Deoxyribokinase from Salmonella typhimurium. Purification and properties

Deoxyribokinase, which catalyzes the ATP-dependent phosphorylation of 2-deoxy-d-ribose to 2-deoxy-d-ribose-5-P as the first step in the inducible fermentation pathway for this sugar in Salmonella typhimurium, was purified approximately 600-fold from deoxyribose-grown cells. Apparent K_m′s for 2-deoxy-d-ribose and ATP were 0.1 and 0.5 mm, respectively. The enzyme had an absolute requirement for divalent cations which was best satisfied by Mg²⁺. Optimal activity was obtained in the presence of 0.5 m NH₄⁺ or Cs⁺. Rb⁺ and K⁺ also stimulated enzyme activity whereas Na⁺ and Li⁺ inhibited. d-Ribose and 2-deoxy-d-ribitol could replace 2-deoxy-d-ribose as phosphoryl acceptor, and several ribo- and deoxyribonucleotides could replace ATP as phosphoryl donor. Molecular weight determinations gave values of 67,800 for the native enzyme and 33,500 for the dissociated enzyme, suggesting the probable existence of two subunits of similar size.     

Reactions of the amino groups of RNAse A: IV. The effects of protein concentration

A study has been made of the effect of ribonuclease (RNAse) concentration on the properties of the amino groups. The biphasic dependence of pK on pH which has been established (Goldfarb and Martin, Bioorg. Chem.5, 137 (1976)). for 5 μM solution of RNAse also have been shown to occur for 50 μM solutions. In the lower pH range (7.5–8.5) the values of pK obtained with 50 μM solutions were similar to those obtained with 5 μM solutions (pK = 7.5) but the intrinsic constants were smaller. In the higher pH range (8.5–10) the pKs in the more concentrated solutions were larger than those found at the smaller concentration and the intrinsic constants were generally smaller. A quantitative study of the concentration vs k_i relation at pH 7.5 indicated a sigmoid relationship for all of the subsets with a constant maximum value equal to, and less than that at 5 μM RNAse and a constant minimum value above that at 20 μM. Parallel studies with oxidized RNAse gave parallel, although not identical, results from which it is proposed that the concentration effect does not arise totally from the three-dimensional structure of native RNAse.     

Missense variant in TPI1 (Arg189Gln) causes neurologic deficits through structural changes in the triosephosphate isomerase catalytic site and reduced enzyme levels in vivo

Mutations in the gene triosephosphate isomerase (TPI) lead to a severe multisystem condition that is characterized by hemolytic anemia, a weakened immune system, and significant neurologic symptoms such as seizures, distal neuropathy, and intellectual disability. No effective therapy is available. Here we report a compound heterozygous patient with a novel TPI pathogenic variant (NM_000365.5:c.569G>A:p.(Arg189Gln)) in combination with the common (NM_000365.5:c.315G>C:p.(Glu104Asp)) allele. We characterized the novel variant by mutating the homologous Arg in Drosophila using a genomic engineering system, demonstrating that missense mutations at this position cause a strong loss of function. Compound heterozygote animals were generated and exhibit motor behavioural deficits and markedly reduced protein levels. Furthermore, examinations of the TPI^Arg189Gln/TPI^Glu104Asp patient fibroblasts confirmed the reduction of TPI levels, suggesting that Arg189Gln may also affect the stability of the protein. The Arg189 residue participates in two salt bridges on the backside of the TPI enzyme dimer, and we reveal that a mutation at this position alters the coordination of the substrate-binding site and important catalytic residues. Collectively, these data reveal a new human pathogenic variant associated with TPI deficiency, identify the Arg189 salt bridge as critical for organizing the catalytic site of the TPI enzyme, and demonstrates that reduced TPI levels are associated with human TPI deficiency. These findings advance our understanding of the molecular pathogenesis of the disease, and suggest new therapeutic avenues for pre-clinical trials.     

Study of copper(II) and nickel(II) ternary complexes involving tertiary amines and phenyl or hydroxylphenyl substituted amino acids

Formation constants of ternary complexes MAL, where M = Cu(II) or Ni(II). A = 2.2′bipyridyl. 1, 10-phenanthroline, and L = 3.4-dihydroxyphenylalanine (dopa), tyrosine, or phenylalanine have been determined by using the computer program SCOGS. It is observed that dopa coordinates with Cu(II)-A and Ni(II)-A through the aminocarboxylate and only over the pH range 3–8, though the ligand coordinates with free Cu(II) ion from the amino carboxylate end in the lower pH range (pH 2–4) and from the catechol end at the higher pH range (pH > 5). The visible spectrum of Cu-A-dopa is similar to that of Cu-A-phenylalanine or Cu-A-tyrosine over the entire pH range, confirming amino carboxylate coordination. Δ log K (K_MAL - log_KML) is found to be positive in all the six Cu(II) complexes. whereas it is negative in Ni(II) complexes. Release in the ternary complexes of the repulsion between the Cu(II) dπ electron and electrons delocalized over the phenyl ring has been proposed as a probable reason for the positive Δ log K.     

experimental in vitro organogensis and its modification by antibody directed to a cell surface antigen.

The hypothesis that cell-surface directed antibody may interfere with organogenesis was investigated by means of an experimental system wherein histotypic in vitro reconstruction of chick embryo mesonephros was modified by an heterologous Forssman antibody. The Forssman antigen is expressed at the surfaces of epithelial cells of the duct and collecting tubules in contrast to its absence from the rest of the nephron. Incubation of suspension of dissociated single cells from mesonephroi allows organotypic reconstruction of both F+ and F? elements when normal serum is added to the culture, while anti-F sera inhibit the reconstruction of F+ structures. It is proposed that these experiments reflect a set of circumstances that may occur in viviparous animals where transplacental antibodies are capable of harming the early embryo.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

Susceptibility of nontarget organisms to Nosema algerae (Microsporida: Nosematidae), a parasite of mosquitoes.

Host response of two penaeid species, Penaeus aztecus and P. setiferus, from the Gulf of Mexico to the pathogenic fungus Fusarium sp. isolated from the California brown shrimp, P. californiensis, was studied in vivo. The hemocytic response to this fungus was traced histologically in the gills. Both species showed complete resistance to infection by the fungal spores when normal or wounded shrimp were held in seawater containing the spores or when spores were injected directly into the shrimp in low concentrations. Complete melanization and encapsulation of the micro- and macroconidia were observed. Spore dosages of 3.2 × 10⁶ or more were lethal, apparently due to mechanical blockage of the blood sinuses of the gills.     

On the non-existence of eloxanthin

Abandonment of the name eloxanthin is proposed. The principal carotenoids in various species of Elodea were (3R, 3′R, 6′R)-lutein (β,ε-carotene-3, 3′-diol) and β, β-carotene. The minor pigments were neoxanthin-X (5′, 6′-epoxy-6, 7-didehydro-5, 6, 5′, 6′-tetrahydro-β, β-carotene-3, 5, 3′-triol), 9′-cis-neoxanthin- X, 9- and 13-cis-violaxanthin (5, 6, 5′, 6′-diepoxy-5, 6, 5′, 6′-tetrahydro-β, β-carotene-3, 3′-diol), antheraxanthin (5, 6-epoxy-5, 6-dihydro-β, β-carotene-3, 3′-diol), neolutein A (13- or 13′-cis-lutein) and neolutein B (9- or 9′-cis-lutein). All attempts to isolate eloxanthin failed.     

Stereochemistry of binding of thiophosphate analogs of ATP and ADP to carbamate kinase, glutamine synthetase, and carbamoyl-phosphate synthetase

Thiophosphate analogs of adenine nucleotides were used to establish the absolute stereochemistry of nucleotide substrates in the reactions of carbamate kinase (Streptococcus faecalis), unadenylylated glutamine synthetase (Escherichia coli), and carbamoyl-phosphate synthetase (E. coli). ³¹P NMR was used to determine that carbamate kinase uses the B isomer of Ado-5′-(2-thioPPP) in the presence of Mg²⁺. The stereospecificity of the reaction with carbamate kinase was not reversed by Cd²⁺ suggesting that the metal ion does not bind to the β-phosphoryl group or that both Mg²⁺ and Cd²⁺ bind to the sulfur atom. Carbamate kinase uses both A and B isomers of Ado-5′-(1-thioPP) with Mg²⁺ and Cd²⁺. We have previously reported that carbamoyl-phosphate synthetase uses the A isomer of Ado-5′-(2-thioPPP) at both ATP sites with Mg²⁺ (Raushel et al., 1978J. Biol. Chem.253, 6627). Current experiments show that the stereospecificity is reversed by Cd^2? and that both A and B isomers are used when Zn²⁺ is present. With Ado-5′-(1-thioPPP), the B isomer is used with Mg²⁺, the A isomer with Cd²⁺, and both isomers with Zn²⁺. Neither carbamate kinase nor carbamoyl-phosphate synthetase utilized Co(III)(NH₃)₄ATP as a substrate and thus we can only speculate that the Δ chelate ring configuration is the chelate structure utilized by carbamoyl-phosphate synthetase (based on the analogy between thiophosphate-ATP analogs and Co³⁺-ATP analogs utilized by hexokinase (E. K. Jaffe, and M. Cohn, 1978Biochemistry17, 652). If the sulfur of the β-phosphoryl of Ado-5′-(2-thioPPP) binds to the metal ion with carbamate kinase, then the Δ chelate ring is also used in this enzyme that catalyzes one of the steps in the overall reaction catalyzed by carbamoyl-phosphate synthetase. Glutamine synthetase reacts with the B isomer of both Ado-5′-(2-thioPPP) and Ado-5′-(1-thioPPP) in the presence of Mg²⁺. When Co²⁺ is used with this enzyme the A and B isomers of both thio-ATP compounds are substrates. Co(III)(NH₃)₄ATP is not a substrate for glutamine synthetase. Glutamine synthetase is therefore different from the two previously mentioned enzymes in that it used the opposite A ring configuration for the metal-ATP chelate.     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Inhibiton of human placental 17 beta-hydroxysteroid dehydrogenase by steroids and nonsteroidal alcohols: aspects of inhibitor structure and binding specificity

Inhibition of human placental 17β-hydroxysteroid dehydrogenase by C₁₈ and C₁₉ steroids and nonsteroidal alcohols was assayed at pH 9.0 with 17β-estradiol 3-methyl ether and NAD⁺ as reactants. The nonstaroidal alcohols tested were poor inhibitors. Cyclopentanol and cyclohexanol had K_i values greater than 5 mm. Nonaromatic C₁₈ and C₁₉ steroids with oxygen functions at both positions 3 and 17 gave no detectable inhibition or had K_i, values greater than or equal to 160 μm. 3μ-Hydroxy-5,16-androstadiene, 5-androsten-3β-ol, 1,3,5(10)-estratrien-3-ol, and 1,3,5(10),16-estratetraen-3-ol, steroids lacking a C(17) oxygen function, had K_i values of 1.8, 6.0, 0.04, and 0.17 μm, respectively, demonstrating that both C₁₈ and C₁₉ steroids can bind at the steroid site. Binding specificity is narrowed and binding affinity for nonaromatic steroids weakened by oxygen functions at C(17) or both C(3) and C(17). The structural implications of the specificity data for steroid recognition and complex formation and in vivo control of enzyme activity are discussed.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b₅ reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b₅ reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b₅ to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b₅ from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b₅ during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b₅ stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b₅ and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b₅ from rat liver microsomes was shown to inhibit erythrocyte cytochrome b₅ reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b₅ preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b₅ of human erythrocytes are immunochemically similar to NADH-cytochrome b₅ reductase and cytochrome b₅ of liver microsomes, respectively.     

Electiveness of rapair of plaque-type mutations induced by hydroxylamine in phage kappa of serratia

Phage ? treated extracellularly with hydroxylamine (HA) was preadsorbed to hcr, exr or wild-type (HY) host cells and plated with HY indicator. 5 plaque mutation types were scored. The frequency distributions of the 5 mutation types (mutation spectra) differed with the hosts, the spectrum in exr especially deviating significantly from those in hcr and HY. This indicates electiveness of repair of certain (pre-) mutations within different genomic regions. HA treatment time (dose) influenced the spectra, too, owing to three mutation types giving linear (one-hit) dose curves and two giving parabolic (about two-hit) curves. The host type did not influence these curve shapes. These findings show that the number of HA hits depends on the genomic region where (pre-) mutations occur.Inactivation of phages as well as cells was strongest in exr compared with hcr and HY hosts (factor 1.3). In contrast, induction of all 5 mutation types was lower in exr (factor about 0.5) and hcr (factor about 0.8) than in HY. This indicates that both repair types (probably post- and pre-replicative) are needed for perfecting part of the HA-induced mutations. The part lacking in repair-defective hosts may be caused by lethality within these hosts of certain premutative lesions. The frequency of mixed compared with pure mutant clones was small. Its dose dependence may be due to recessive lethal lesions within the non-premutated DNA strand.     

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

BACKGROUND: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes. METHODS: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV). RESULTS: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected. CONCLUSIONS: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.     

Simultaneous analysis of phenylglycols and phenylethanols in human urine by gas chromatography—mass spectrometry

A method is described for the determination of the neutral metabolites formed from catecholamines and various other structurally related phenylethylamines by using gas chromatography—chemical ionization—mass spectrometry. These metabolites (phenylglycols and phenylethanols) were extracted from urine specimens and converted to pentafluoropropionyl derivatives which were separated on either 3% OV-1, 3% SP-2250, or 3% QF-1 packed columns. Our results demonstrate the presence in human urine of p-hydroxyphenylglycol, a metabolite of octopamine. One patient excreted 13 and 91 μg/day of free and total (free + conjugated) p-hydroxyphenylglycol, respectively. Treatment with a monoamine oxidase inhibitor reduced the excretion of total p-hydroxyphenylglycol to 30% of baseline level.     

Occurrence of 15-cis-violaxanthin in Viola tricolor

A new cis isomer in the violaxanthin series has been isolated from the blossoms of Viola tricolor and identified by MS, IR and UV as the central-monocis form. It was converted to all-trans-violaxanthin by stereomutation. The CD correlation between 15-cis-violaxanthin and natural violaxanthin (5,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro- β,β-caroten-3,3′-diol) provided the basis for assignment of the absolute configurations 3S, 5R, 6S, 3′S, 5′R, 6′S. Trans—cis isomerization of all-trans-violaxanthin also resulted in 15- cis-violaxanthin. In addition a quantitative determination of the carotenoids was conducted.     

Synthesis of 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose and its 1,2,4-triacetate

Methyl phenylphosphonite or dimethyl phosphite underwent acid-catalyzed addition reactions with some hexofuranos-5-ulose 5-(p-tolylsulfonylhydrazones) (7, 9, and 16), to give the corresponding adducts, 17, 18, 19, and 21. The isomer ratios of the adducts were affected by a 3-substituent in the hydrazones. Treatment of adduct 21 with sodium borohydride and sodium dihydrobis(2-methoxyethoxy)-aluminate (SDMA), followed by acid hydrolysis, gave 5,6-dideoxy-3-O-methyl-5-C-(phenylphosphinyl)-d-glucopyranose (26), which was acetylated to give the 1,2,4-tri-O-acetyl derivatives 27a and 27b. Conformational analysis of compound 27a by X-ray crystallography revealed that the compound was 1,2,4-tri-O-acetyl-5,6-dideoxy-3-O-methyl-5-C-[(S)-phenylphosphinyl]-β-d-glucopyranose in the ⁴C₁(d) form having all substituents equatorial.     

Selenium derivatives of L-arabinose, D-ribose,and D-xylose

Attempted cyclization of 2,3,4-tri-O-methyl-5-seleno-L-arabinose dimethyl acetal in acidic solution gave the corresponding diselenide. Intramolecular attack by the selenobenzyl group at C-5 of 5-O-p-tolylsulfonyl-L-arabinose dibenzyl diseleno-acetal resulted in the formation of benzyl 1,5-diseleno-L-arabinopyranoside. Similarly, 2,3,5-tri-O-methyl-4-O-p-tolylsulfonyl-D-xylose dibenzyl diselenoacetal gave benzyl 2,3,5-tri-O-methyl-1,4-diseleno-L-arabinofuranoside, and 2,3,4-tri-O-acetyl-5-O-p-tolylsulfonyl-D-xylose (or ribose) dibenzyl diselenoacetal gave benzyl 2,3,4-tri-O-acetyl-1,5-diseleno-D-xylo- (or ribo-)pyranoside. The glycosylic benzylseleno group was removed from the pyranoside with mercuric acetate, but attempted deacetylation of the product led to decomposition and not to the expected 5-seleno-D-xylopyranose.     

Some characteristics of Δ1-pyrroline-5-carboxylate dehydrogenase in rat cerebellum

Δ¹-Pyrroline-5-carboxylate dehydrogenase (P5CDH) in rat cerebellum was assayed with a simple spectrophotometric method using high-speed supernatants of whole tissue homogenates. Kinetic analysis showed that the enzyme has K_m values of 0.83 ± 0.21 mM for Δ¹-pyrroline-5-carboxylate and 0.47 ± 0.02 mM for NAD⁺. Various cations inhibited P5CDH but only at relatively high concentrations. Several amino acids were strongly inhibitory in the order $\text{GABA}\text{>}\text{glycine}\text{>}\text{hydroxyproline}\text{>}\text{cysteine}\text{?}\text{proline}\text{>}\text{glutamine}\text{>}\text{glutamate}\text{>}\text{alanine}$.