Determination of successful reperfusion after thrombolysis for acute myocardial infarction: a noninvasive method using ultrasonic tissue characterization that can be applied clinically.

BACKGROUND: The aim of the present study was to determine the use of cyclic variation in ultrasonic integrated backscatter (IBS), which is reduced in ischemic myocardium, to predict an occluded infarct-related artery (IRA) after thrombolysis for acute myocardial infarction (AMI). This is important, because patency of the IRA 90 minutes after thrombolysis has been shown to predict outcome. METHODS AND RESULTS: One hundred thirteen patients with AMI had peak-to-peak cyclic IBS measured in the myocardial territory supplied by their IRA as well as a remote territory with normal function from the parasternal long- or short-axis view. This analysis took 5 to 10 minutes. Wall motion score index was assessed, and coronary angiography, to determine patency of the IRA, was performed in all patients. Cyclic IBS in the IRA territory was much lower in segments supplied by an occluded IRA (3.3 versus 4.6 dB, P<0.00001). Using a difference in cyclic IBS between infarcted and normal segments of 15% (or 1.5 dB) as a cutoff, the sensitivity, specificity, positive and negative predictive values to determine an occluded IRA were 92%, 75%, 81%, and 89%, respectively. CONCLUSIONS: The difference in cyclic IBS between IRA and remote normal segments, which can be analyzed rapidly, can be used to predict patency of the IRA in patients with AMI. This provides a noninvasive method to determine those patients who may require urgent invasive investigation.

Synthesis and in vitro evaluation of an 111In-labeled ST-peptide enterotoxin (ST) analogue for specific targeting of guanylin receptors on human colonic cancers.

BACKGROUND: Human colonic cancer cells are known to express guanylate cyclase C (GC-C) receptors for guanylin and uroguanylin. E. coli ST is a peptide with high metabolic stability that specifically binds to GC-C receptors. An in vitro evaluation of a new synthetic indium-111 labeled ST conjugate for specific targeting of human colonic cancers that express GC-C receptors was performed. MATERIALS AND METHODS: A DOTA conjugated ST analogue DOTA-NCS-6-Ahx-Phe19-ST[1-19] (DOTA-NCS-ST) was synthesized and labeled with indium-111. The non-radioactive indium analogue (In-DOTA-NCS-ST) was also prepared in macroscopic quantities. 111In-DOTA-NCS-ST was produced as a single species (>80% RCP) and purified by HPLC. Human colon cancer CaCO-2 and T-84 cells were used to evaluate the in vitro IC50 values for GC-C receptor binding and determine the cell uptake and retention of radioactivity. RESULTS: The DOTA-NCS-ST and In-DOTA-NCS-ST conjugates exhibit high in vitro binding affinity for GC-C receptors with IC50 values <10 nM. The in vitro cell binding studies with the 111In-DOTA-NCS-ST conjugate demonstrated that 111In-label ST internalizes in human colon cancer cells and exhibits long-term retention. CONCLUSION: The combination of radiolabeling efficacy and specific in vitro cell uptake and retention suggests that the DOTA-NCS-ST construct holds potential for the development of diagnostic or therapeutic radiopharmaceuticals labeled with trivalent radiometals for specific targeting of human colonic cancers.

In vivo evaluation of an 111In-labeled ST-peptide analog for specific-targeting of human colon cancers.

In vitro competitive binding studies of In-DOTA-NCS-6-Ahx-Phe(19)-ST[1-19] vs. 125I-Tyr(5)-6-Ahx-Phe(19)-ST[1-19] with guanylate cyclase -C (GC-C) receptors on human colon cancer LS-180 cells revealed an IC(50) value of 7.7 +/- 0.1.6 nM. The in vitro cellular residualization studies of the 111In-DOTA-NCS-ST peptide and GC-C receptor mediated stimulated cGMP production with LS-180 cells demonstrates that this peptide selectively binds to LS-180 cells in an agonistic fashion. In vivo biodistribution studies in LS-180 tumor bearing SCID mice demonstrates that the 111In-DOTA-NCS-ST peptide targets the tumor with a specific uptake of 0.94 +/- 0.31%ID/g at 1 hr p.i. and approximately 23% was retained by the tumor at 4 hrs p.i. The radioactivity cleared rapidly from the blood stream with 84.5 +/- 3.4%ID at 1h p.i. found in the urine. High activity in urine and kidney, and minimal activity in liver and intestines, demonstrates preferential clearance of the radioactivity through the renal/urinary pathway. The specific in vitro and in vivo accumulation of the radioactivity by LS-180 human colonic cancer cells highlights the potential of radiometallated-DOTA-ST analogs as diagnostic/therapeutic radiopharmaceuticals.

Lymphoguanylin: cloning and characterization of a unique member of the guanylin peptide family.

Guanylin and uroguanylin are small peptides containing two disulfide bonds that activate membrane guanylate cyclase-receptors in the intestine, kidney and other epithelia. Hybridization assays with a uroguanylin complementary DNA (cDNA) detected uroguanylin-like messenger RNAs (mRNAs) in the opossum spleen and testis, but these transcripts are larger than uroguanylin mRNAs. RT of RNA from spleen to produce cDNAs for amplification in the PCR followed by cloning and sequencing revealed a novel lymphoid-derived cDNA containing an open reading frame encoding a 109-amino acid polypeptide. This protein shares 84% and 40% of its residues with preprouroguanylin and preproguanylin, respectively. A 15-amino acid, uroguanylin-like peptide occurs at the COOH-terminus of the precursor polypeptide. However, this peptide is unique in having only three cysteine residues. We named the gene and its peptide product lymphoguanylin because the source of the first cDNA isolated was spleen and its mRNA is expressed in all of the lymphoid tissues tested. A 15-amino acid form of lymphoguanylin containing a single disulfide bond was synthesized that activates the guanylate cyclase receptors of human T84 intestinal and opossum kidney (OK) cells, although with less potency than uroguanylin and guanylin. Northern and/or RT-PCR assays detected lymphoguanylin mRNA transcripts in many tissues and organs of opossums, including those within the lymphoid/immune, cardiovascular/renal, reproductive, and central nervous organ systems. Lymphoguanylin joins guanylin and uroguanylin in a growing family of peptide agonists that activate transmembrane guanylate cyclase receptors, thus influencing target cell function via the intracellular second messenger, cGMP.

Late-infantile ceroid-lipofuscinosis: lysine methylation of mitochondrial ATP synthase subunit c from lysosomal storage bodies.

Late-infantile ceroid-lipofuscinosis is a fatal autosomal recessively inherited disease characterized by massive accumulations of lysosomal storage bodies in many tissues. A major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase. Juvenile ceroid-lipofuscinosis, a disease that is similar to but genetically distinct from the late-infantile disorder, also involves lysosomal accumulation of the subunit c protein. In the juvenile disease, the stored form of the protein contains an epsilon-N-trimethyllysine (TML) residue at position 43. Analyses were performed to determine whether subunit c protein stored in the late-infantile disease is also trimethylated at lysine residue 43. Amino acid composition analysis of the subunit c protein stored in brains from subjects with the late-infantile disease indicated that one of the two lysine residues in the protein is trimethylated. Data from molecular mass analysis of the protein was consistent with the presence of three methyl groups not present in the unmodified protein. The TML in the storage body subunit c protein was found by amino acid sequence analysis to occur exclusively at residue 43. The lysine at this position in the stored protein was completely methylated. Recent studies suggest that the subunit c protein from normal mitochondria may also have the same amino acid modification. Thus, it appears that specific methylation of lysine residue 43 of mitochondrial ATP synthase subunit c is probably a normal post-translational modification, and that the lysosomal storage of this protein in late-infantile, as well as in juvenile ceroid-lipofuscinosis, does not result from a defect in its methylation.

Regulation of intestinal uroguanylin/guanylin receptor-mediated responses by mucosal acidity.

Guanylin and uroguanylin are intestinal peptides that stimulate chloride secretion by activating a common set of receptor-guanylate cyclase signaling molecules located on the mucosal surface of enterocytes. High mucosal acidity, similar to the pH occurring within the fluid microclimate domain at the mucosal surface of the intestine, markedly enhances the cGMP accumulation responses of T84 human intestinal cells to uroguanylin. In contrast, a mucosal acidity of pH 5.0 renders guanylin essentially inactive. T84 cells were used as a model epithelium to further explore the concept that mucosal acidity imposes agonist selectivity for activation of the intestinal receptors for uroguanylin and guanylin, thus providing a rationale for the evolution of these related peptides. At an acidic mucosal pH of 5.0, uroguanylin is 100-fold more potent than guanylin, but at an alkaline pH of 8.0 guanylin is more potent than uroguanylin in stimulating intracellular cGMP accumulation and transepithelial chloride secretion. The relative affinities of uroguanylin and guanylin for binding to receptors on the mucosal surface of T84 cells is influenced dramatically by mucosal acidity, which explains the strong pH dependency of the cGMP and chloride secretion responses to these peptides. The guanylin-binding affinities for peptide-receptor interaction were reduced by 100-fold at pH 5 versus pH 8, whereas the affinities of uroguanylin for these receptors were increased 10-fold by acidic pH conditions. Deletion of the N-terminal acidic amino acids in uroguanylin demonstrated that these residues are responsible for the increase in binding affinities that are observed for uroguanylin at acidic pH. We conclude that guanylin and uroguanylin evolved distinctly different structures, which enables both peptides to regulate, in a pH-dependent fashion, the activity of receptors that control intestinal salt and water transport via cGMP.

Prouroguanylin and proguanylin: purification from colon, structure, and modulation of bioactivity by proteases.

Uroguanylin and guanylin are peptides isolated from urine and intestinal mucosa, which regulate cyclic GMP production in enterocytes by activating an apical membrane, receptor-guanylate cyclase. This study extended our previous findings, which showed that colonic mucosa of opossums contained uroguanylin and guanylin peptides, by purifying prouroguanylin and proguanylin from this tissue. Prouroguanylin and proguanylin coeluted from Sephadex G-75 gelfiltration columns with a similar molecular size between 6 and 12 kDa. Mass spectrometry indicated that proguanylin (approximately 8.7 kDa) had a 10% lower molecular mass than prouroguanylin (approximately 9.7 kDa). Isoelectric focusing separated prouroguanylin (pI approximately 4.5) from proguanylin (pI approximately 7.5). N-terminal sequence analysis of reverse phrase-HPLC purified prohormones revealed 13 amino acids in opossum proguanylin that shared 77-85% identity with human and rat proguanylin, but only 23% identity with opossum prouroguanylin. The N-terminal 19 residues obtained for opossum prouroguanylin shared 32-42% identity with rat and human proguanylin. Prouroguanylin and proguanylin were both inactive and required pretreatment with proteases to elicit cyclic GMP responses in T84 cells. V8 protease treatment of proguanylin liberated a bioactive, 16-amino acid form of guanylin. Chymotrypsin treatment activated prouroguanylin, but inactivated the bioactive peptide domain within proguanylin. In summary, colonic mucosa contains the bioactive peptide and inactive prohormone forms of uroguanylin and guanylin. Thus, after proteolytic processing of prouroguanylin and proguanylin, bioactive uroguanylin and guanylin could both function to regulate guanylate cyclase activity by autocrine and/or paracrine actions on enterocytes. Also, these peptide hormones are implicated in an intestinal-renal axis for the endocrine regulation of salt and water homeostasis.

Mitochondrial ATP synthase subunit c stored in hereditary ceroid-lipofuscinosis contains trimethyl-lysine.

The subunit c protein of mitochondrial ATP synthase accumulates in lysosomal storage bodies of numerous tissues in human subjects with certain forms of ceroid-lipofuscinosis, a degenerative hereditary disease. Subunit c appears to constitute a major fraction of the total storage-body protein. Lysosomal accumulation of subunit c has also been reported in putative animal models (dogs, sheep and mice) for ceroid-lipofuscinosis. In humans with the juvenile form of the disease, hydrolysates of total storage-body protein have been found to contain significant amounts of epsilon-N-trimethyl-lysine (TML). TML is also abundant in storage-body protein hydrolysates from affected dogs and sheep. These findings suggested that one or both of the two lysine residues of subunit c might be methylated in the stored form of the protein. The normal subunit c protein from mitochondria does not appear to be methylated. In a putative canine model for human juvenile ceroid-lipofuscinosis, residue 43 of the storage-body subunit c was previously found to be TML. In the present study, subunit c was isolated from the storage bodies of humans with juvenile ceroid-lipofuscinosis, and from sheep and mice with apparently analogous diseases. In all three species, partial amino acid sequence analysis of the stored subunit c indicated that the protein contained TML at residue 43. These findings strongly suggest that specific methylation of lysine residue 43 of mitochondrial ATP synthase plays a central role in the lysosomal storage of this protein.

Adrenaline-responsive electromechanical dissociation.

Electromechanical dissociation (EMD) occurs when there is no detectable blood pressure in the presence of organised electrocardiographic activity. This condition excludes pulseless ventricular tachycardia, and traditionally was thought to result from a failure of electrical depolarisation in the myocardium to elicit mechanical contraction. It is now understood from echocardiographic evidence that 'pseudo-EMD' is more common-where electrical activity is associated with myocardial contractions too weak to sustain a detectable blood pressure [1]. Three cases of EMD in association with subarachnoid haemorrhage (SAH) are described. This form of cardiovascular collapse appears to respond readily to the use of adrenaline. To our knowledge, this has not been reported previously. We suggest that this form of EMD occurs despite adequate myocardial function and results from sympathetic failure with loss of peripheral vascular resistance.

Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase.

The intestinal hormone guanylin and bacterial heat-stable enterotoxins (STs) are members of a peptide family that activates intestinal membrane guanylate cyclase. Two different peptides that activate the human intestinal T84 cell guanylate cyclase have been purified from urine and intestinal mucosa of opossums (Didelphis virginiana). The highly acidic peptide, QEDCELCINVACTGC, was named uroguanylin because it was isolated from urine and shares 53% identity with guanylin. A second peptide, SHTCEICAFAACAGC, was purified from urine and intestinal mucosa. This alanine-rich peptide was 47% identical to uroguanylin and 73% identical to human guanylin, suggesting that it may be an opossum homologue of guanylin. Synthetic uroguanylin-(2-15) (i.e., EDCELCINVACTGC) was 10-fold more potent than synthetic rat guanylin, but both peptides were less potent than Escherichia coli ST in the T84 cell cGMP bioassay. Uroguanylin-(2-15) and guanylin inhibited 125I-ST binding to T84 cell receptors in competitive radioligand binding assays. Transepithelial Cl- secretion was stimulated by 1 microM uroguanylin, indicated by an increase in the short circuit current of T84 cells. Thus, uroguanylin is another paracrine hormone in the emerging peptide family that activates intestinal membrane guanylate cyclase. The second peptide may be the opossum form of guanylin, or perhaps, it is still another member of this peptide family. The presence of uroguanylin and guanylin in urine and receptors in proximal tubules suggests that these peptides may also originate from renal tissue and may regulate kidney function.

Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase). Similarities to IgE binding proteins and the S-type animal lectin superfamily.

We have isolated and sequenced a 598-bp full length cDNA clone for the human Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase), the unique and prominent constituent of human eosinophils and basophils that forms the hexagonal bipyramidal crystals classically observed in tissues and secretions from sites of eosinophil-associated inflammation. A 426-bp open reading frame encoded a 142-amino acid polypeptide with a predicted molecular mass of 16.5 kDa and isoelectric point of 7.28. The deduced amino acid sequence of CLC protein showed 20 to 30% similarity over regions of approximately 100 amino acids with the carboxyl-terminal domains of four IgE-binding proteins, including the 31-kDa human and rat IgE-binding proteins, the 35-kDa mouse carbohydrate binding protein (CBP35), Mac-2, the murine macrophage cell surface protein that is identical to CBP35, and the human homologue of Mac-2. These proteins are members of a superfamily of beta-galactoside binding S-type animal lectins, which includes a group of highly conserved 14-kDa lectins isolated from human lung, heart, placenta, bovine heart, chicken skin, mouse fibroblasts, and the electric organ of the electric eel; CLC protein also showed sequence similarities to these 14-kDa animal lectins, including conservation of 7 of 16 invariant amino acid residues thought to comprise the carbohydrate-binding domain of these proteins, with conservative amino acid changes at others; thus, CLC protein could potentially possess carbohydrate or IgE-binding activities. Northern analyses revealed an approximately 900-bp mRNA species that was present in peripheral blood eosinophils from patients with eosinophilia, basophils from patients with chronic myelogenous leukemia, and in HL-60 cells induced towards eosinophilic differentiation with B cell growth factor-II (IL-5) or granulocytic differentiation with DMSO, but was absent in neutrophils, monocytes, T cells, B cells, or HL-60 cells induced towards monocytic differentiation with vitamin D3. Southern analyses revealed a gene of approximately 5 to 6 kb in length. The cDNA clone and complete amino acid sequence data for CLC protein will facilitate structure-function analyses of its unusual hydrophobic properties, unique propensity for crystallization, lysophospholipase, and potential lectin-like activities.

Effects of sulfate ions on Alzheimer beta/A4 peptide assemblies: implications for amyloid fibril-proteoglycan interactions.

To model the possible involvement of sulfated proteoglycans in amyloidogenesis, we examined the influence of sulfate ions, heparan, and Congo red on the conformation and morphology of peptides derived from the Alzheimer beta/A4 amyloid protein. The peptides included residues 11-28, 13-28, 15-28, and 11-25 of beta/A4. Negative-stain electron microscopy revealed a sulfate-specific tendency of the preformed peptide fibrillar assemblies of beta(11-28), beta(13-28), and beta(11-25), but not beta(15-28), to undergo extensive lateral aggregation and axial growth into "macrofibers" that were approximately 0.1-0.2 micron wide by approximately 20-30 microns long. Such effects were observed at low sulfate concentrations (e.g., 5-50 mM) and could not be reproduced under comparable conditions with Na2HPO4, Na2SeO4, or NaCl. Macrofibers in NaCl were only observed at 1,000 mM. At physiological ionic strength of NaCl, fibril aggregation was observed only with addition of sulfate ions at 5-50 mM. Selenate ions, by contrast with sulfate ions, induced only axial and not substantial lateral aggregation of fibrils. X-ray diffraction indicated that the original cross-beta peptide conformation remained unchanged; however, sulfate binding did produce an intense approximately 65 A meridional reflection not recorded with control peptides. This new reflection probably arises from the periodic deposition of the electron-dense sulfate along the (long) axis of the fibril. The sulfate binding could provide sites for the binding of additional fibrils that generate the observed lateral and axial aggregation. The binding of heparan to beta(11-28) also produced extensive aggregation, suggesting that in vivo sulfated compounds can promote macrofibers. The amyloid-specific, sulfonated dye Congo red, even in the presence of sulfate ions, produced limited aggregation and reduced axial growth of the fibrils. Therefore, electrostatic interactions are important in the binding of exogenous compounds to amyloid fibrils. Our findings suggest that the sulfate moieties of certain molecules, such as glycosaminoglycans, may affect the aggregation and deposition of amyloid fibrils that are observed as extensive deposits in senile plaques and cerebrovascular amyloid.

A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases.

We identified a 60-kDa diisopropylfluorophosphate-(DFP) reactive protein in human bronchoalveolar lavage fluid, at a yield of 50-100 pmol/lavage. The protein is associated with the cell-free lavage fluid sediment, which consists mainly of surfactant. [3H]DFP labeling is inhibited by heating to 56 degrees C, 2 mM phenylmethylsulfonylfluoride and 1 mM bis(4-nitrophenyl)-phosphate. An identical 60-kDa [3H]DFP-reactive protein is present in the insoluble fraction of alveolar macrophage-conditioned culture medium and in total membrane preparations of alveolar macrophages. The [3H]DFP-labeled protein was purified approximately 30-fold from lavage fluid sediment by size-exclusion (Sephacryl S-200) and ion-exchange (Mono-Q) chromatography. Cyanogen bromide treatment of the partially purified protein produced a major labeled peptide of 14 kDa with an NH2-terminal sequence 90% identical to a region of form 1 rabbit liver microsomal carboxylesterase. Esterase activity in unlabeled starting material, detected using p-nitrophenyl valerate as substrate, copurified with the [3H]DFP-labeled enzyme. Degenerate oligonucleotide primers were designed based on the partial amino acid sequence and on a highly conserved region of known liver carboxylesterase sequences. Polymerase chain reaction using these primers and reverse-transcribed human alveolar macrophage mRNA yielded a 354-base pair product which was then used to screen a human alveolar macrophage cDNA library. A complete esterase sequence was obtained from two incomplete, overlapping clones, and is virtually identical to human liver carboxylesterase partial sequences. Northern blot analysis demonstrated a single approximately 1.7-kilobase transcript in human monocytes and alveolar macrophages, with much higher levels in the latter. These data indicate that human alveolar macrophages both contain and release a serine esterase that is apparently identical to liver microsomal carboxylesterase. Its enzymatic profile suggests it is a major component of alveolar macrophage-nonspecific esterase activity. We hypothesize that it acts as a detoxication enzyme in the lung.

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

The lactose carrier of Klebsiella pneumoniae M5a1; the physiology of transport and the nucleotide sequence of the lacY gene.

A comparison has been made between the physiology and amino acid sequence of the lactose carriers of Klebsiella pneumoniae M5a1 and Escherichia coli K-12. The membrane transport of lactose was much weaker in Klebsiella than in E. coli. On the other hand o-nitrophenylgalactoside uptake by Klebsiella was distinctly greater than with E. coli. In spite of the differences in sugar transport between the two organisms, the amino acid sequences of the respective lactose carriers were remarkably similar (60% of the amino acids are identical).

Sequence of the lon gene in Escherichia coli. A heat-shock gene which encodes the ATP-dependent protease La.

To learn more about the mechanism and regulation of the ATP-dependent protease La in Escherichia coli, the lon gene was completely sequenced using the dideoxy method on fragments generated by Bal31 digestion. The predicted amino acid composition based on the DNA sequence agreed well with the composition of the acid-hydrolyzed protease. The predicted NH2-terminal amino acid sequence, tryptophan content, and the carboxyl terminus also agreed with experimental data. However, the molecular weight of 87,000 (783 amino acids) calculated from the DNA sequence was lower than prior estimates. The tetrameric enzyme contains four binding sites for ATP, a DNA-binding domain, a proteolytic site, and a regulatory site that binds unfolded polypeptides. An ATP-binding pocket exists on each subunit as shown by consensus sequences and elements of secondary structure resembling those on other nucleotide-binding proteins (e.g. adenylate kinase, RecA). For this purpose, improved consensus patterns for identifying ATP-binding domains were developed. Computer-assisted comparisons, however, failed to demonstrate any regions homologous to sequences in other polypeptides including proteases or DNA-binding proteins. This enzyme also contains an unusual highly acidic domain surrounded by very basic sequences. Protease La is the first ATP-dependent protease sequenced and seems to represent a new type of enzyme. The promoter sequence was similar to consensus sequences for other heat-shock promoters. Using site-directed mutagenesis, alterations were introduced into the putative promoter sequence. Mutations upstream of -35 had little effect, but alterations immediately upstream of -10 lowered basal transcription of a lon-lacZ operon fusion and reduced its response to inducers of the heat-shock response.