The contributions of Stein and Moore to protein science.
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Biochemistry↗
Biomedical subjects
J M Manning
Publications and source records attributed to J M Manning.
Random chemical modification of the oxygen-linked chloride-binding sites of hemoglobin: those in the central dyad axis may influence the transition between deoxy- and oxy-hemoglobin.
The features of random chemical modification are defined with reference to acetylation of bovine hemoglobin, which has been performed in a random manner so that all of the amino groups that participate in functional chloride binding (i.e., those that are oxygen-linked) could be identified. Random chemical modification, which has objectives different from those of both specific (selective) and extensive chemical modification, has been achieved for bovine hemoglobin with the mild reagent, 14C-methyl acetate phosphate; retention of function was demonstrated by a Hill coefficient of n = 2.2 for the modified hemoglobin. After removal of unmodified Hb chains, the mixture of randomly modified acetylated alpha or beta chains was subjected to tandem treatment with trypsin and chymotrypsin. Peptides were purified by HPLC and identified by amino acid analysis. The amount of radioactivity in the acetylated amino group of a purified peptide was taken as an estimate of the degree of chloride binding. For bovine Hb, two amino groups of the alpha-chain (Val-1 and Lys-99) and three amino groups of the beta-chain (Met-1, Lys-81, and Lys-103) were shown to be oxygen-linked (i.e., to have incorporated significantly more radioactivity in the deoxy conformation compared to the same site in the oxy conformation). Three of these sites were already known chloride-binding sites [i.e., Val-1(alpha), the N-terminus of the alpha-chain, and two sites between the 2 beta-chains of bovine hemoglobin, Met-1(beta) and Lys-81(beta); these findings support the conclusions of the random modification approach. Two other chloride-binding sites, Lys-99(alpha) and Lys-103(beta), align the sides of the central dyad axis connecting the two well-known major chloride-binding sites of bovine Hb. The interrelationship of these five chloride-binding sites was assessed by improved molecular graphics. When viewed through the central dyad axis, the functional chloride-binding sites in the central cavity appear to be symmetrically related and to connect the two major chloride-binding sites. Modifiers or mutants that are directed at these regions in the central dyad axis may favor the deoxy conformation to provide a lower oxygen affinity by preventing the constriction of the central cavity that normally occurs upon oxygenation.
Acetylation↗
Seasonal changes in gonadotropin-releasing hormone secretion in the ewe: alteration in response to the negative feedback action of estradiol.
Two experiments were performed to test the hypothesis that there is a seasonal change in the negative feedback effect of estradiol on episodic secretion of GnRH in the ewe. The first experiment identified a specific estradiol treatment (delivered by s.c. Silastic implant) that produced a 50% decrease in the frequency of pulsatile secretion of LH in ovariectomized ewes during the anestrous season. In the second experiment, this estradiol treatment was administered to ovariectomized ewes during the mid-breeding and anestrous seasons. Separate groups of ovariectomized ewes not treated with estradiol were included during each season to test for a seasonal difference in the effect of estradiol on episodic GnRH and LH secretion. Samples of hypophyseal portal blood (for GnRH) and jugular blood (for LH) were obtained at 5-min intervals approximately one month after placement of the estradiol implants. During the breeding season, no effect of estradiol was observed on either the frequency or size of GnRH and LH pulses. During anestrus, however, estradiol produced a profound suppression of the frequency of GnRH and LH pulses, and an increase in GnRH pulse size. No significant seasonal change was observed in the characteristics of GnRH and LH pulses in ovariectomized ewes in the absence of estradiol treatment. These findings lead to the conclusion that there is a marked seasonal change in the negative feedback effect of estradiol on episodic GnRH secretion in the ewe, with the steroid being maximally effective during anestrus.
Anestrus↗
Partial reactions of bacterial D-amino acid transaminase with asparagine substituted for the lysine that binds coenzyme pyridoxal 5'-phosphate.
In bacterial D-amino acid transaminase (EC 2.6.1.21) replacement of Lys-145, which is covalently linked to the coenzyme pyridoxal 5'-phosphate in the wild-type enzyme, by an Asn residue gave a mutant enzyme (K145N) that slowly performed each half-reaction, as determined by spectral measurements. With the wild-type enzyme, the kinetics of these events were so rapid that pre-steady-state conditions were needed for their determination. The internal aldimine between coenzyme and Lys-145 was rapidly reduced with NaCNBH3 in the wild-type enzyme, whereas in the mutant enzyme the coenzyme, which is not covalently linked to the protein, was more resistant to reduction; the reduced forms of both wild-type and mutant enzymes were inactive. With large amounts of the K145N mutant enzyme and either amino acid or keto acid substrate alone, the formation of some reaction intermediates, i.e., the external aldimine with D-alanine and the ketimine with alpha-ketoglutarate, can be measured by conventional spectroscopy. Suicide substrates also induced slow spectral shifts of the E-PLP form of the enzyme. For the K145N enzyme, exogenous amines affected only the rate of the transaldimination but not the removal of the alpha-proton of the substrate. These results suggest that in the mutant enzyme some amino acid side chain other than Lys-145 performs this function. In order to identify this site, the K145N mutant enzyme was completely inactivated by the radiolabeled suicide substrate D-serine. Peptide mapping of tryptic digests showed that Lys-267 was the modified site.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Inactivation of dimeric D-amino acid transaminase by a normal substrate through formation of an unproductive coenzyme adduct in one subunit.
D-amino acid transaminase, which contains pyridoxal 5'-phosphate (vitamin B6) as coenzyme, catalyzes the formation of D-alanine and D-glutamate from their corresponding alpha-keto acids; these D-amino acids are required for bacterial cell wall biosynthesis. Under conditions usually used for kinetic assay of enzyme activity, i.e., short incubation times with dilute enzyme concentrations, D-alanine behaves as one of the best substrates. However, the enzyme slowly loses activity over a period of hours when exposed to substrates, intermediates, and products at equilibrium. The rate of inactivation is dependent on enzyme concentration but independent of substrate concentration greater than Km values. Continuous removal of the product pyruvate by enzymic reduction precludes the establishment of equilibrium and prevents inactivation. The formation of small but detectable amounts of a quinonoid intermediate absorbing at 493 nm is proportional to inactivation. Studies with [14C]-D-alanine labeled on different carbon atoms indicate that the alpha-carboxyl group of the substrate is absent in the inactive enzyme; such decarboxylation is not a usual function of this enzyme. The inactive transaminase contains 1.1 mol of [14C]-D-alanine-derived adduct per mole of dimeric enzyme; this finding is consistent with the 50% reduction in the fluorescence intensity at 390 nm (due to the PMP form of the coenzyme) for the inactive enzyme. Thus, inactivation of one subunit of the dimeric enzyme renders the entire molecule inactive. Inactivation may occur when a coenzyme intermediate, perhaps the ketimine, is slowly decarboxylated and then undergoes a conformational change from its catalytically competent location.(ABSTRACT TRUNCATED AT 250 WORDS)
Alanine↗
Nitric oxide can modify amino acid residues in proteins.
Nitric oxide derived from sodium nitroprusside binds to the heme moiety of hemoglobin and also modifies some functional groups in the protein. As hemoglobin concentration is increased, globin modification is decreased presumably due to formation of the NO complex with heme. The SH groups of hemoglobin are probably not involved in the formation of the stable product formed by NO. In the presence of inositol hexaphosphate, which binds preferentially in the cleft between the two beta-chains of hemoglobin, formation of one modified derivative was selectively reduced. With hemoglobin specifically blocked on its N-terminal residues, globin modification was also significantly reduced. Carbonic anhydrase, which is blocked at its N-terminus, was also refractory to modification. The results suggest that the N-terminal groups of some proteins can be modified by nitric oxide, perhaps by deamination.
Allosteric Regulation↗
Acylpeptide hydrolase: inhibitors and some active site residues of the human enzyme.
Acylpeptide hydrolase may be involved in N-terminal deacetylation of nascent polypeptide chains and of bioactive peptides. The activity of this enzyme from human erythrocytes is sensitive to anions such as chloride, nitrate, and fluoride. Furthermore, blocked amino acids act as competitive inhibitors of the enzyme. Acetyl leucine chloromethyl ketone has been employed to identify one active site residue as His-707. Diisopropylfluorophosphate has been used to identify a second active site residue as Ser-587. Chemical modification studies with a water-soluble carbodiimide implicate a carboxyl group in catalytic activity. These results and the sequence around these active site residues, especially near Ser-587, suggest that acylpeptide hydrolase contains a catalytic triad. The presence of a cysteine residue in the vicinity of the active site is suggested by the inactivation of the enzyme by sulfhydryl-modifying agents and also by a low amount of modification by the peptide chloromethyl ketone inhibitor. Ebelactone A, an inhibitor of the formyl aminopeptidase, the bacterial counterpart of eukaryotic acylpeptide hydrolase, was found to be an effective inhibitor of this enzyme. These findings suggest that acylpeptidase hydrolase is a member of a family of enzymes with extremely diverse functions.
Acetylation↗
The functional, oxygen-linked chloride binding sites of hemoglobin are contiguous within a channel in the central cavity.
Chloride ion is a major allosteric regulator for many hemoglobins and particularly for bovine hemoglobin. A site-directed reagent for amino groups, methyl acetyl phosphate, when used for global rather than selective modification of R (oxy) and T (deoxy) state bovine hemoglobin, can acetylate those functional amino groups involved in binding of chloride; the extensively acetylated hemoglobin tetramer retains nearly full cooperativity. The chloride-induced decrease in the oxygen affinity parallels the acetylation of bovine hemoglobin (i.e., their effects are mutually exclusive), suggesting that methyl acetyl phosphate is a good probe for the functional chloride binding sites in hemoglobins. Studies on the overall alkaline Bohr effect indicates that the part of the contribution dependent on chloride and reduced by 60% after acetylation is due to amino groups, Val-1(alpha) and Lys-81(beta); the remaining 40% is contributed by the imidazole side chain of His-146(beta), which is not acetylated by methyl acetyl phosphate, and is not dependent on chloride. The five amino groups--Val-1(alpha), Lys-99(alpha), Met-1(beta), Lys-81(beta), and Lys-103(beta)--of bovine hemoglobin that are acetylated in an oxygen-linked fashion are considered functional chloride binding sites. Molecular modeling indicates that these functional chloride binding sites are contiguous from one end of the central cavity of hemoglobin to the other; some of them are aligned within a chloride channel connecting each end of the dyad axis.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylation↗
Deficiency of acylpeptide hydrolase in small-cell lung carcinoma cell lines.
During protein biosynthesis, processing of the N terminus of many proteins may occur through acetylation and deacetylation. The enzyme acylpeptide hydrolase is likely involved in deacetylation of nascent peptide chains or of bioactive peptides. The related enzyme, acylase, hydrolyzes the acetyl amino acid product of the acylpeptide hydrolase reaction to acetate and a free amino acid. There is a reciprocal relationship between the substrates for these enzymes (i.e., substrates for one enzyme are competitive inhibitors for the other). In several cultured cell lines, including normal and malignant cells, the ratio of acylpeptide hydrolase to acylase enzyme activities appears to be coordinated and characteristic for a given cell type. Thus, in normal cultured lung cells, hamster ovary cells, hepatoma cells, and lymphocyte cells, nearly equal amounts of these enzymes are expressed, conducive to optimal processing of acetylated N-terminal residues. Four lines of erythroleukemic cell lines were found to express nearly twice as much acylase as acylpeptide hydrolase activity. In the Ehrlich ascites tumor cell line, where 80% of the proteins have been reported to remain acetylated at their N terminus, acylpeptide hydrolase is hardly expressed but acylase activity is not reduced. The 3p21 region of human chromosome 3, which contains the DNF15S2 locus that encodes acylpeptide hydrolase (Jones et al., Proc Natl Acad Sci USA 1991;88:2194), undergoes deletion in some carcinoma cells; the gene that encodes for the acylase is also present on region 3p of the same chromosome. We found that both acylpeptide hydrolase and acylase activities are practically absent in six small-cell lung carcinoma cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Amidohydrolases↗
Effects of methyl acetyl phosphate, a covalent antisickling agent, on the density profiles of sickle erythrocytes.
Methyl acetyl phosphate specifically acetylates valine-1, lysine-82, and lysine-144 in the 2,3-diphosphoglycerate binding cleft of hemoglobin S, thereby inhibiting its gelation (greater than 32 gm/dl) at pH 7.4. To extend these findings, the effect of methyl acetyl phosphate on the density of sickle cells has been evaluated by phthalate ester gradient centrifugation and by Larex-Percoll density centrifugation. After treatment with methyl acetyl phosphate (40% modification of the intracellular hemoglobin S), oxygenated sickle erythrocytes had a lowered density profile, as measured in a phthalate ester gradient. Thus 83% of untreated oxygenated sickle cells had densities greater than 1.098 gm/ml, whereas after treatment with methyl acetyl phosphate, 52% of the cells were in this density range. Under anaerobic conditions, methyl acetyl phosphate was even more effective in lowering cell density. For example, 50% of untreated deoxygenated cells had densities greater than 1.098 gm/ml, but none of the cells treated with methyl acetyl phosphate were this dense. For studies with Larex-Percoll density gradients, sickle erythrocytes were first separated into two fractions (densities greater than and less than 1.1 gm/ml) by Percoll-Hypaque centrifugation. The amount of oxygenated sickle cells exhibiting densities greater than 1.074 gm/ml decreased by about 32% on treatment with methyl acetyl phosphate. For deoxygenated sickle cells, treatment with methyl acetyl phosphate resulted in an average decrease of approximately 24% in the number of cells with densities greater than 1.074 gm/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Anemia, Sickle Cell↗
Effect of substitution of a lysyl residue that binds pyridoxal phosphate in thermostable D-amino acid aminotransferase by arginine and alanine.
Lys-145 of the thermostable D-amino acid aminotransferase, which binds pyridoxal phosphate, was replaced by Ala or Arg by site-directed mutagenesis. Both mutant enzymes were purified to homogeneity; their absorption spectra indicated that both mutant enzymes contained pyridoxal phosphate bound non-covalently. Even though the standard assay method did not indicate any activity with either mutant, addition of an amino donor, D-alanine, to the Arg-145 mutant enzyme led to a slow decrease in absorption at 392 nm with a concomitant increase in absorption at 333 nm. This result suggests that the enzyme was converted into the pyridoxamine phosphate form. The amount of pyruvate formed was almost equivalent to that of the reactive pyridoxal phosphate in the mutant enzyme. Thus, the Arg-145 mutant enzyme is able to catalyze slowly the half-reaction of transamination. Exogenous amines, such as methylamine, had no effect on the half-reaction with the Arg-145 mutant enzyme. In contrast, the Ala-145 mutant enzyme neither underwent the spectral change by addition of D-alanine nor catalyzed pyruvate formation, in the absence of added amine. However, the Ala-145 mutant enzyme catalyzed the half-reaction significantly in the presence of added amine. These findings suggest that a basic amino acid residue, such as lysine or arginine, is required at position 145 for catalysis of the half-reaction. The role of the exogenous amines differs with various active-site mutant enzymes.
Alanine↗
Preparation, properties, and plasma retention of human hemoglobin derivatives: comparison of uncrosslinked carboxymethylated hemoglobin with crosslinked tetrameric hemoglobin.
Human hemoglobin A has been crosslinked by diisothiocyanatobenzenesulfonate to give a limited number of products in a yield of approximately 70%. The predominant product was crosslinked between subunits within a tetramer and had a Mr of 64,000; no higher Mr species were formed. This product had one crosslink per tetramer located between the NH2 termini of its alpha chains, as established by HPLC analysis, amino acid analysis, Edman degradation, and mass spectrometry. This crosslinked derivative had a slightly increased oxygen affinity [P50 = 9 mmHg (1 mmHg = 133 Pa); P50 for unmodified hemoglobin = 11 mmHg], and the retention time of this derivative in the circulation of rats was 2.9 and 3.3 hr at two hemoglobin concentrations (7 g/dl and 14 g/dl, respectively). The half-life of an uncrosslinked carboxymethylated derivative, which has a low oxygen affinity (P50 = 28 mmHg), was 0.6 and 0.7 hr under the same conditions. Therefore, prolongation of the plasma-retention time of infused hemoglobin is dependent on the crosslinking of the tetramer but independent of the oxygen affinity of the derivative.
Allosteric Regulation↗
Genetic relationship between acylpeptide hydrolase and acylase, two hydrolytic enzymes with similar binding but different catalytic specificities.
An 87% identity has been found between the reported cDNA sequence that encodes acylpeptide hydrolase (EC 3.4.19.1) [Mitta, M., Asada, K., Uchimura, Y., Kimizuka, F., Kato, I., Sakiyama, F. & Tsunasawa, S. (1989) J. Biochem. 106, 548-551] and a cDNA transcribed from a locus (DNF15S2) on the short arm of human chromosome 3, reported by Naylor et al. [Naylor, S.L., Marshall, A., Hensel, C., Martinez, P.F., Holley, B. & Sakaguchi, A.Y. (1989) Genomics 4, 355-361]; the DNF15S2 locus suffers deletions in small cell lung carcinoma associated with a reduction or loss of acylase activity (EC 3.5.1.14). Acylpeptide hydrolase catalyzes the hydrolysis of the terminal acetylated amino acid preferentially from small acetylated peptides. The acetylamino acid formed by acylpeptide hydrolase is further processed to acetate and a free amino acid by an acylase. The substrates for the acylpeptide hydrolase and the acylase behave in a reciprocal manner since acylpeptide hydrolase binds but does not process acetylamino acids and the acylase binds acetylpeptides but does not hydrolyze them; however, the two enzymes share the same specificity for the acyl group. These findings indicate some common functional features in the protein structures of these two enzymes. Since the gene coding for acylpeptide hydrolase is within the same region of human chromosome 3 (3p21) that codes for the acylase and deletions at this locus are also associated with a decrease in acylase activity, there is a close genetic relationship between the two enzymes. There could also be a relationship between the expression of these two enzymes and acetylated peptide growth factors in some carcinomas.
Alanine↗
Covalent inhibitors of the gelation of sickle cell hemoglobin and their effects on function.
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Antisickling Agents↗
The energetic regulation of ovulation: a realistic role for body fat.
This review weighs the evidence for and against the hypothesis that ovulation is regulated by a critical amount of body fat. The evidence supporting this hypothesis is correlative, and most of it stems from observations made in humans. On balance, the evidence from human studies does not support the hypothesis, however, and the results of animal studies argue strongly against it. In the latter regard, a variety of experimental approaches have been tried in both adult and peripubertal females of several species, and the results almost uniformly show little relationship between fatness and ovulation. There is no doubt that ovulation can be regulated somehow in relation to whole-body energy balance and that fat stores are an important component of energy balance, but there is no reason to accord body fat a direct causal role in regulating ovulation.
Adipose Tissue↗
Suppression of puberty in rats by exercise: effects on hormone levels and reversal with GnRH infusion.
In an effort to better understand the effects of prolonged exercise on the hypothalamic-pituitary-ovarian axis, we made a variety of comparisons between young female rats in two treatment groups: 1) prolonged exercise, in which growth and reproductive development were arrested at a peripubertal stage by requiring rats to run for long periods of time in order to obtain food; and 2) voluntary exercise, in which same-aged control rats were fed ad libitum and given free access to a running wheel. The pulsatile secretion of luteinizing hormone (LH) and growth hormone were completely suppressed by the prolonged exercise treatment. Mean levels of follicle-stimulating hormone, prolactin, and thyroid-stimulating hormone were not affected. Prolonged exercise elevated corticosterone titers, and the secretory pattern of this steroid was changed out of phase with running activity. Tissue levels of gonadotropin-releasing hormone (GnRH) in the hypothalamus and LH in the pituitary were enhanced, not suppressed, by prolonged exercise. Most importantly, pulsatile infusions of GnRH reinstated normal pubertal development and ovulation in rats still growth restricted by the prolonged exercise treatment. The results of this study indicate that the suppressive effects of prolonged exercise somehow affect the production of the hypothalamic GnRH-pulse generator signal.
Animals↗
Effects of a resistive training program on lipoprotein--lipid levels in obese women.
The purpose of this study was to determine the effects of a resistive training program on the time course of changes in strength, body mass index, lipids, lipoproteins, and apolipoproteins in sedentary obese women. Sixteen sedentary obese women strength trained 3 times . wk-1 for 12 wk performing three sets of six to eight repetitions per set with sets 1 and 2 at 60-70% of one-repetition maximum. During set 3, the subjects used the greatest weight possible so that failure occurred between six to eight repetitions. Six sedentary obese women served as controls. Blood samples for serum total cholesterol (TC), high-density lipoproteins (HDL-C), low-density lipoproteins (LDL-C), triglycerides (TG), TC/HDL-C ratio, apolipoprotein A-I (apo A-I), and apolipoprotein B-100 (apo B-100) were obtained pre, and after 4, 8, and 12 wk of training and approximately 3-4 d following the last training session. A 3-d dietary record was obtained on all subjects pre and post, and subjects were instructed not to alter their diet. The 12 wk of resistive training did not result in a significant change in body weight, BMI, or total kilocalories consumed per day but did show a mean improvement of 58% in muscular strength (P less than 0.05). The training program did not significantly alter the TC, HDL-C, LDL-C, TG, TC/HDL-C ratio, apo A-I, or apo B-100 levels, which suggests that this increase in strength owing to resistive training in the absence of body weight loss did not alter the lipid profiles in these sedentary obese women.
Adult↗
Substitution of glutamine for lysine at the pyridoxal phosphate binding site of bacterial D-amino acid transaminase. Effects of exogenous amines on the slow formation of intermediates.
In bacterial D-amino acid transaminase, Lys-145, which binds the coenzyme pyridoxal 5'-phosphate in Schiff base linkage, was changed to Gln-145 by site-directed mutagenesis (K145Q). The mutant enzyme had 0.015% the activity of the wild-type enzyme and was capable of forming a Schiff base with D-alanine; this external aldimine was formed over a period of minutes depending upon the D-alanine concentration. The transformation of the pyridoxal-5'-phosphate form of the enzyme to the pyridoxamine-5'-phosphate form (i.e. the half-reaction of transamination) occurred over a period of hours with this mutant enzyme. Thus, information on these two steps in the reaction and on the factors that influence them can readily be obtained with this mutant enzyme. In contrast, these reactions with the wild-type enzyme occur at much faster rates and are not easily studied separately. The mutant enzyme shows distinct preference for D- over L-alanine as substrates but it does so about 50-fold less effectively than the wild-type enzyme. Thus, Lys-145 probably acts in concert with the coenzyme and other functional side chain(s) to lead to efficient and stereochemically precise transamination in the wild-type enzyme. The addition of exogenous amines, ethanolamine or methyl amine, increased the rate of external aldimine formation with D-alanine and the mutant enzyme but the subsequent transformation to the pyridoxamine-5'-phosphate form of the enzyme was unaffected by exogenous amines. The wild-type enzyme displayed a large negative trough in the circular dichroic spectrum at 420 nm, which was practically absent in the mutant enzyme. However, addition of D-alanine to the mutant enzyme generated this negative Cotton effect (due to formation of the external aldimine with D-alanine). This circular dichroism band gradually collapsed in parallel with the transformation to the pyridoxamine-5'-phosphate enzyme. Further studies on this mutant enzyme, which displays the characteristics of the wild-type enzyme but at attenuated rates, may yield information on the factors controlling the stereochemistry of the reaction as well as on the catalytic steps of the transaminase pathway.
Base Sequence↗