A T cell receptor V alpha domain expressed in bacteria: does it dimerize in solution?

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.

The orphan nuclear receptor Ear-2 is a negative coregulator for thyroid hormone nuclear receptor function.

Thyroid hormone (T3) nuclear receptors (TR) are ligand-dependent transcription factors which regulate growth, differentiation, and development. One emerging hypothesis suggests that TR mediate these diverse effects via a large network of coregulators. Recently, we found that TR-mediated transcriptional responses varied in six cell lines derived from different tissues. We therefore used human TR subtype beta1 (TRbeta1) as bait to search for coregulators in human colon carcinoma RKO cells with a yeast two-hybrid system. RKO cells exhibited T3-dependent and -independent transcriptional activation. One of the three positive clones was identified as Ear-2, which is a distant member of the chick ovalbumin upstream promoter-transcription factors of the orphan nuclear receptor family. The physical interaction between Ear-2 and TRbeta1 was further confirmed by specific binding of Ear-2 to glutathione S-transferase-TRbeta1. In addition, Ear-2 was found to associate with TRbeta1 in cells. As a result of this physical interaction, binding of TRbeta1 to the T3 response elements was inhibited. Using reporter systems, we found that both the basal activation and the T3-dependent activation mediated by TRbeta1 were repressed by Ear-2 in CV1 cells. In RKO cells, however, the T3-independent transcriptional activity was more sensitive to the repression effect of Ear-2 than the T3-dependent transcriptional activity. The repression effect of Ear-2 was reversed by steroid hormone receptor coactivator 1. These results suggest that TR-mediated responses reflect a balance of corepressors and coactivators in cells. These findings further strengthen the hypothesis that the diverse activities of TR are achieved via a large network of coregulators that includes Ear-2.

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.

Physical characterization of histidine-rich protein from Plasmodium lophurae.

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43,000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97-120) predicted that 82% of its residues would be found in three long alpha-helices. The protein's CD spectrum has a strong resemblance to that of poly(L-histidine) at pH 4-5, where the homopolymer is thought to be in a right-handed alpha-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

On the one-sided action of amphotericin B on lipid bilayer membranes.

The one-sided action of the polyene antibiotic, amphotericin B, on phospholipid bilayer membranes formed from synthetic phosphatidylcholines (DOPC and DPhPC) and sterols (ergosterol and cholesterol), has been investigated. We found formation of well-defined ionic channels for both sterols and not only for ergosterol-containing membranes (Bolard, J., P. Legrand, F. Heitz, and B. Cybulska. 1991. Biochemistry. 30:5707-5715). Characteristics of these channels were studied in the presence of different salts. It was found that the channels have comparable conductances but different lifetimes that are approximately 100-fold less in cholesterol-containing membranes than in ergosterol-containing ones. Channel blocking by tetraethylammonium (TEA) ions shows that TEA blockage of channels in the presence of cholesterol increases their lifetimes in analogy to the lengthening of lifetimes of protein channels blocked by local anesthetics (Neher, E., and J. H. Steinbach. 1978. J. Physiol. 277: 153-176). However, the effect of the blocker on single-channel conductance is very close for both sterols. The data support the classical model of amphotericin B pore formation from complexes initially lying on the membrane surface as nonconducting prepores. We explain the antibiotic's cytotoxic selectivity by differences in the lifetimes of the channels formed with different sterols and suggest that phosphatidylcholine-sterol membranes can be used as a tool for rapid estimation of polyene antibiotic cytotoxicity.

CRP-DNA complexes: inducing the A-like form in the binding sites with an extended central spacer.

The consensus DNA sequence for binding of the Escherichia coli cyclic AMP receptor protein (CRP) has two symmetrically related inverted recognition elements TGTGA:TCACA, separated by a variable spacer, normally 6 bp long. We have shown that the CRP-cAMP complex, when bound to synthetic binding sites with an extended 8 bp spacer segment, induces an increase in the DNA circular dichroism (CD). The CD change at lambda > 275 nm agrees with the shift of approximately one helical turn of DNA into A-like form. The B-conformation is preserved for CRP binding sites similar to that in the lac and uxaCA promoters with 6 bp spacers. Another effect accompanying DNA binding is a dramatic increase of the negative CD magnitude in the spectral region of the ligand cAMP, at lambda < 272 nm. This effect is observed when CRP binds to specific sites with 6 or 8 bp spacers as well as to non-specific DNA. We reason that the A-like form arises by compressing and unwinding the DNA in CRP-DNA complexes having 8 bp central spacers. This serves to maintain a fixed length and twisting angle and is controlled by the protein's relatively rigid frame. This model is consistent with the observation that some binding sites with 6 bp spacers may also show the CD increase inherent to the sites with the extended 8 bp spacers. These 6 bp spacers are characterized by an increased twisting angle that requires their unwinding to bind to CRP. We propose that a mutual adaptation between CRP and binding sites by local untwisting and a B-->A-like transition in the DNA is of general importance and may occur in other protein-DNA complexes, such as the complex of RNA polymerase with promoter DNA.

An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor.

By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of thyroid hormone receptor isoform homodimers and mutant heterodimers to hormone-induced dissociation from deoxyribonucleic acid: its role in dominant negative action.

General resistance to thyroid hormone is an inheritable disease with resistance of peripheral tissues to elevated levels of thyroid hormone. Genetic studies have shown that it is due to interference in the functions of wild-type thyroid hormone nuclear receptors (wTRs) via the dominant negative effect of mutant TRs (mTRs). The present study compared the heterodimerization of the two TR isoforms, TR beta1 and TR alpha1, with mutant TRs to understand if mTRs had isoform-dependent dominant negative action. Using electrophoresis gel mobility shift assay, we have demonstrated that mutant PV, S, ED, and OK form heterodimers with wTR alpha1 and deltaTR beta1 (in which the A/B domain of wTR beta1 has been deleted), on the F2-thyroid hormone response element (TRE). In the presence of T3, both homo- and heterodimer complexes are dissociated in a T3 concentration dependent manner. The ED50 for deltaTR beta1 homodimers was 3-fold higher than that of wTR alpha1 homodimers. ED50s for deltaTR beta1/mTR heterodimers were 10- to 40-fold higher than the corresponding wTR alpha1/mTR heterodimers. Mutant ED and OK homodimers were only partially dissociated at the highest T3 concentrations used (100 nM), whereas no dissociation could be detected for PV and S homodimers, indicating differential sensitivity of the F2-bound TR dimers to the T3-induced dissociation. We presented a model that indicates the dissociation of any particular TR dimer from F2 is determined by competition of T3 for both of its constituent TRs. By transfection assays, we showed that the potency of the dominant negative action of PV on TR alpha1 and TR beta1 inversely correlated with the sensitivity of the appropriate mTR/wTR heterodimer to T3-induced dissociation from F2. The differential dominant negative action of mutants on the two TR isoforms could play an important role in the heterogeneity of tissue-specific manifestations in patients with resistance to thyroid hormone.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild-type/mutant receptor heterodimer.

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Dominant negative activity of mutant thyroid hormone alpha1 receptors from patients with hepatocellular carcinoma.

Complementary DNAs for two mutant thyroid hormone alpha1 receptors (TR alpha1) were isolated from hepatocellular carcinomas of two patients. Sequence analyses of the complementary DNAs showed a single Val390Ala and double Pro398Ser/Glu350Lys mutations in mutants H and L, respectively. We characterized their hormone-binding, DNA-binding, and dominant negative activities. Mutants H and L did not bind the hormone T3. Their DNA-binding activities were analyzed using three types of thyroid hormone response elements (TREs) in which the half-site binding motifs are arranged in an everted repeat (Lys), an inverted repeat (Pal), or a direct repeat separated by four nucleotides (DR4). Compared with wild-type TR alpha1 (w-TR alpha1), which bound these TREs with different homodimer/monomer ratios, binding of mutant L to the three TREs as homodimers was reduced by approximately 90%. However, binding of mutant H to these TREs was more complex. Although it bound normally to DR4 as homodimers, its binding to Lys as homodimers was reduced by approximately 80%. Surprisingly, its binding to Pal was markedly enhanced compared with w-TR alpha1. The binding of these two mutants to the three TREs as heterodimers with retinoid X receptors (RXR alpha and -beta) was not significantly affected. Consistent with the lack of T3-binding activity, both mutants had lost their trans-activation capacity. Mutants H and L exhibited dominant negative activity, but differed in their TRE dependency. The dominant negative potency of mutant H was in the rank order of Pal > DR4 > Lys, whereas no TRE dependency was observed for mutant L. The present study indicates that mutations of the TR alpha gene do occur in patients and that these novel TR alpha1 mutants provide a valuable tool to further understand the molecular basis of the dominant negative action of mutant TRs.

Identification of naturally occurring dominant negative mutants of thyroid hormone alpha 1 and beta 1 receptors in a human hepatocellular carcinoma cell line.

To understand the function of thyroid hormone nuclear receptors (TRs) in human hepatocellular carcinoma cells (HCC), we characterized the hormone binding and transactivational activity of TRs in a HCC cell line, J7. TR alpha 1 (J7-TR alpha 1) and TR beta 1 (J7-TR beta 1) complementary DNAs were cloned from this cell line, and the binding activity to the hormone response elements (TREs) and to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3) of the expressed TR proteins were evaluated. J7-TR alpha 1 and J7-TR beta 1 bound to TREs similarly as the TRs isolated from other tissues. However, J7-TR alpha 1 did not bind to T3, and J7-TR beta 1 bound to T3 with only about 10% the affinity of the wild-type TR beta 1. Sequencing of the complementary DNAs shows a single Met259Ile mutation in J7-TR alpha 1 and Met334Val in J7-TR beta 1. Using reporters containing TREs, we found that J7-TR alpha 1 and J7-TR beta 1 had virtually lost their transactivational activity. Moreover, these two mutants inhibited the transactivational activity of the wild-type TRs by a dominant negative effect not only on the transfected TRs, but also on endogenous TRs in other two HCC cell lines, SK-Hep-1 and HepG2. The potency of the dominant negative effect of these two mutants inversely correlated with the expression level of endogenous TRs. The present studies identified two novel naturally occurring TR mutants that have potent dominant negative action. The identification of both the alpha and beta dominant negative mutants in J7 made this cell line a useful model system to further understand the molecular mechanism of the dominant negative action of TR mutants.

Purification and partial characterization of an unusual protein of Plasmodium falciparum: histidine-rich protein II.

The human malarial parasite Plasmodium falciparum secretes a histidine-rich protein (HRP-II) from infected erythrocytes. HRP-II has a very high content of histidine (H) (34%), alanine (A) (37%) and aspartic acid (D) (10%) and many contiguous repeats of the sequences AHH and AHHAAD. The histidine content of the protein suggested the potential to bind metal ions. We have demonstrated by metal chelate chromatography an extraordinary capacity of HRP-II to bind zinc ions (Zn2+) and employed this characteristic to isolate the extracellular protein. The HRP-II was further purified by antibody affinity chromatography. The identity of the purified protein was verified by relative molecular weight on denaturing polyacrylamide gels, by reactivity with monoclonal antibodies and monospecific rabbit antiserum, and by comparison of the amino-acid analysis with that derived from the cloned gene sequence. Analysis of the sequence for periodicities using the hydrophobic moment method indicated that HRP-II may potentially form a 3/10 helix. Immunoprecipitation of HRP-II from culture supernatants of parasites metabolically labeled with tritiated sugars showed that the extracellular form of HRP-II is a glycoprotein containing galactose.

The alkaline transition of swine pepsinogen.

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.

The alkaline transition of swine pepsinogen.

At alkaline pH, swine pepsinogen is reversibly inactivated in a transition which involves the cooperative release of two protons from the molecule and is governed by a pK = 9. Stopped flow kinetic studies on the absorbance changes accompanying this reaction show that it can be resolved into two steps, with increasing pH; a slow conformational change, whose amplitude follows the ionisation curve of one group of pK = 9.9, followed by a rapid pH dependent conformational change, linked to a group of pK = 8.2. The pH dependence of the rate of the slow step is interpreted to show the presence of a protonated group which cannot ionise in the neutral form of the zymogen, but is in slow equilibrium with a form where it titrates with a pK = 6.8. At the same time, a histidine in the amino terminal region of the protein becomes reactive to diethyl pyrocarbonate, suggesting this to be the group which triggers the reaction.

A review of the effects of manipulation of the cysteine residues of rat aryl sulfotransferase IV.

Aryl sulfotransferase IV from rat liver has the broad substrate range that is characteristic of the enzymes of detoxication. With the standard assay substrates, 4-nitrophenol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), sulfation is optimum at pH 5.4 whereas the reaction is minimal in the physiological pH range. These properties preclude a physiological function for this cytosolic enzyme. Partial oxidation of the enzyme, however, results not only in an increase in the rate of sulfation but also in a shift of the pH optimum to the physiological pH range. The mechanism for this dependence on the redox environment involves oxidation at Cys66, the cysteine residue that is conserved throughout the phenol sulfotransferase family. As documented by mass spectroscopic methods, oxidation by GSSG leads to the formation of an internal disulfide between Cys66 and Cys232; for mutants at Cys232, the oxidation product is a mixed disulfide of Cys66 and glutathione. Both of these disulfide species activate the enzyme and allow it to function at a pH optimum in the physiological range. The activated enzyme differs from the reduced form by a more circumscribed substrate spectrum. All five mutants, in which each of the cysteines of the sulfotransferase subunit have been changed to serine, are catalytically active. Only Cys66 is required for the redox response.

Changes in substrate specificity of the recombinant form of phenol sulfotransferase IV (tyrosine-ester sulfotransferase).

The over-expression of mammalian enzymes in bacterial systems by means of recombinant DNA technology has provided the enzymologist with a supply of catalyst sufficiently abundant to identify suboptimal substrates. Such large quantities are particularly useful when working with the enzymes of detoxication, a family of proteins that are distinguished by their broad substrate specificity for generally lipophilic compounds, i.e., by their very low specificity for features other than the functional group [1]. We have achieved bountiful expression of a sulfotransferase active with phenols [2], an enzyme originally purified and characterized from rat liver [3], and classified as tyrosine-ester sulfotransferase, EC 2.8.2.9 [4,5], but usually referred to as rat liver phenol or aryl sulfotransferase IV. Having improved the sensitivity and versatility of some of the assays for sulfotransferases, we examined the substrate spectrum of this enzyme. As presented here, the results of this examination point to the limitations of enzyme nomenclature and to the danger of equating enzymes isolated from their normal habitat with those formed by recombinant technology in a foreign cell. Our experiments also establish a greater catalytic scope for the natural rat liver enzyme than that previously described.

Effect of burn patient serum on fibroblast and keratinocyte cell morphology.

The effect of burn patient serum on fibroblast and keratinocyte cell morphology in culture was investigated using the scanning electron microscope. Serum was taken from five patients with burn injuries ranging from 8 to 65 per cent TBSA (10-65 per cent full-thickness). One patient had superficial burns. Pooled serum from 23 volunteers was used as the control serum. The cells were seeded onto collagen-coated glass coverslips and incubated for 5 days with culture medium containing 10 per cent (v/v) control serum or patient serum taken during the early postburn period. Scanning electron micrographs demonstrated a reduction in fibroblast cell density with serum from patients with full-thickness burns. Furthermore, the spindle shape of the fibroblast cell was greatly exaggerated compared with control cultures. The integrity of the keratinocyte sheet was destroyed when keratinocyte cells were incubated with serum fron patients with full-thickness burns. Globular-like structures or membrane protrusions were present in concentrated areas on keratinocyte cells which were not present in control cultures. This study demonstrated the vulnerability of cutaneous cells to systemic factors present in the early postburn serum. The extent of the effect appears to be related to the presence of full-thickness injury. This effect may further explain the frequent aberrant wound healing response to burn injury.

The cellular mechanism of ossicular erosion in chronic suppurative otitis media.

This is the first report of the application of a new examination technique for the assessment of cellular activity during bone resorption in chronic suppurative otitis media (CSOM). A total of nineteen includes removed during the course of tympanomastoid surgery were studied (retraction pocket: 2; tubo-tympanic CSOM: 4; attico-antral CSOM: 13). The microscopic surface topography of each specimen was examined using the scanning electron microscopy (SEM), and the appearances are interpreted in terms of cortical cellular activity. The results suggest that the mechanism of ossicular erosion in CSOM is similar regardless of the exact type of disease. Extensively pitted areas were seen in all specimens. These pits are morphologically indistinguishable from those characteristic of osteoclastic activity (Howship's lacunae). We conclude that in all causes the surface topography of eroded incudes is consistent with the activity of osteoclasts.

A reversible unfolding reaction of swine pepsin; implications for pepsinogen's folding mechanism.

Above pH 6, swine pepsin undergoes a conformational change to a neutral form which has 80% of the secondary structure of the native protein. In contrast to native pepsin, this form of the enzyme can be reversibly unfolded by urea in a rapid, cooperative reaction. Since all of pepsin's sequence is present in its precursor pepsinogen, it is likely that this neutral structure is present in one or more of the transient intermediates previously detected in the reversible unfolding reaction of the zymogen. The mechanism of this rapid reaction may resemble early steps in protein folding.

On the apparent inhibition of intramolecular activation of pepsinogen by pepsin substrates.

Marciniszyn et al. (Marciniszyn, J., Huang, J. S. Hartsuch, J. A., Tang, J. (1976) J. Biol. Chem. 251, 7095-7102) have recently suggested an intermediate in the intramolecular activation of pepsinogen. As evidence, they showed apparent competitive inhibition of activation by globin, indication a pepsinogen-globin complex. Previous work had shown pepsinogen activation to occur very rapidly in the presence of high concentrations of hemoglobin, a very similar pepsin substrate (McPhie, P. (1974) Biochem. Biophys. Res. Commun. 56, 789-792). This contradiction has been resolved by a re-evaluation of the techniques used in the two investigations. The experimental conditions of Marciniszyn et al. Were inadequately defined to ensure denaturation of pepsin, a prerequisite of their method. A small decrease in pH, caused by the presence of extraneous protein, prevents this denaturation and leads to consistent underestimates of the rate of zymogen activation.