Specific 13C reductive methylation of glycophorin A. Possible relation of the N-terminal amino acid and the lysine residues to MN blood group specificities

Heterozygous and homozygous glycophorin A were partially and fully reductively methylated with 13C-enriched formaldehyde in the presence of sodium cyanoborohydride. Total reductive methylation modified the five lysine residues (to produce N epsilon,N-[13C]dimethyl lysine) and the N-terminal amino acid residues (N alpha,N-[13C]dimethyl serine and leucine) of glycophorins AM and AN, respectively. 13C-NMR spectra of these species indicated that the 13C-enriched methyl carbons of the five lysyl derivatives all occur at 44.1 ppm downfield from Me4Si. Titration results indicate that the pK alpha of these methylated lysines is greater than 10. The chemical shift equivalent methyl resonances of the 13C-enriched methylated N-terminal Leu derivative were found to occur at 42.8 ppm downfield from Me4Si and exhibited a normal pH titration behavior (pK alpha approximately 7.4). The methyl resonances of the N alpha,N-[13C]dimethyl Ser derivative, on the other hand, were found to exhibit chemical shift nonequivalence, indicating rotational constraints about the C alpha-N bond. The linewidths of the two methyl resonances were also found to be considerably different; this phenomenon could be eliminated by running spectra of the sample (pH approximately 5.0) at elevated temperatures (75 degrees C). This result suggested that for the N alpha,N-[13C]dimethyl Ser derivative of glycophorin AM, hindered rotation must occur about one of the N alpha-13CH3 bonds. This structural difference at the N-terminal residue of glycophorins AM and AN may be related to the MN blood group determinants displayed by these related glycoproteins.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Human liver acid phosphatases: Cysteine residues of the low-molecular-weight enzyme

The stoichiometry and the reactivity of the sulfhydryl groups of a human liver acid phosphatase have been studied. The smallest (M_r = 14,400) of the three molecular-weight forms of acid phosphatase from human liver, recently purified and characterized in our laboratory, was treated with various sulfhydryl group-specific reagents: p-hydroxymercuribenzoate, p-hydroxymercuriphenylsulfonate, fluorescein mercuriacetate, methyl methanethiosulfonate, p-nitrophenoxycarbonyl methyl disulfide, and thiosulfate. A total loss of enzymatic activity was obtained in each case. By spectrophotometric titration with 5,5′-dithiobis(2-nitrobenzoate) and p-hydroxymercuriphenylsulfonate it was shown that there are six free sulfhydryls per protein molecule, consistent with the amino acid analysis of this enzyme. The same number was deduced as a result of inactivation studies carried out with p-hydroxymercuribenzoate and p-hydroxymercuriphenylsulfonate. A total loss of activity was obtained at reagent to enzyme ratios of 6:1 in both cases. Similar results were obtained upon inactivation by p-nitrophenoxycarbonyl methyl disulfide, where the enzyme was found to possess only 10% residual activity at an inhibitor-to-enzyme ratio of 6:1. With fluorescein mercuriacetate as an inactivator, total loss of activity was found at a 2.5 times molar excess of this reagent over protein. Both the stoichiometry of inactivation and fluorescence titration experiments suggest that fluorescein mercuriacetate can function as a bifunctional sulfhydryl group reagent. The activity of a totally inactivated enzyme preparation obtained following reaction with excess of p-nitrophenoxycarbonyl methyl disulfide or with methyl methanethiolsulfonate could be almost completely restored upon treatment with dithiothreitol. These data are consistent with the interpretation that in each enzyme molecule, there are six free sulfhydryl groups of almost equal reactivity, at least one of which is essential for enzymatic activity.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

Quantitative structure-activity relationships of chymotrypsin-ligand interactions: An analysis of interactions in p3 space.

Fourteen derivatives of l-alanine of the type CH₃CH(NHCO-3-C₅H₄N)COOR₃ have been synthesized and their hydrolysis by chymotrypsin was studied with the object of characterizing enzymic space (?₃) to which R₃ binds. The binding of R₃ ($\text{log}\text{1}\text{K}{}_{\mathrm{m}}$) was shown via correlation analysis to correlate with molar refractivity (MR) of R₃ rather than hydrophobicity (π). The results confirmed our earlier predictions. A correlation equation for the hydrolysis of 77 acyl-amino acid esters of the general formula R₂CH(NHCOR₁)COOR₃ relating $\text{log}\text{(k}{}_{\mathrm{<mtext>cat</mtext>}}\text{k}{}_{\mathrm{m}}\text{)}$ to molar refractivity of R₁, R₂, and R₃ and to $\text{σ}{}^1$ (Taft's polar parameter) of R₃ was formulated. The general picture of ligand interactions with chymotrypsin as seen with correlation analysis is discussed.     

Thrombin-platelet interactions: an assessment of the roles of saturable and nonsaturable binding in platelet activation

Thrombin binds to platelets and induces platelet activation, but the relationship of binding to activation is not clear. To better define this relationship, we have analyzed parameters of binding and activation by alpha-thrombin and by three analogous proteases that activate platelets somewhat differently. The proteases were nitro-alpha-thrombin, a derivative with nitrated tyrosine, gamma-thrombin, a product of partial proteolysis of alpha-thrombin, and trypsin, a homologous protease. Nitro-alpha-thrombin and native alpha-thrombin activated platelets similarly, whereas gamma-thrombin and trypsin activated to a slightly lesser extent than alpha-thrombin and only after a distinctive delay. alpha-Thrombin and nitro-alpha-thrombin bound to platelets to about the same extent, but only alpha-thrombin showed evidence of saturable binding. Hirudin, a thrombin inhibitor, blocked both platelet activation and saturable binding by alpha-thrombin. With nitro-alpha-thrombin, hirudin blocked platelet activation, but it had no effect on binding. gamma-Thrombin and trypsin bound less than alpha-thrombin and with no evidence of saturable binding. There were identical relationships between the total amount bound and the extent of platelet activation for the four proteases (some show no saturable binding) but distinct differences in the relationships of total amount bound and the rate of activation; similar rates of activation required the binding of three to five times more gamma-thrombin or trypsin than alpha-thrombin. That is, without saturable binding, activation was slower. These data thus show a correlation between total amount bound and extent of activation but no correlation between amount saturably bound and the extent of platelet activation. Conversely, the rate of activation is more closely correlated with saturable binding than with total binding. We conclude that high-affinity saturable binding is not essential for thrombin-induced platelet activation but that it may accelerate the reaction.     

Studies on the structure of rabbit muscle aldolase: Ordering of the tryptic peptides; Sequence of 164 amino acid residues in the NH2-terminal BrCN peptide

The sequence of 164 amino acid residues in the NH₂-terminal BrCN peptide of rabbit muscle aldolase has been determined. The information has permitted location of the following amino acid residues involved in the catalytic activity or in maintaining the structural integrity of the enzyme: Cys-72, forms a disulfide bridge with Cys-336 in the COOH-terminal segment on inactivation of the enzyme by oxidation; Lys-107, forms a Schiff base with pyridoxal phosphate upon inactivation of aldolase by this reagent; Cys-134 and Cys-177, buried, do not react with SH-reagents in the native enzyme.     

gamma-Glutamyl transpeptidase: relation to immunoglobulin A and free secretory component.

The experiments reported show that bovine γ-glutamyl transpeptidase can be separated from free secretory component. An ion-exchange Chromatographic procedure was developed to analyze the incubation mixtures of the enzyme with glutathione or S-(2-acetamido)-glutathione and glycylglycine. Using this system or the γ-glutamyl p-nitroanilide assay, no significant transpeptidase activity could be detected in the free secretory component-containing fractions of DEAE-cellulose chromatography. Gel filtration on Biogel A-5M showed that the bovine whey transpeptidase chromatographed in the void volume suggesting an aggregate of a minimum molecular weight of about 5 × 10⁶. The transpeptidase could be separated from all immunoglobulins in bovine whey and human colostrum by a combination of agarose gel filtration and immunoadsorption. Concentrated samples of human and sheep saliva showed normal amounts of secretory component, but no detectable γ-glutamyl transpeptidase activity. These experiments show that (1) the transpeptidase and secretory component are two different proteins, and (2) the transpeptidase is present in bovine and human milk as a high molecular weight aggregate which does not include any of the immunoglobulins.     

Aminolysis of thiazolinones in manual amino acid sequence analysis : Direct detection of carboxyl, amide, and tryptophan residues in conjunction with the dansyl-Edman method

Aminolysis of thiazolinones with methylamine and identification of the resulting phenylthiocarbamyl-amino acid methylamides were shown to be highly useful steps in manual sequence degradations. The effects of different conditions in the degradative steps on the detectability of phenylthiocarbamyl-amino acid methylamides were studied, and the influence of aminolysis on subsequent dansylations was examined. Phenylthiocarbamyl-amino acid methylamides may be identified by thin-layer chromatography or high-performance liquid chromatography without interference from thioureas or other background material. Aminolysis is especially useful when used in conjunction with the dansyl-Edman method. It provides a way to distinguish aspartic acid from asparagine, and glutamic acid from glutamine as well as a means of identification of tryptophan residues.     

A specific micromethod for determination of acetyl residues in proteins

A method is described for the separation and concentration of rat liver lysosomes from mitochondria in a one-step procedure by zonal centrifugation. Some of the practical problems associated with the use of the B-XX1X rotor are discussed.     

Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein

In this investigation, the interaction of mouse sperm with unfertilized eggs and embryos, solubilized zonae pellucidae isolated from eggs and embryos, and purified zona pellucida glycoproteins ZP1, 2, and 3 (J. D. Bleil, and P. M. Wassarman, (1980b) Dev. Biol. 76, 185-202) has been examined in vitro by light and electron microscopy. The experiments described were carried out in order to determine the temporal sequence of events during sperm-egg interaction in vitro and to identify the component(s) of zonae pellucidae responsible for inducing mouse sperm to undergo the acrosome reaction. "Pulse-chase" analysis of the sequence of sperm-egg interactions revealed that mouse sperm first "attach" loosely and then "bind" tightly to the unfertilized egg's zona pellucida. Binding of sperm to egg zonae pellucidae is followed by induction of the acrosome reaction. Induction of the acrosome reaction can be mediated by the zona pellucida, since solubilized zonae pellucidae isolated from unfertilized eggs were found to be just as effective as the calcium ionophore A23187 in inducing the reaction in vitro. Furthermore, ZP3 purified from zonae pellucidae isolated from unfertilized eggs, but not from two-cell embryos, was also just as effective as either solubilized zonae pellucidae from eggs or ionophore A23187 in inducing the acrosome reaction. ZP1 and 2 from both eggs and embryos, and ZP3 from embryos, had little effect on the extent of the acrosome reaction as compared to control samples. The results of these and other experiments (J. D. Bleil, and P. M. Wassarman, (1980b) Cell 20, 873-882) strongly suggest that, at least in vitro, mouse sperm recognize and bind to ZP3 of egg zonae pellucidae, and that such binding leads to the induction of the acrosome reaction. Modification of ZP3 following fertilization eliminates sperm binding to zonae pellucidae and, consequently, induction of the acrosome reaction is precluded.     

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride.

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Biosynthesis and amino terminal acetylation of cat hemoglobin B

Acetylation of the amino-terminal serine of the β chains of cat hemoglobin B (HbB) occurs during synthesis of hemoglobin in a mRNA-dependent protein synthesizing system from rabbit reticulocyte lysate in the presence of acetyl-CoA and cat reticulocyte mRNA. The process occurs after peptide chain growth of about 30 amino acid residues. When endogenous acetyl-CoA was removed from the rabbit reticulocyte lysate by pretreatment with oxalacetate and citrate synthase, nonacetylated HbB (HbB′) was synthesized. Thus, β^B globin chain synthesis goes to completion in the absence of acetylation even though the latter normally occurs during nascent chain growth. When HbB′ was incubated with acetyl-CoA in a rabbit reticulocyte lysate, hemoglobin with properties identical to those of HbB was produced. Thus, the selective amino terminal acetylation of β^B globin also occurs in the completed hemoglobin.