Human tryptase-beta (HTbeta) is a serine protease with an atypical tetrameric structure and an unusual dependence on heparin binding or high salt for functional and structural stability. In the absence of heparin and at physiological salt, pH, and temperature, HTbeta rapidly loses activity by a reversible process that we have called spontaneous inactivation. The role of tetramer dissociation in this process is controversial. Using small irreversible or competitive inhibitors of HTbeta as stabilizing ligands, we were able to examine tetramer stability under inactivating (decay) conditions in the absence of heparin and to define further the process of spontaneous inactivation. Size exclusion chromatography showed that interaction with inhibitors stabilized the tetramer. Using sedimentation equilibrium, spontaneously inactivated HTbeta (si-HTbeta) was shown to be a destabilized tetramer that dissociates upon dilution and which in the presence of a competitive inhibitor re-formed a stable tetramer. Addition of inhibitors to si-HTbeta rescued catalytic activity as was shown after inhibitor displacement. At high concentrations of si-HTbeta (4-5 microM), the binding of inhibitor alone provided sufficient free energy for complete reactivation and tetramer stabilization, whereas at low si-HTbeta concentration (0.1 microM) where the destabilized tetramer would be mostly dissociated, reactivation required more free energy which was provided by the binding of both an inhibitor and heparin. The results demonstrate that HTbeta is a tetramer in the absence of heparin and that tetramer dissociation is a consequence of and not a prerequisite for inactivation. Heparin binding likely stabilizes the tetramer by favoring a functionally active conformation with stable intersubunit contacts, rather than by simply cross-linking active monomers.

The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.

Recombinant human tryptases (rHTs) corresponding to alpha and beta isoforms were characterized. rHTbeta was similar to tryptase isolated from skin (HST); it was a tetramer, hydrolyzed model substrates efficiently, and was functionally unstable when incubated under physiological conditions. Activity was lost rapidly (t(1/2) approximately 1 min) by a reversible process similar to that observed for the spontaneous inactivation of HST. Circular dichroism (CD) and intrinsic fluorescence emission (IFE) spectra of active rHTbeta corresponded to those of active HST and upon spontaneous inactivation IFE decreased in parallel to activity loss. rHTalpha differed from HST in catalytic ability and stability. rHTalpha did not react with model substrates, an active site titrant, or a competitive inhibitor of HST/rHTbeta. IFE and CD spectra were similar to those of the active and not the spontaneously inactivated form of HST. Under physiological conditions, rHTalpha IFE decreased at a rate 900-fold slower than that observed for HST, and rHTalpha remained tetrameric when examined by size exclusion chromatography at physiological salt concentration. Thus, rHTalpha is a stable "inactive" form of HT. Three active site variants of rHTalpha, K192Q, D216G, and K192Q-D216G were characterized. Residues 192 and 216 (chymotrypsinogen numbers for residues 191 and 215 of rHTalpha) lie at the entrance to the primary specificity (S1) pocket, and the mutations converted them to the residues of HTbeta. While K192Q displayed the same properties as rHTalpha, the catalytic and stability characteristics of D216G and K192Q-D216G progressively approached those of HST. Thus, the contrasting stability/activity properties of rHTalpha and rHTbeta are largely related to differences at the S1 pocket. On the basis of the properties of the variants, we suggest that the side chain of Asp216 is blocking and stabilizing the S1 pocket and that this stabilization is sufficient to prevent spontaneous inactivation.

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches, Detergent purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels, Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product, DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers, The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger; than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

Human skin tryptase, a serine proteinase stored within mast cell secretory granules, rapidly loses enzymatic activity in solutions of physiological salt concentration, pH, and temperature. The inactivation of tryptase can be slowed and even reversed by addition of heparin, a highly sulfated glycosaminoglycan also found in the secretory granules. These properties may be relevant to tryptase regulation after secretion from mast cells. To further characterize the molecular changes underlying the functional instability of tryptase, circular dichroism (CD) and analytical ultracentrifugation were used to investigate structural changes during spontaneous inactivation. The CD spectra of active and spontaneously inactivated tryptase are different, particularly in the region around 230 nm where active tryptase displays a distinct negative peak. This peak is also observed in the CD spectrum of bovine chymotrypsin but not in trypsin, elastase, or chymotrypsinogen. Loss of activity resulting from spontaneous inactivation was accompanied by a diminution of the 230-nm signal. The kinetics for the signal loss appeared to be first-order and closely paralleled the rate of enzymatic activity loss. Dextran sulfate, a highly sulfated polysaccharide, was capable of reactivating tryptase and restoring the CD signal. After 2 h of decay (> 90% loss of activity), addition of dextran sulfate resulted in an almost immediate return of the CD signal to that of active tryptase. The return of the CD signal appeared to be more rapid than the return of enzymatic activity, thereby suggesting the presence of an unidentified step which is rate-limiting for activity return (and loss) and subsequent (prior) to the CD change accompanying activity loss. Ultracentrifugation analysis of tryptase showed a marked change in its association state upon inactivation. Sedimentation equilibrium under stabilizing conditions demonstrated the presence of a single species with the molecular weight of a tetramer. After spontaneous inactivation, a mixture of species was evident, which was characterized as monomers and tetramers in equilibrium. These results demonstrate that spontaneous inactivation of tryptase is associated with reversible conformational changes and that a consequence of inactivation is the formation of a destabilized tetrameric form. Although the molecular mechanism initiating these changes remains unclear, possible insights into the process are discussed on the basis of the similarity between the CD spectra of tryptase and chymotrypsin.

The spontaneous loss of human tryptase hydrolytic activity was investigated. Time course studies monitoring the loss in catalytic activity were biphasic and correlated with a reduction in the concentration of catalytic sites. There was an initial rapid phase leading to greater than 85% loss in activity. The remaining activity gradually decayed toward completion over a 40-h period. The initial phase could be described as a first-order process with a t1/2 of approximately 6.0 min in 0.2 M NaCl (pH 6.8, 30 degrees C). The rate constant for this phase showed little, if any, sensitivity to changes in enzyme concentration, consistent with a first-order process, and analysis of the reaction as a function of temperature was consistent with a single rate-determining step. The rate of this process, however, showed marked sensitivity to changes in NaCl concentration and pH. Increasing the NaCl concentration as well as decreasing the pH below the pI (pH 6.3) reduced the rate of activity loss, whereas increasing the pH above pH 8.0 markedly increased the rate of activity loss. The effect of NaCl concentration and pH on the rate of activity loss suggests that the rate-limiting step governing the fast phase of the reaction involves electrostatic interactions. The presence of a fast and a slow phase in the decay process may suggest heterogeneity in the sample or the rapid formation of an inactive, but reversible, intermediate. A reversible intermediate was demonstrated when "inactivated tryptase" was incubated in the presence of heparin, and an increase in tryptase catalytic activity was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of Staphylococcus aureus V8 protease (V8) on the multimeric structure of human von Willebrand factor (vWF) were studied to test and expand our model for the substructure of vWF. Electron microscopy of V8 digests of vWF revealed that the multimers were cleaved where the flexible rod (R) domains join the large elongated globular (G) domains. The resulting two major fragments, which were purified by affinity and hydrophobic interaction chromatography and by glycerol-gradient ultracentrifugation, are disulfide-linked homodimers of these domains (i.e. RR and GG) and are morphologically identical to the alternating RR and GG domains of intact vWF. The glycoprotein fragment GG (6.5 X 35 nm) has mass 343 kDa by sedimentation equilibrium and the amino-terminal sequence of intact plasma vWF. It contains the binding site for heparin within 300 residues of its amino terminus and a separate site for the platelet GPIb receptor responsible for platelet agglutination in the presence of ristocetin. With approximately 18% alpha-helix and approximately 15% beta-pleated sheet, fragment GG accounts for most of the ordered secondary structure present in whole vWF. The two thin flexible rod domains (1.8-2.0 X 30-34 nm) of fragment RR are joined at a small central nodule (approximately 5 nm diameter) and also have a small nodule at each free end. Fragment RR contains an extraordinarily high cystine content, lower than average amounts of other hydrophobic residues, and essentially no alpha-helix, as judged by circular dichroism. The amino-terminal sequence and amino acid composition of fragment RR corresponded to that of the COOH-terminal 685 residues of the intact vWF subunit (Titani, K., Kumar, S., Takio, K., Ericsson, L. H., Wade, R. D., Ashida, K., Walsh, K. A., Chopek, M. W., Sadler, J. E., and Fujikawa, K. (1986) Biochemistry 25, 3171-3184). This sequence analysis gives a mass of 180 kDa for glycosylated fragment RR, somewhat higher than the 130 kDa we obtained by sedimentation equilibrium. Our sequence analysis of a 110-kDa plasmic vWF peptide also permitted identification of a major plasmin cleavage site 705 residues from the COOH terminus and a half-cystine residue (1360) involved in maintaining the multimeric structure of plasmin-degraded vWF.(ABSTRACT TRUNCATED AT 400 WORDS)

This chapter is concerned with the use of sedimentation equilibrium, a procedure that is generally applicable to proteins in the molecular weight range 10,000 to 500,000 and can be used conveniently for protein-amphiphile complexes without interference from non-protein-containing detergent micelles. This chapter presents a summary of the theoretical treatment of sedimentation equilibrium in a centrifugal field for systems containing two or more components and discusses the effects of specific interactions between the protein and solute molecules. It then proceeds to a description of experimental procedures and present examples of the application of this technique to protein-amphiphile complexes. Partial specific volumes are tabulated for amino acid residues, carbohydrates, and a number of detergents and lipids. Atomic and group partial molar volumes that are used to calculate partial specific volumes for chemical compounds are also provided. The chapter's consideration is the determination of protein molecular weight in a homogeneous system by a rigorous thermodynamic method.

The beta-adrenergic receptor from several tissues has been purified to homogeneity or photoaffinity radiolabeled and its subunit molecular weight determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. In this study we have examined the oligomeric structure of nondenatured beta 1- and beta 2-adrenergic receptor proteins, as solubilized with the detergent digitonin. Model systems used were frog and turkey red blood cell as well as rat, rabbit, and bovine lung plasma membrane preparations. To correct for the effects of detergent binding, sedimentation equilibrium analysis in various solvents, as adapted for the air-driven ultracentrifuge, was used. With this approach an estimate of 6 g of digitonin/g of protein binding was determined, corresponding to a ratio of 180 mol of digitonin/mol of protein. Protein molecular weights estimated by this method were 43 500 for the turkey red blood cell beta 1 receptor and 54 000 for the frog red blood cell beta 2 receptor. Molecular weights of 60 000-65 000 were estimated for beta 1 and beta 2 receptors present in mammalian lungs. These values agree with estimates of subunit molecular weight obtained by SDS gel electrophoresis of purified or photoradiolabeled preparations and suggest beta-adrenergic receptors to be digitonin solubilized from the membrane as single polypeptide chains.