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R B Freedman

Publications and source records attributed to R B Freedman.

At least 19 recordsLinked to original sources
PubMed1992

Site-directed mutations that alter the inhibitory activity of the tissue inhibitor of metalloproteinases-1: importance of the N-terminal region between cysteine 3 and cysteine 13.

The tissue inhibitor of metalloproteinases-1 (TIMP-1) was subjected to single-site mutations within the N-terminal three loops using an oligonucleotide-directed polymerase chain reaction method. All the histidines, and a number of other residues conserved between TIMP-1 and TIMP-2, were individually modified and the mutant TIMPs expressed in mammalian cells. Purified mutant TIMPs were shown to be correctly folded by measuring the effect of guanidine hydrochloride on intrinsic fluorescence. Kinetic analyses of mutants using a quenched fluorescent peptide substrate and the metalloproteinase PUMP indicated that mutation of His7 and Gln9 caused an increase in the apparent dissociation constant, largely due to an increase in the rate of dissociation of complexes. The data indicate that the anchored sequence between Cys 3 and Cys 13 is a key region for interaction of TIMP-1 with metalloproteinases.

Amino Acid Sequence↗
PubMed1992

Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis.

Amino Acid Sequence↗
PubMed1992

Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.

Adenosine Triphosphate↗
PubMed1991

Protein disulfide isomerase is essential for viability in Saccharomyces cerevisiae.

Protein disulfide isomerase (PDI) is an enzyme involved in the catalysis of disulfide bond formation in secretory and cell-surface proteins. Using an oligodeoxyribonucleotide designed to detect the conserved 'thioredoxin-like' active site of vertebrate PDIs, we have isolated a gene encoding PDI from the lower eukaryote, Saccharomyces cerevisiae. The nucleotide sequence and deduced open reading frame of the cloned gene predict a 530-amino-acid (aa) protein of Mr 59,082 and a pI of 4.1, physical properties characteristic of mammalian PDIs. Furthermore, the aa sequence shows 30-32% identity with mammalian and avian PDI sequences and has a very similar overall organisation, namely the presence of two approx. 100-aa segments, each of which is repeated, with the most significant homologies to mammalian and avian PDIs being in the regions (a, a') that contain the conserved 'thioredoxin-like' active site. The N-terminal region has the characteristics of a cleavable secretory signal sequence and the C-terminal four aa (-His-Asp-Glu-Leu) are consistent with the protein being a component of the S. cerevisiae endoplasmic reticulum. Transformants carrying multiple copies of this gene (designated PDI1) have tenfold higher levels of PDI activity and overproduce a protein of the predicted Mr. The PDI1 gene is unique in the yeast genome and encodes a single 1.8-kb transcript that is not found in stationary phase cells. Disruption of the PDI1 gene is haplo-lethal indicating that the product of this gene is essential for viability.

Amino Acid Sequence↗
PubMed1991

The reactivities and ionization properties of the active-site dithiol groups of mammalian protein disulphide-isomerase.

1. The number of reactive thiol groups in mammalian liver protein disulphide-isomerase (PDI) in various conditions was investigated by alkylation with iodo[14C]acetate. 2. Both the native enzyme, as isolated, and the urea-denatured enzyme contained negligible reactive thiol groups; the enzyme reduced with dithiothreitol contained two groups reactive towards iodoacetic acid at pH 7.5, and up to five reactive groups were detectable in the reduced denatured enzyme. 3. Modification of the two reactive groups in the reduced native enzyme led to complete inactivation, and the relationship between the loss of activity and the extent of modification was approximately linear. 4. Inactivation of PDI by alkylation of the reduced enzyme followed pseudo-first-order kinetics; a plot of the pH-dependence of the second-order rate constant for inactivation indicated that the essential reactive groups had a pK of 6.7 and a limiting second-order rate constant at high pH of 11 M-1.s-1. 5. Since sequence data on PDI show the presence within the polypeptide of two regions closely similar to thioredoxin, the data strongly indicate that these regions are chemically and functionally equivalent to thioredoxin. 6. The activity of PDI in thiol/disulphide interchange derives from the presence of vicinal dithiol groups in which one thiol group of each pair has an unusually low pK and high nucleophilic reactivity at physiological pH.

Alkylation↗
PubMed1991

Redox properties and cross-linking of the dithiol/disulphide active sites of mammalian protein disulphide-isomerase.

1. The redox properties of the active-site dithiol/disulphide groups of PDI were determined by equilibrating the enzyme with an excess of GSH + GSSG, rapidly alkylating the dithiol form of the enzyme to inactivate it irreversibly, and determining the proportion of the disulphide form by measuring the residual activity under standard conditions. 2. The extent of reduction varied with the applied redox potential; to a first approximation, the data fitted a model in which all the enzyme dithiol/disulphide groups are independent and equivalent and the equilibrium constant between these sites and the GSH/GSSG redox couple is 42 microM at pH 7.5. 3. The standard redox potential for PDI active-site dithiol/disulphide couples was calculated from this result and found to be -0.11 V; hence PDI is a stronger oxidant and weaker reductant than GSH, nicotinamide cofactors, thioredoxin and dithiothreitol. 4. The redox equilibrium data for PDI with the GSH/GSSG redox couple showed sigmoidal deviations from linearity. The sigmoidicity could be modelled closely by assuming a Hill coefficient of 1.5. 5. This evidence of co-operative interactions between the four active sites in a PDI dimer was extended by studying the reaction between PDI and homobifunctional alkylating agents with various lengths between the reactive groups. A species whose electrophoretic mobility suggested it contained an intrachain cross-link was observed in all cases, whereas there was no evidence for cross-linking between the chains of the PDI homodimer. Most effective cross-linking was achieved with reagents containing five or more methylene spacer groups, implying a minimum distance of 1.6 nm (16 A) between the active-site reactive groups within the two thioredoxin-like domains of the PDI polypeptide.

Acetamides↗
PubMed1991

Comparison of the activities of protein disulphide-isomerase and thioredoxin in catalysing disulphide isomerization in a protein substrate.

1. The activities of protein disulphide-isomerase (PDI) and thioredoxin in catalysing disulphide bond isomerization in a protein substrate were compared by using the standard assay, namely the re-activation of 'scrambled' RNAase. 2. The specific activity of PDI was 25-fold greater than that of thioredoxin. 3. The greater efficiency of PDI compared with thioredoxin is considered to be due more to the presence of multiple catalytic domains in PDI than to differences in their active-site sequences. 4. Data and procedures were defined for expressing enzyme activity in standard units, i.e. mumol of active RNAase generated/min.

Amino Acid Sequence↗
PubMed1990

Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation.

1. The cDNA coding for preprochymosin has been sub-cloned into the transcription/translation vector pGEM-3Z, the T7 promoter used to transcribe the gene and the product expressed in an 'in vitro' cell-free system comprising rabbit reticulocyte lysate and dog pancreatic microsomes. 2. Translations in various conditions, and analyses of the translation product in reducing and non-reducing conditions, indicate that oxidizing translation conditions and the cleavage of the N-terminal 'pre-' sequence are essential for generation of a disulphide-bonded translation product. 3. The disulphide-bonded translation product was resistant to proteinases, as expected for a translation product segregated within microsomal vesicles; in the presence of detergent to solubilize the membranes, the product was not readily susceptible to proteolysis, and was converted to a proteinase-resistant core fragment. 4. Segregated prochymosin, synthesized in reducing conditions, was completely degraded by proteinases under similar conditions. 5. Proteinase treatment of purified recombinant prochymosin gave rise to a proteinase-resistant fragment of similar Mr, suggesting that the disulphide-bonded product of translation in vitro was correctly folded. 6. The translocated, disulphide-bonded and folded prochymosin could be converted into pseudochymosin at pH 2.0, and addition of chymosin to the activation mixture resulted in increased pseudochymosin production.

Animals↗
PubMed1990

Denaturation studies on natural and recombinant bovine prochymosin (prorennin).

1. Prochymosin in solution in the presence of 8 M-urea is fully unfolded, as indicated by its fluorescence spectrum, fluorescence quenching behaviour and far-u.v.c.d. spectrum. 2. Equilibrium studies on the unfolding of prochymosin and pepsinogen by urea were carried out at pH 7.5 and pH 9.0. The results indicate that the stabilization energies of the two proteins are identical at pH 7.5, but that at pH 9.0 pepsinogen is significantly less stable than prochymosin. 3. Kinetic studies on the unfolding of prochymosin and pepsinogen indicate that the processes can be described by a single first-order rate constant, and that at any given value of denaturant concentration and pH the rate of unfolding of prochymosin is significantly greater than that of pepsinogen. 4. Unfolding of prochymosin by concentrated urea is not fully reversible, unlike that of pepsinogen. Kinetic analysis of the refolding of the proteins suggests the presence of a slow process following unfolding in urea; for pepsinogen this process leads to a slowly refolding form, whereas for prochymosin the slow process in urea leads to a form that cannot refold on dilution of the denaturant. 5. The results provide a rationale for an empirical process for recovery of recombinant prochymosin after solubilization of inclusion bodies in concentrated urea. 6. In all respects studied here, natural and recombinant bovine prochymosin were indistinguishable, indicating that the refolding protocol yields a recombinant product identical with natural prochymosin.

Animals↗
PubMed1990

Source of heterogeneity in secreted interferon-gamma. A study on products of translation in vitro.

A cDNA clone coding for human interferon-gamma (IFN-gamma) was subcloned into a transcription-translation vector. When the mRNA transcribed in vitro was added to a rabbit reticulocyte-lysate system, two polypeptides were synthesized: one corresponding in Mr to pre-IFN-gamma (18,000) and one with a lower Mr (12,000) which corresponds to a polypeptide arising from incorrect initiation of translation. When microsomal vesicles isolated from dog pancreas or Chinese-hamster ovary (CHO) cells were added to the translation system, translocation of the pre-IFN-gamma occurred, as judged by protection from exogenous proteinases. The resultant changes in the Mr of the translation products were indicative of signal-peptide cleavage and heterogeneous core glycosylation. When translation products were treated with N-glycanase, the higher-Mr products were no longer observed, consistent with removal of all oligosaccharide side chains, leaving a single core polypeptide. Glycosylation of the synthesized protein yielded both singly and doubly glycosylated products compatible with the glycosylation variants seen in secreted IFN-gamma. Quantitative differences were seen in the relative amounts of singly and doubly glycosylated products synthesized by dog pancreatic compared with CHO-derived microsomes. These data indicate that the relative amounts of IFN-gamma glycosylation variants are determined at an early stage in protein synthesis and that product variants may occur when IFN-gamma is expressed in cells derived from different tissues.

Animals↗
PubMed1990

Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.

Amino Acid Sequence↗
PubMed1990

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.

Animals↗
PubMed1989

Conformational differences between two wheat (Triticum aestivum) 'high-molecular-weight' glutenin subunits are due to a short region containing six amino acid differences.

'High-molecular-weight' (HMW, high-Mr) glutenin subunits are protein constituents of wheat (Triticum aestivum) seeds and are responsible in part for the viscoelasticity of the dough used to make bread. Two subunits, numbered 10 and 12, are the products of allelic genes. Their amino acid sequences have been derived from the nucleic acid sequences of the respective genes. Subunit 10 has fewer amino acids than subunit 12, but migrates more slowly on SDS/PAGE (polyacrylamide-gel electrophoresis). This anomaly is due to between one and six of the amino acid differences between the subunits, localized towards the C-terminal end of the proteins. This has been established by making chimaeric genes between the genes for subunits 10 and 12, transcribing and translating them in vitro and analysing the products by SDS/PAGE. The postulated conformational differences between subunits 10 and 12 are discussed in relation to current hypotheses for the structure of HMW glutenin subunits.

Amino Acid Sequence↗