200 micrograms/injection (IGFBP-1/IGF-I molar ratio of 5:1) resulted in 78% or greater inhibition of rhIGF-I-stimulated growth (P < 0.05). rhIGFBP-1 doses of 48 and 240 micrograms/injection (IGFBP-1/IGF-I molar ratios of 0.2:1 and 1:1, respectively) had intermediate inhibitory effects. None of the rhIGFBP-1 doses potentiated the growth-promoting effects of rhIGF-I. Rats treated with rhIGFBP-1 alone (twice daily injections of 9.8, 48, 240, or 1200 micrograms) showed no significant differences in growth parameters compared to rats treated with vehicle. Coadministration of rhIGFBP-1 (1200 micrograms/injection, given twice daily) with GH (15 mU/injection, given twice daily) inhibited weight gain and tibial epiphysis enlargement stimulated by GH by at least 50% in each of two experiments (P < 0.05). These studies demonstrate that nonphosphorylated rhIGFBP-1 can inhibit the growth-promoting effects of rhIGF-I and GH in vivo. The results suggest that in addition to its proposed role in glucose homeostasis, IGFBP-1 may play a role in inhibiting somatic growth and other physiological functions stimulated by IGF-I and GH.

Recombinant insulin-like growth factor binding protein-1 inhibits IGF-I, serum, and estrogen-dependent growth of MCF-7 human breast cancer cells.

The insulin-like growth factors (IGFs) are potent mitogens for breast cancer cells and their activity is modulated by high affinity binding proteins (IGFBPs). We have recently shown that IGFBP-1 purified from human amniotic fluid neutralizes IGF-I-dependent growth of MCF-7 cells. In this study we examined the effects of recombinant IGFBP-1 (rBP-1) on IGF-I, estradiol (E2), and serum-induced monolayer and anchorage independent growth (AIG) of MCF-7 cells. Under serum-free conditions, rBP-1 had no effect on MCF-7 basal monolayer growth. However, 40 nM rBP-1 completely blocked the mitogenic action of both IGF-I and 5% charcoal stripped serum (CSS). This concentration of rBP-1 partially inhibited E2-induced growth, while 80 nM rBP-1 completely abolished E2 mitogenicity. The addition of either excess IGF-I or 5 nM [Arg3]IGF-I, a species that does not bind IGFBPs, neutralized rBP-1 inhibitory effects. In AIG assays, 80 nM rBP-1 reduced colony number by at least 70% and decreased colony size in all treatment groups compared to control. We examined rBP-1 effects on both IGF-I binding to MCF-7 membranes and activation of type I IGF receptor (IGFR1) and found that 80 nM rBP-1 reduced IGF-I receptor binding to levels of nonspecific binding and completely abolished ligand-dependent IGFR1 phosphorylation. However, neither treatment with 5% CSS nor exposure to E2 resulted in IGFR1 phosphorylation suggesting that different mechanism(s) are responsible for rBP-1 inhibitory action under this condition. Our data suggest rBP-1 may serve as an antagonist of human breast cancer growth by interfering with growth factor-mediated cell proliferation.

Phosphorylation of alpha-crystallin B in Alexander's disease brain.

The phosphorylation of alpha-crystallin B was studied in homogenates of autopsy samples of brain tissue from patients with Alexander's disease, a condition characterized by over-expression of this protein. After incubation in the presence of [gamma-32P]ATP and cAMP the homogenates were analyzed by two-dimensional electrophoresis, (isoelectric focusing followed by SDS-PAGE). Three major polypeptides having the same molecular weight as bovine lens alpha-crystallin B and pIs 7.1, 6.9 and 6.7 were detected in the Coomassie blue stained gels. These three polypeptides were recognized by an alpha-crystallin B-specific antiserum in Western blots. The polypeptides with pIs 7.1 and 6.7 co-migrated in isoelectric focusing gels with bovine lens alpha B and its phosphorylated form alpha Bp, respectively. Radioautography of the two-dimensional gels demonstrated the presence of 32P in the most acidic polypeptide. The results demonstrate the occurrence of alpha B phosphorylation in Alexander's disease brain tissue.

The calf gamma crystallins--a Raman spectroscopic study.

The solution structures of the four major components of bovine lens gamma-crystallin, gamma s, gamma II, gamma III and gamma IV are compared using Raman spectroscopy. The spectral region sensitive to the vibrational frequencies of aromatic and sulfur containing residues and to the backbone skeletal stretching modes (500-1000 cm-1), and that reflecting secondary structure (1,000-1,700 cm-1) are strikingly similar in all four gamma-crystallin fractions. These similarities are indicative of the dominant anti-parallel beta sheet structure common to all the gamma-crystallins. A comparison of the ratios of the Raman intensities at 850 cm-1 and 830 cm-1 (I850/I830), an empirical measure of the degree of hydrogen bonding of phenolic hydroxyl groups, suggests that the tyrosine residues in all the gamma-crystallin fractions are moderately hydrogen bonded. Distinct differences in the solution structures of the gamma-crystallins were observed in the higher energy end of the vibrational Raman spectra. The sulfhydryl stretching frequencies for the gamma-crystallins exhibit complex splitting patterns in the 2,500-2,600 cm-1 region. These patterns are due to the competing effects of hydrogen bonding and S-pi interactions with neighboring aromatic residues. All five proteins exhibit multiple, but distinct, thiol frequencies, suggesting that the microenvironments of the cysteine residues in these proteins are significantly different.

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues.

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.

Purification of a 43,000 dalton aggregate generated from alpha-crystallin.

A procedure is presented for the purification of an aggregate of covalently linked polypeptides of alpha-crystallin. Using either iron catalyzed oxidation or UV irradiation, 6-12% of calf lens alpha-crystallin can be converted to a 43,000 Da aggregate containing non-reducible cross-linked polypeptides as indicated by SDS-PAGE under reducing conditions. The 43,000 Da aggregate generated by Fe2+ oxidation can be isolated to approximately 85% homogeneity with respect to its relative molecular weight on SDS-PAGE. The procedure can be performed in two steps; 1) gel filtration of the oxidized alpha-crystallin under deaggregating and reducing conditions, and 2) preparative SDS-PAGE of the 43,000 Da aggregate-enriched peak obtained by filtration. The 43,000 Da aggregate band can be recovered from the polyacrylamide gels using a new preparative electroelution technique. 1.0 +/- 0.3 mg of purified 43,000 Da aggregate can be obtained from 100 mg of Fe2+ oxidized alpha-crystallin. The described methodology will facilitate further characterization of this 43,000 Da alpha-crystallin aggregate.

Differential synthesis and phosphorylation of the alpha-crystallin A and B chains during bovine lens fiber cell differentiation.

[14C]-amino acids and [32P]-orthophosphate incorporation experiments were carried out in bovine lenses in culture to study the synthesis and phosphorylation of alpha-crystallin A and B polypeptides during differentiation of the lens fiber cells. Following culture, the [14C] or [32P]-labelled alpha-crystallin was isolated by gel filtration chromatography from four regions of the lens corresponding to: A) quiescent epithelial cells, B) dividing epithelial cells and early stages of elongation, C) young elongating fibers, and D) mature fibers from the superficial cortex. The incorporation of label into the alpha-crystallin primary gene products alpha A2 and alpha B2 and their respective phosphorylated forms alpha A1 and alpha B1 was determined by isoelectric focusing and radioautography. Different synthesis and phosphorylation patterns were observed in alpha A and alpha B polypeptides. Synthesis and phosphorylation of the alpha B chain occurs most actively in the epithelial cells, both processes decrease during differentiation and there is no net accumulation of the phosphorylated form alpha B1 in the mature fiber cell. In contrast to the B chain, the A chain synthesis, minimal in the epithelial cell, increases with differentiation. Most striking, the A chain phosphorylation, not detectable in the epithelial cells, increases with differentiation. In the mature fiber cell, the phosphorylated form alpha A1 accounts for one third of the A chain. These observations indicate that the two chains may have different functions. the synthesis and phosphorylation patterns of alpha A suggest a lens-specific function of this polypeptide in the fiber cell and in the terminal differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)

Raman spectroscopic evidence for a disulfide bridge in calf gamma II crystallin.

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical gamma II crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-1), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of gamma II as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-1), the native form shows a line near 511 cm-1 which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:1 computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-1. In every other spectral region, both native and reduced gamma II forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution.

Fe2+ oxidation of alpha-crystallin produces a 43,000 Da aggregate composed of A and B chains cross-linked by non-reducible covalent bonds.

The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.

The disulfide content of calf gamma-crystallin.

The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.

The effect of H2O2 upon thioredoxin-enriched lens epithelial cells.

Thioredoxin, a dithiol polypeptide, has been examined as a potential contributor to the recovery of lens epithelial cells from oxidative insult. It is reported that Escherichia coli thioredoxin can (a) effectively reduce lens-soluble protein disulfide bonds generated by H2O2, (b) restore to its initial activity H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase, (c) act as an effective source of reducing potential for lens methionine sulfoxide peptide reductase, and (d) act as a free radical quencher based on studies with a stable free radical system generated by ascorbic acid and 2,6-dimethoxy-p-benzoquinone. Thioredoxin is much more effective than dithiothreitol in restoring glyceraldehyde-3-phosphate dehydrogenase activity and as a cofactor for methionine sulfoxide peptide reductase. Upon incubation with epithelial cells, thioredoxin can be observed in the cell using rocket immunoelectrophoresis. These cells recover from H2O2 insult more rapidly than control cell preparations based upon 1) analyses of plasma membrane-related activities: leucine and 86Rb uptake and 2) analyses of parameters primarily related to the internal cell metabolism: ATP concentration and glyceraldehyde-3-phosphate dehydrogenase activity. Analysis of thioredoxin in cell preparations indicates that only about 9% is in the reduced state implying a low effective concentration of the polypeptide. The experiments suggest that low levels of thioredoxin may significantly increase the ability of lens epithelial cells to recover from exposure to H2O2.

The effects of antimineralocorticoids on synthesis of aldosterone metabolites in target tissues.

[3H]Aldosterone is transformed into several metabolites by subcellular fractions of rat kidney. 80-90% of the metabolites synthesized by nuclei and plasma membranes are 5 alpha-DHAldo and 3 alpha,5 alpha-THAldo in ratios of 1:2 and 1:1 respectively; small quantities of 3 beta,5 alpha-THAldo are also synthesized. In contrast, kidney cytosol metabolizes Aldo principally to 5 beta-reduced products with co-chromatograph with 5 beta-DHAldo and 3 alpha,5 beta-THAldo. Several polar neutral metabolites, as well as sulfate and acidic metabolites are also synthesized by the cytosol fraction. Similar 5 alpha-reduced metabolites, 5 alpha-DHAldo, 3 alpha,5 alpha-THAldo and 3 beta,5 alpha-THAldo are also synthesized when [3H]aldosterone is incubated in vitro with toad urinary bladder for 1 and 5 h. Significant quantities of 5 beta- and 20 beta-reduced products and sulfate and acidic metabolites are also synthesized. The metabolism of [3H]aldosterone in both target tissues is significantly inhibited by aldosterone antagonists. Several of the reduced metabolites of aldosterone synthesized in kidney and toad bladder possess significant mineralocorticoid activity. 5 alpha-DHAldo and 3 alpha,5 alpha-THAldo possess 1/10 and 1/30 and 3 alpha,5 beta possesses 1/80-1/100 of the antinatriuretic activity of Aldo. It is suggested that the metabolism of Aldo in its target tissues may be linked to regulation or expression of the hormone's actions.

Purification and properties of rabbit liver cathepsin M and cathepsin B.

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.