Modulation of the amino acid control of hepatic protein degradation by caloric deprivation. Two modes of alanine co-regulation

Intracellular protein degradation in perfused livers of fed rats has been shown to be directly regulated by 7 amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) and co-regulated by alanine. Responses to graded increases of regulatory amino acids (individually or combined) are multiphasic and include (a) an initial inhibition at 0.5 times normal plasma concentrations, (b) a localized, zonal loss of inhibition at normal levels, and (c) suppression to basal rates at 4 times normal concentrations or greater; the zonal loss of inhibition is prevented by 0.5 mM (normal) alanine. In further perfusion studies carried out at the usual time (1100 h), we have occasionally observed a sharp decrease in proteolytic responsiveness at normal amino acid concentrations. The decrease, which occurred spontaneously in normal fed rats, was attributed to a nearly 90% loss in the sensitivity of alanine co-regulation. In all instances, alanine sensitivity was restored after 4 to 24 h of starvation. The cause of the insensitivity and the mechanism of its reversal by caloric deprivation are not presently known. Starvation for 24 h also appeared to alter the individual inhibitory effectiveness of Leu, Tyr, and Gln. On the other hand, inhibition by the full regulatory group at 4 times normal plasma levels was unchanged when compared with the complete plasma mixture except for a concentration shift in the peak zonal loss of proteolytic inhibition from 1.25 to 0.6 times plasma levels. Since the shift paralleled known changes in portal vein regulatory amino acids, it may have been adaptive in nature. As with fed animals, the zonal loss in starvation was abolished by 0.5 mM alanine, but not with high levels of lactate and pyruvate (10 mM), a finding consistent with the view that co-regulation is mediated by the recognition of alanine per se rather than its metabolism.     

Multiphasic control of hepatic protein degradation by regulatory amino acids. General features and hormonal modulation

Previous studies with livers from fed rats perfused in the single-pass mode have shown that regulatory amino acids (Leu, Tyr, Gln, Pro, Met, His, and Trp) as a group as well as leucine alone inhibit deprivation-induced protein degradation optimally at 0.5 and 4 times (X) normal plasma amino acid concentrations. However, they lose inhibitory effectiveness almost completely within a narrow zone centered at normal (1 X) levels (P?s?, A. R., Wert, J. J., Jr., and Mortimore, G.E. (1982) J. Biol. Chem. 257, 12114-12120; P?s?, A. R., and Mortimore, G. E. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4270-4274). We now report similar effects for tyrosine and glutamine and suggest that this multiphasic dose response is a general feature of the regulatory group. Insulin (2.4 micrograms h-1) selectively modulated the response by abolishing the zonal loss, whereas glucagon (10 micrograms h-1) blocked the initial inhibition (0.5 X); proteolytic suppression was restored at 4 X normal plasma levels. Although the zonal loss of inhibition at 1 X was associated with a near maximal increase in the volume density of macroautophagy, the vacuoles differed from those induced by stringent amino acid deprivation in containing 4.5-fold more smooth than rough endoplasmic reticulum and thus represented a separate population. Surprisingly, the leucine analog, L-alpha-hydroxyisocaproate, elicited multiphasic responses identical to those of L-leucine, including inhibition at 0.1 mM (equivalent to 0.5 X Leu). Inasmuch as alpha-ketoisocaproate is not effective at this concentration, the initial suppression of protein degradation could be mediated from a site that recognizes structural features common to leucine and its hydroxyl analog.     

Parallel control of hepatic proteolysis by phenylalanine and phenylpyruvate through independent inhibitory sites at the plasma membrane.

Intracellular protein degradation in the rat hepatocyte is regulated by 7 amino acids of which Leu, Gln, and Tyr play major roles. Although Phe is known to inhibit proteolysis as effectively as Tyr at high concentrations, it has not been regarded as an active regulator because of its rapid hydroxylation to Tyr. We now show that proteolytic responses to Phe during liver perfusion differ strikingly from effects of the multiphasic regulators Leu, Gln, and Tyr in eliciting mirror image responses at half-normal and normal plasma concentrations. Since response curves to phenylpyruvate and Phe were identical, we considered the possibility that phenylpyruvate mediated its anomalous inhibition intracellularly. However, when phenylpyruvate was produced from phenyllactate intracellularly at a rate providing the same rate of transamination (and intracellular concentration) as that derived from the uptake of phenylpyruvate, no response was obtained. Hence, the effect of phenylpyruvate was not initiated within the cell but more likely from the plasma membrane. Comparable evidence for Phe is less direct. Recent findings indicate that recognition sites for Leu and Gln are located at the plasma membrane. Since Phe augments the concerted inhibition by Leu and Gln at 4-fold normal levels, Phe is probably recognized in close proximity to them. However, the failure of phenylpyruvate to substitute for Phe in this interaction suggests that proteolytic inhibition by phenylpyruvate and Phe is mediated through similar, although independent, plasma membrane sites.     

Amino acid and hormonal control of macromolecular turnover in perfused rat liver. Evidence for selective autophagy

The control of RNA degradation by amino acids, insulin, and glucagon was investigated in perfused livers of fed rats previously labeled in vivo with [6-14C] orotic acid; rates were determined from the release of [14C]cytidine in the presence of 0.5 mM cytidine to suppress reutilization. Studies with cyclically perfused livers showed that plasma amino acids at 10 times (10X) normal concentrations inhibited RNA breakdown by 85%. Similar inhibition was obtained with a known regulatory amino acid mixture (Leu, Met, Pro, Trp, and His), whereas leucine alone (0.8 mM) decreased degradation by 47%. Perfusions carried out in the single-pass mode with graded levels of plasma amino acids revealed that the acceleration of RNA degradation over the full range of amino acid deprivation (0 to 10X normal levels) was the same as that for protein breakdown (3.19 and 3.15% h-1, respectively), and both were equally suppressed by insulin (2.4 micrograms h-1). Glucagon (10 micrograms h-1), though, was far less effective in stimulating RNA than protein turnover. A direct comparison of the two dose responses revealed a strong dissociation at 1 and 2 times normal amino acid levels. These findings support the notion that RNA and protein are degraded within a single macroautophagic compartment during amino acid and insulin deprivation. Glucagon, however, appeared to induce a second pathway in which the proportion of sequestered RNA to protein was selectively reduced. Electron micrographs showed that the ratio of vacuoles containing rough as compared with smooth endoplasmic reticulum was decreased by nearly 80% under these conditions.     

Amino acid control of proteolysis in perfused livers of synchronously fed rats. Mechanism and specificity of alanine co-regulation

The primary control of autophagically mediated proteolysis in perfused rat liver is carried out via two alternate mechanisms in response to specific regulatory amino acids. One (L) elicits direct inhibition at low and high plasma levels, but requires a co-regulatory amino acid to express inhibition at normal concentrations. The second (H) is ineffective at normal levels and below, but active at higher concentrations. Because regulation is subject to unpredictable variability with ad libitum feeding, we have utilized rats synchronously fed 4 h day-1 to stabilize responses. Proteolytic control is seen to evolve in stages: H appears 12 h after the start of feeding; by 18 h L emerges, alternating with H in a statistically predictable way; with omission of the 24-h feeding, H disappears and L remains constant through 42 h. In both 18- and 42-h rats, alanine, glutamate, and aspartate exhibit similar inhibitory activity when added singly to the regulatory group at normal plasma concentrations. However, since alanine, but not glutamate or aspartate, evokes proteolytic acceleration when it is deleted from a full plasma mixture, alanine appears to be the sole co-regulator. Alanine yields co-regulatory effects with normal plasma leucine (0.2 mM) in 18- and 42-h animals and interacts synergistically with 0.8 mM leucine in 42-h but not in 18-h rats where leucine alone inhibits strongly. Because the inactivation of alanine amino-transferase by aminooxyacetate (determined from the conversion of [14C]alanine to glucose) does not alter the co-regulatory and synergistic effects of alanine, regulation by alanine must be mediated from a site of recognition before transamination.     

From amino acid to glucosinolate biosynthesis: protein sequence changes in the evolution of methylthioalkylmalate synthase in Arabidopsis

Methylthioalkylmalate synthase (MAM) catalyzes the committed step in the side chain elongation of Met, yielding important precursors for glucosinolate biosynthesis in Arabidopsis thaliana and other Brassicaceae species. MAM is believed to have evolved from isopropylmalate synthase (IPMS), an enzyme involved in Leu biosynthesis, based on phylogenetic analyses and an overlap of catalytic abilities. Here, we investigated the changes in protein structure that have occurred during the recruitment of IPMS from amino acid to glucosinolate metabolism. The major sequence difference between IPMS and MAM is the absence of 120 amino acids at the C-terminal end of MAM that constitute a regulatory domain for Leu-mediated feedback inhibition. Truncation of this domain in Arabidopsis IPMS2 results in loss of Leu feedback inhibition and quaternary structure, two features common to MAM enzymes, plus an 8.4-fold increase in the k(cat)/K(m) for a MAM substrate. Additional exchange of two amino acids in the active site resulted in a MAM-like enzyme that had little residual IPMS activity. Hence, combination of the loss of the regulatory domain and a few additional amino acid exchanges can explain the evolution of MAM from IPMS during its recruitment from primary to secondary metabolism.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Leucine in food mediates some of the postprandial rise in plasma leptin concentrations

In vitro, leptin secretion is regulated at the level of mRNA translation by the rapamycin-sensitive mammalian target of rapamycin (mTOR) and its agonist leucine (Leu). Studies were conducted on meal-trained rats to evaluate the potential physiological relevance of these in vitro findings and the role of Leu in affecting rises in plasma leptin observed after a meal. In the first study, we correlated changes in plasma insulin and Leu to mTOR-signaling pathway activation and plasma leptin at different times during meal feeding. Rapid rises in plasma insulin and Leu, along with mTOR signaling (phosphorylation of eIF4G, S6K1, rpS6, and 4E-BP1) in adipose tissue were observed during the 3-h meal and declined thereafter. Plasma leptin rose more slowly, peaking at 3 h, and was inhibited by rapamycin (0.75 mg/kg) pretreatment. In another experiment, oral Leu or norleucine was provided instead of a meal. Leu and norleucine stimulated a rise in plasma leptin; however, the magnitude was less than the response to a complete meal. In a third study, rats were provided a meal that lacked Leu, branched-chain amino acids, or all amino acids. Stimulation of leptin secretion was reduced approximately 40% in animals provided the Leu-deficient meal. Further reductions were not observed by removing the other amino acids. Thus Leu appears to regulate most of the effects of dietary amino acids on the postprandial rise in plasma leptin but is responsible only for part of the leptin response to meal feeding.     

The specific receptor site for aliphatic carboxylate anion in the labellar sugar receptor of the fleshfly

A two minute treatment of a single sugar receptor cell with 10 mg/ml pronase did not affect its response to d-fructose, but depressed markedly its response to l-valine. This is the first direct evidence for a specific site for certain aliphatic amino acids.All six amino acids that can stimulate the sugar receptor were examined and classified into two groups according to the presence or absence of the inhibitory effects of pronase treatment. Responses to certain aliphatic amino acids and a corresponding fatty acid were depressed whereas responses to phenylalanine and trytophan were not. Further evidence for the existence of two classes of amino acids comes from the fact that the α amino group of valine is not essential whereas that of phenylalanine is. It was concluded that the first class of amino acids react with a specific receptive site for carboxylate anions whereas the second react with the furanose site.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

Summary The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (t_m) for poly(A) poly(U) and poly(I)poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of t_m at low concentrations (1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala>Gly>Ser>Asn>Pro>Met, Val>Ile, Leu for poly(A) poly(U) and Ala, Gly, Ser>Asn>Pro>Val>Ile, Met, Leu for poly(I)poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

Identification of an amino acid residue involved in the substrate-binding site of rat liver uricase by site-directed mutagenesis.

Computer analysis has shown that a conserved amino acid sequence (Leu 160 to Lys 164) of rat liver uricase is also present in other enzymes with purine substrates. The significances of the amino acids in this sequence were studied by site-directed mutagenesis. Replacement of Lys 164 by Glu or Ile resulted in loss of uricase activity and decrease in binding of the competitive inhibitor xanthine. The far ultraviolet circular dichroic spectra of the mutant uricases were identical to that of the wild type protein, indicating that the replacement of Lys 164 by other amino acids did not result in serious modification of the conformation of uricase. These findings suggest that this amino acid is involved in the substrate-binding site of the enzyme.     

Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin

The ability of myoglobin to bind oxygen reversibly depends critically on retention of the heme prosthetic group. Globin side chains at the Leu(89)(F4), His(97)(FG3), Ile(99)(FG5), and Leu(104)(G5) positions on the proximal side of the heme pocket strongly influence heme affinity. The roles of these amino acids in preventing heme loss have been examined by determining high resolution structures of 14 different mutants at these positions using x-ray crystallography. Leu(89) and His(97) are important surface amino acids that interact either sterically or electrostatically with the edges of the porphyrin ring. Ile(99) and Leu(104) are located in the interior region of the proximal pocket beneath ring C of the heme prosthetic group. The apolar amino acids Leu(89), Ile(99), and Leu(104) "waterproof" the heme pocket by forming a barrier to solvent penetration, minimizing the size of the proximal cavity, and maintaining a hydrophobic environment. Substitutions with smaller or polar side chains at these positions result in exposure of the heme to solvent, the appearance of crystallographically defined water molecules in or near the proximal pocket, and large increases in the rate of hemin loss. Thus, the naturally occurring amino acid side chains at these positions serve to prevent hydration of the His(93)-Fe(III) bond and are highly conserved in all known myoglobins and hemoglobins.     

The site of amino acid addition to posttranslationally modified proteins of regenerating rat sciatic nerves

The posttranslational modification of proteins by amino acids has been described in a variety of biological systems. These reactions occur at low levels in intact sciatic nerves of rats but are increased 10-fold following nerve injury and during subsequent regeneration of the nerve. While it has been shown in brain and liver that the site of addition of Arg is to the N-terminus, there is no information on the location at which the other amino acids add on to targeted proteins nor the site of addition of Arg in regenerating nerves. In the present study, we have used manual micro-Edman degradation combined with HPLC, and digestion with carboxypeptidase A and B to determine the site of addition of various amino acids to targeted proteins. Of the 3H-labelled amino acids incorporated posttranslationally into proteins of regenerating sciatic nerves (Arg, Lys, Leu, Phe, Val, Ala, Pro and Ser), only [3H]Arg was found to be present at the N-terminus. To determine whether amino acid additions were occurring at the C-terminus, proteins modified by two of the amino acids incorporated in greatest amounts (Lys and Leu) were incubated with specific carboxypeptidases. [3H]Leucine was not liberated following incubation with carboxypeptidase, suggesting that Leu is not added at the C-terminus of modified proteins. Under similar conditions, some [3H]Lys was liberated, but in amounts not significantly different from controls incubated without carboxypeptidase, indicating a non-specific degradation of Lys modified proteins rather than a specific release of Lys from the C-terminus. These experiments show that in regenerating sciatic nerves of rats, Arg is the only amino acid added posttranslationally to the amino terminus of target proteins, and that Leu, and probably Lys, are not conjugated to proteins at the C-terminus.     

Regulation of branched-chain amino acid oxidation in isolated muscles, nerves and aortas of rats.

1. The oxidation of the three branched-chain amino acids was regulated in parallel fashion in rat tissues studied in vitro. 2. With 0.1 mM-[1-14C]isoleucine as substrate in the presence of 5.5 mM-glucose, 14CO2 production was 0.6 mumol/2 h per g in the aorta, 0.3 in peripheral nerve, 0.2 in muscle and 0.13 in spinal cord. 3. The ratio 14C oxidized/14C incorporated into proteins with 0.1 mM-[1-14C]leucine was 1.3 in hemidiaphragms, 3.3 in sciatic nerve and 1.0 in nerves undergoing Wallerian degeneration. Leucine oxidation decreased only slightly during degeneration, but protein synthesis doubled. 4. Hemidiaphragms incubated with [1-14C]leucine or 4-methyl-2-oxo[1-14C]pentanoate increased 14CO2 production 7-9-fold as substrate concentration was increased from 0.1 to 0.5 mM; under the same conditions 14CO2 production by nerves increased only 2-3-fold. 5. 2-Oxoglutarate stimulated the oxidation of the branched-chain amino acids by muscles and peripheral nerves and the oxidation of 4-methyl-2-oxopentanoate by hemidiaphragms but not by nerves. 6. Octanoate (0.1-1.0 mM) markedly stimulated the oxidation of branched-chain amino acids and of 4-methyl-2-oxopentanoate in hemidiaphragms, but inhibited oxidation of both by peripheral nerves and spinal cord. In aortas, oxidation of isoleucine (the only substance tested) was inhibited by octanoate. 7. The effects of octanoate and 2-oxoglutarate on leucine oxidation by hemidiaphragms were additive at low concentrations. When maximally stimulating concentrations of either agent were used, addition of the other was ineffective. 8. Pyruvate inhibited the oxidation of branched-chain amino acids and 4-methyl-2-oxopentanoate in all tissues tested. 9. Insulin did not affect the oxidation of 4-methyl-2-oxopentanoate by muscles or nerves. 10. The oxidative decarboxylation of the branched-chain alpha-oxo acids is suggested as a regulatory site of branched-chain amino acid oxidation. Differences in regulation between muscle on the one hand, and nerve and aorta on the other, are discussed.     

Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: a single amino acid change converts a phosphatidylinositol- glycan-anchored protein to a secreted protein

Placental alkaline phosphatase (PLAP) is anchored in the plasma membrane by a phosphatidylinositol-glycan moiety (PI-glycan). PI-glycan is added posttranslationally to the nascent peptide chain after the removal of 29 amino acids from the COOH-terminus. The contribution of selected COOH-terminal amino acids to the signal for PI-glycan addition was tested by creating a fusion protein with the COOH-terminus of PLAP and a secreted protein and by mutagenesis of specific PLAP COOH-terminal amino acids. The cDNA encoding the COOH-terminus of PLAP was fused in frame to the cDNA for human clotting Factor X and expressed in transfected COS-1 cells. Fusion proteins containing 32 amino acids of the PLAP COOH-terminus were modified by PI-glycan addition. Thus, the signal for PI-glycan modification must reside in these amino acids. Next, the region between the hydrophobic domain and the cleavage site was examined for additional determinants. Mutations of the hydrophilic residues in the spacer region demonstrated that these amino acids do not contribute to the signal for PI-glycan addition. Deletion of amino acids in the spacer region prevented the addition of PI-glycan suggesting that the length of the spacer domain or the amino acids around the cleavage site are important determinants. Finally, we demonstrated that interruption of the hydrophobic domain by a charged residue prevents PI-glycan addition and results in a protein that is secreted into the medium. The finding that a single Leu to Arg substitution in the hydrophobic domain converts a PI-glycan anchored, membrane protein to a secreted protein suggests that an essential signal for the correct sorting of PI-glycan anchored proteins versus secreted proteins resides in the hydrophobic domain. Substitution of a charged amino acid for a hydrophobic amino acid may be a mechanism for producing membrane bound and secreted forms of the same protein.     

The mechanism of action of dipeptidyl aminopeptidase. Inhibition by amino acid derivatives and amines; activation by aromatic compounds.

A variety of amino acid and peptide amides have been shown to be inhibitors of dipeptidyl aminopeptidase. Among these compounds derivatives of strongly hydrophobic amino acids are the strongest inhibitors (Phe-NH2, Ki = 1.0 +/- 0.2 mM), while amides of basic amino acids were somewhat less effective (Lys-NH2, Ki = 36 +/- 3 mM). Short chain amino acid amides are notably weaker inhibitors (Gly-NH2, Ki = 293 +/- 50 mM). The interaction of the side chains of compounds with the enzyme appears to be at a site other than that at which the side chain of the amino-penultimate residue of the substrate interacts since the specificity of binding is different. Primary amines have been shown to inhibit, e.g., butylamine, Ki = 340 +/- 40 mM, and aromatic compounds have been shown to stimulate activity toward Gly-Gly-NH2 and Gly-Gly-OEt (phenol, 35% stimulation of activity at a 1:1 molar ratio with the substrate). The data suggest that inhibition involves binding at the site occupied by the free alpha-amino group and the N-terminal amino acid.     

Synergistic Effects of High Hydrostatic Pressure,Mild Heating,and Amino Acids on Germination and Inactivation of Clostridium sporogenes Spores

The synergistic effects of high hydrostatic pressure (HHP), mild heating, and amino acids on the germination of Clostridium sporogenes spores were examined by determining the number of surviving spores that returned to vegetative growth after pasteurization following these treatments. Pressurization at 200 MPa at a temperature higher than 40°C and treatment with some of the 19 l-amino acids at 10 mM or higher synergistically facilitated germination. When one of these factors was omitted, the level of germination was insignificant. Pressures of 100 and 400 MPa were less effective than 200 MPa. The spores were effectively inactivated by between 1.8 and 4.8 logs by pasteurization at 80°C after pressurization at 200 MPa at 45°C for 120 min with one of the amino acids with moderate hydrophobicity, such as Leu, Phe, Cys Met, Ala, Gly, or Ser. However, other amino acids showed poor inactivation effects of less than 0.9 logs. Spores in solutions containing 80 mM of either Leu, Phe, Cys, Met, Ala, Gly, or Ser were successfully inactivated by pasteurization by more than 5.4 logs after pressurization at 200 MPa at 70°C for 15 to 120 min. Ala and Met reduced the spore viability by 2.8 and 1.8 logs, respectively, by pasteurization at a concentration of 1 mM under 200 MPa at 70°C. These results indicate that germination of the spores is facilitated by a combination of high hydrostatic pressure, mild heating, and amino acids.     

Immunochemistry of the dominating antigenic region Ala582 to Cys604 in the transmembranous protein of simian and human immunodeficiency virus

The immunochemistry of two homologous uniquely antigenic peptides representing Ala582 to Cys604 in the transmembrane proteins of simian immunodeficiency virus of rhesus macaque origin, SIVmac (closely related to HIV-2) and HIV-1 (strain HTLV-IIIB) was characterized at the resolution of single amino acids. Five different antigenic sites were identified in the SIVmac peptide by use of 34 mAb against this peptide and two different sites were similarly demonstrated in the HIV-1 peptide by use of 10 peptide-specific mAb. Within some sites the mAb could be subgrouped to show a progressively more narrow epitopic dependence on amino acids in the central part of the site. Three SIVmac peptide mAbs had a remarkably narrow amino acid dependence, Glu584 and Tyr586. Anti-peptide mAbs reacting with the site Trp596 to Gln602 effectively blocked the capacity of the peptide to react with human postinfection HIV-2 antibodies previously demonstrated to have a restricted reactivity involving this site. No similar blocking was seen when mAb specific for Leu587 to Gln590 were used except with a single broadly reacting HIV-2 serum, which depended on an amino acid at a distance of only 6 residues, Trp596. A cross-reacting site involving amino acids Ala582 to Glu588/Lys588 was identified with mAb and rabbit hyperimmune sera against the two peptides. This site was not accessible in the intact transmembrane proteins as determined by ELISA and Western blot tests. Antipeptide mAb against other sites as well as rabbit sera reacted strongly in these tests and can be used as type-specific, component-unique reagents.     

Induction by nucleotide triphosphate hydrolysis of a form of sarcoplasmic reticulum ATPase capable of medium phosphate-oxygen exchange in presence of calcium.

Sarcoplasmic reticulum vesicles rendered leaky by exposure to alkaline pH, like intact vesicles, catalyze a rapid Mg2+-dependent exchange of oxygens of medium Pi with water. The exchange with 10 mM Pi is strongly inhibited by 0.15 mM Ca2+. Upon addition and hydrolysis of ITP or ATP, a rapid phosphate-oxygen exchange is observed even with 0.15 mM Ca2+ present and a definite but smaller exchange at 8 mM Ca2+. Oxygen exchange per Pi formed is greater with ITP than with ATP. When no Pi is initially present, the extent of oxygen exchange is increased with time of incubation as Pi is formed. With 18O-labeled Pi present, ATP hydrolysis accelerates 18O loss. The results show that much of the oxygen exchange occurs as a result of reversible binding of medium Pi. Thus the binding and cleavage of ITP or ATP overcomes the Ca2+ inhibition of the medium Pi in equilibrium HOH exchange. Such findings support the concept that the cleavage cycle includes a transient conformational form which can reversibly react with Pi to give a phosphoryl enzyme and resultant oxygen exchange or in a rate-limiting step decay to a form with high Ca2+ and NTP affinity.     

Modulation of the sensitivity of FimB recombination to branched-chain amino acids and alanine in Escherichia coli K-12

Phase variation of type 1 fimbriae of Escherichia coli requires the site-specific recombination of a short invertible element. Inversion is catalyzed by FimB (switching in either direction) or FimE (inversion mainly from on to off) and is influenced by auxiliary factors integration host factor (IHF) and leucine-responsive regulatory protein (Lrp). These proteins bind to sites (IHF site II and Lrp sites 1 and 2) within the invertible element to stimulate recombination, presumably by bending the DNA to enhance synapses. Interaction of Lrp with a third site (site 3) cooperatively with sites 1 and 2 (termed complex 1) impedes recombination. Inversion is stimulated by the branched-chain amino acids (particularly leucine) and alanine, and according to a current model, the amino acids promote the selective loss of Lrp from site 3 (complex 2). Here we show that the central portion of the fim invertible element, situated between Lrp site 3 and IHF site II, is dispensable for FimB recombination but that this region is also required for full amino acid stimulation of inversion. Further work reveals that the region is likely to contain multiple regulatory elements. Lrp site 3 is shown to bind the regulatory protein with low affinity, and a mutation that enhances binding to this element is found both to diminish the stimulatory effects of IVLA on FimB recombination and to inhibit recombination in the absence of the amino acids. The results obtained emphasize the importance of Lrp site 3 as a control element but also highlight the complexity of the regulatory system that affects this site.