The origin of multiply sigmoid curves of pH-dependence. The partitioning of groups among titration pK values.

An acid, HnA, with n ionizing groups is known to have the same titration curve as an equimolar mixture of n hypothetical monobasic acids, whose dissociation constants are known as the 'titration constants' of the real acid. We show that the pH-dependence of any property of HnA is also represented by the sum of one-site titration curves, characterized by these same titration constants. Since one such property is the degree of dissociation of one of the dissociating groups, a fraction of each group shows each of the various titration pK values, so that the group partitions among them. The n groups therefore share the same n titration pK values but differ in the fractions belonging to each. The one H+ ion per molecule that titrates with each pK is thus made up of the fractions, one from each group, that share this pK value. A group may possess a single pK value, in that it contributes virtually all of this pK and almost nothing to the others, only if either (1) in titrates in a different pH range from the other groups or (2) its affinity for H+ is unaffected by their ionization state.

Cloning, genetic organization, and characterization of a structural gene encoding bacillopeptidase F from Bacillus subtilis.

Bacillopeptidase F is an extracellular serine protease that is expressed at the beginning of the stationary phase. To study its structure, regulation of expression, and physiological roles, we have cloned and characterized the structural gene (bpf) encoding this protease from Bacillus subtilis. DNA sequence analysis suggests this protease is synthesized as a preproenzyme (Mr = 92,000). Through processing at both the NH2 and COOH termini, it is gradually converted into various forms with molecular mass ranging from 80 to 48 kDa. Shortening the 3' end of bpf demonstrates that at least 290 amino acid residues from the COOH-terminus of bacillopeptidase F are not required for either catalytic activity or secretion. Bacillopeptidase F exhibits sequence similarity with several serine proteases. Its gene is found immediately downstream from the fts operon which was mapped at 135 degrees on the B. subtilis genetic linkage map. Inactivation of the chromosomal copy of bpf shows no effect on cell growth and sporulation. A triple protease-deficient strain (WB300 with the structural genes for bacillopeptidase F and two other major proteases inactivated) was constructed to serve as a better expression host for the production and secretion of foreign proteins.

Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure.

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

Studies on the mechanism of the iodination of tyrosine by lactoperoxidase.

Studies with lactoperoxidase showed that a highly reactive intermediate is produced (on the enzyme) from I- and H2O2 which then diffuses from the enzyme and very rapidly and indiscriminately iodinates any Tyr or peptides containing Tyr which are in the same solution. The evidence supporting these conclusions follows. 1) The rate followed the Michaelis-Menten pattern with I- and H2O2 while the concentration of Tyr peptides had no measurable effect on the rate; 2) the rates of reaction were independent of the type of peptide in which Tyr was located; 3) the amount of iodination which had occurred after the reaction had gone to completion and the amounts of monoiodination and diiodination after completion of the reaction were independent of the peptide type, the pH, the solvent polarity, or the ionic strength; 4) competition for reaction by two very different Tyr peptides depended only on their initial concentrations; and 5) iodination of a large protein occurred through a dialysis membrane. Free Tyr was iodinated at the same rate as Tyr peptides by lactoperoxidase, but monoiodotyrosine and m-fluorotyrosine were iodinated at one-half that rate. The results also showed that one can choose ratios of [peptide] to [H2O2] such that monoiodination is maximized relative to diiodination. It was also found that the iodination capacity of a mixture of I- and H2O2 with lactoperoxidase (when Tyr was absent) was only slowly dissipated. Finally, the results showed that lactoperoxidase can be used to brominate and chlorinate Tyr peptides at a slow rate.

Synthetic peptides as substrates for processing enzymes in the bovine pituitary.

Synthetic peptides, based on sequences of proopiomelanocortin (POMC) cleaved in both the bovine anterior and intermediate pituitaries (-Phe-Pro-Leu-Gly-Phe-Lys-Arg-Glu-Leu-Thr-Gly-) and only in the intermediate lobe (-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-), were used as substrates for the enzymes that process POMC to active hormones in the anterior and intermediate lobes of the pituitary. Cleavage of these peptides at the dibasic pair of residues, the expected cleavage site, was observed with a lysate from bovine pituitary secretory granules. Cleavage occurred optimally at a pH between 4 and 5 and was inhibited with sulfhydryl reagents, pepstatin, and leupeptin. Little specificity for the nature of the basic residues at the cleavage site was observed. An additional cleavage, following glutamic acid residues, was also seen.

Analysis of fluorine and iodine derivatives of tyrosine.

Separation of tyrosine, fluorotyrosine, monoiodotyrosine and diiodotyrosine was achieved by reversed-phase high-performance liquid chromatography (HPLC) using a gradient of acetonitrile with water and using trifluoroacetic acid for ion pairing. No derivatization of the amino acids, prior to separation, was needed. The spectral properties of Tyr and its fluorine and iodine derivatives and the dependence of their absorbance maxima on pH, made it possible to analyze and differentiate between these derivatives in the free amino acid form or in peptides. This analysis was accomplished by adjusting the post column HPLC eluate from two identical runs to different pH values and then comparing the spectra of the peaks from these two runs with a diode array detector. Hydrolysis in 6 M hydrochloric acid was totally destructive to mono- and diiodotyrosine. However, base hydrolysis in 13.5 M sodium hydroxide for 30 min at 121 degrees C in an autoclave caused no destruction and allowed excellent recovery of all of the Tyr derivatives. This is the first report of simple methods for the detection and analysis of these amino acids and of a hydrolytic method which protects against their loss. A method of storage was also proposed.

Evidence that the Ya and Yc subunits of glutathione transferase B (ligandin) are the products of separate genes.

A study of the structure of glutathione transferase B (ligandin) has been made with a view to understanding the relationship between the structures of the subunits of which it is composed. It consists of a mixture of a homodimer (YaYa) and a heterodimer (YaYc) in which the monomers are defined by their apparent molecular weights, that of Ya being 22000 and Yc 25000. Soluble tryptic peptides from the native homodimer YaYa have been compared with those from an artificial homodimer YcYc produced by rehybridization of native YaYc. Approximately 10 peptides specific to YaYa, 12 specific to YcYc and 21 common to both have been detected. Some of the above peptides are derived from variants of the monomers themselves. YaYa and YcYc have two C termini which are the same in both dimers, namely phenylalanine and lysine. Also there are four cysteinyl peptides, of which three are common to YaYa and YcYc and one specific to each. These results suggest that Ya and Yc are derived from at least two different but related genes.

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins.

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.

Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane.

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 mug/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.

Glutathione adducts of N-methyl-4-aminoazobenzene formed in vivo and by reaction of N-benzoyloxy-N-methyl-4-aminoazobenzene with glutathione.

N-Benzoyloxy-N-methyl-4-aminoazobenzene (N-BzO-MAB) is believed to be an analogue of the ultimate carcinogenic form of N,N-dimethyl-4-aminoazobenzene (DAB). The reaction of N-BzO-MAB with glutathione in vitro yielded one major and two minor aminoazo dye-glutathione adducts. After purification by ion exchange chromatography and high pressure liquid chromatography, analysis of chemical properties, and the measurement of ultraviolet, visible, proton magnetic resonance, and mass spectra, the major and one minor adduct were identified as 3-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (3-GS-MAB) and 2'-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (2'-GS-MAB) respectively. The other minor adduct was tentatively identified as 4'-(glutathion-S-yl)-N-methyl-4-aminoazobenzene (4'-GS-MAB). Fractionation and analyses of biliary metabolites from rats given DAB revealed the presence of two aminoazo dye-glutathione adducts. One of these was identical to 3-GS-MAB in its chromatographic and chemical properties and its visible and ultraviolet spectra. The other adduct was partially characterized and judged to be a 4-aminoazobenzene-glutathione adduct. The role of glutathione in the detoxification of carcinogenic aminoazo dyes is discussed.

The binding and catalytic activities of forms of ligandin after modification of its thiol groups.

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.

Studies on phosphoglyceromutase from chicken breast muscle: chemical modification of lysyl residues.

To examine the role of lysyl residues in the activity of the enzyme, phosphoglyceromutase (PGM) from chicken breast muscle was chemically modified with trinitrobenzenesulfonate (TNBS) and pyridoxal 5'-phosphate. Trinitrophenylation resulted in modification of about nine lysines per mole of PGM with almost complete activity loss. Substrate (3-PGA) offered some protection to TNBS inactivation but cofactor (2,3-DPGA) did not. Reduction of the Schiff's base complex between pyridoxal 5'-phosphate and PGM gave irreversible inactivation of the enzyme. Inactivation was due to incorporation of 1 mol of pyridoxal 5'-phosphate per mole of PGM dimer through the epsilon-amino group of a lysyl residue. The effect of pyridoxal 5'-phosphate was specific for intact native enzyme and reaction with only one lysine per dimer was not due to induced conformational changes nor to dissociation of the reacted enzyme. 3-PGA prevented much of the reaction with pyridoxal 5'-phosphate with preservation of 70% of the activity and was a competitive inhibitor of the active site directed reagent. Cofactor (2,3-DPGA) acting noncompetitively, reduced the rate at which inactivation occurred with pyridoxal 5'-phosphate. Incorporation of 2,3-[32P]DPGA into PGM irreversibly inactivated with pyridoxal 5'-phosphate and NaBH4 was incomplete indicating hindrance to phosphorylation in the modified enzyme. The results indicate that a lysyl residue is located at or near the active site of PGM and that it is probably involved in the binding of 3-PGA.

Studies on phosphoglyceromutase from chicken breast muscle: number and reactivity of sulfhydryl groups.

Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5'-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB.