Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Technetium-99m amino acid chelates: correlation of their physico-chemical and physiological parameters. Part I

Ten alpha-aminocarboxylic acids were chelated with 99mTc and purified by sephadex gel chromatography. Their hepatobiliary and renal excretion patterns were determined in rats. It was observed that lipophilicity of these chelates is the only determinant in governing their hepatobiliary excretion, whereas their renal excretion is dependent on some other factors in addition to lipophilicity. Depression of renal excretion in presence of probenecid indicated an interaction of renal tubular transport enzymes with these chelates, which was explained from their hexacoordinated dioxotechnetium (V) chelate structure (II).     

ΔμH+ dependency of in vitro protein translocation into Escherichia coli inner-membrane vesicles varies with the signal-sequence core-region composition

Signal sequences frequently contain α-helix-destabilizing amino acids in the hydrophobic core. Nuclear magnetic resonance studies on the conformation of signal sequences in membrane mimetic environments revealed that these residues cause a break in the α-helix. In the precursor of the Escherichia coli outer membrane protein PhoE (pre-PhoE), a glycine residue at position -10 (Gly^?10) is thought to be responsible for the break in the α-helix. We investigated the role of this glycine residue in the translocation process by employing site-directed mutagenesis. SDS-PAGE analysis showed drastic variations in the electrophoretic mobilities of the mutant precursor proteins, suggesting an important role of the glycine residue in determining the conformation of the signal sequence. In vivo, no drastic differences in the translocation kinetics were observed as compared with wild-type PhoE, except when a charged residue (Arg) was substituted for Gly^?10. However, the in vitro translocation of all mutant proteins into inverted inner-membrane vesicles was affected. Two classes of precursors could be distinguished. Translocation of one class of mutant proteins (Ala, Cys and Leu for Gly^?10) was almost independent of the presence of a ΔμH⁺, whereas translocation of the other class of precursors (wild type or Ser) was strongly decreased in the absence of the ΔμH⁺. Apparently, the ΔμH⁺ dependency of in vitro protein translocation varies with the signal-sequence core-region composition. Furthermore, a proline residue at position -10 resulted in a signal sequence that did not prevent the folding of the precursor in an in vitro trimerization assay.     

Bmi‐1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance

To determine whether Bmi-1 deficiency could lead to renal tubulointerstitial injury by mitochondrial dysfunction and increased oxidative stress in the kidney, 3-week-old Bmi-1^-/- mice were treated with the antioxidant N-acetylcysteine (NAC, 1 mg mL⁻¹) in their drinking water, or pyrro-quinoline quinone (PQQ, 4 mg kg⁻¹ diet) in their diet for 2 weeks, and their renal phenotypes were compared with vehicle-treated Bmi1^-/- and wild-type mice. Bmi-1 was knocked down in human renal proximal tubular epithelial (HK2) cells which were treated with 1 mm NAC for 72 or 96 h, and their phenotypes were compared with control cells. Five-week-old vehicle-treated Bmi-1^-/- mice displayed renal interstitial fibrosis, tubular atrophy, and severe renal function impairment with decreased renal cell proliferation, increased renal cell apoptosis and senescence, and inflammatory cell infiltration. Impaired mitochondrial structure, decreased mitochondrial numbers, and increased oxidative stress occurred in Bmi-1^-/- mice; subsequently, this caused DNA damage, the activation of TGF-β1/Smad signaling, and the imbalance between extracellular matrix synthesis and degradation. Oxidative stress-induced epithelial-to-mesenchymal transition of renal tubular epithelial cells was enhanced in Bmi-1 knocked down HK2 cells. All phenotypic alterations caused by Bmi-1 deficiency were ameliorated by antioxidant treatment. These findings indicate that Bmi-1 plays a critical role in protection from renal tubulointerstitial injury by maintaining redox balance and will be a novel therapeutic target for preventing renal tubulointerstitial injury.     

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K⁺ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K⁺ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.     

Conformational behaviour of A cyclolinopeptide a analogue: Two-dimensional NMR study of cyclo(Pro1-Pro-Phe-Phe-Ac6c-IIe-ala-Val8)

The cyclic octapeptide cyclo[-Pro¹-Pro-Phe-Phe-Ac₆c-Ile-ala-Val⁸-] [C₈-Ac₆c], containing the Pro¹-Pro-Phe-Phe sequence, followed by a bulky helicogenic C^α,α-dialkylated glycine residue Ac₆c (1-aminocyclohexane-1-carboxylic acid), and a _D-Ala residue at position 7 has been synthesized. This cyclic peptide is a deletion analogue of the naturally occurring cyclic nonapeptide cyclolinopeptide A (CLA). It has been designed with the aim of studying the role that the Ac₆c and _D-Ala residues play on the conformational behaviour of the whole molecule and their influence on the conformation of the Pro¹-Pro-Phe-Phe sequence when compared with cyclolinopeptide A. C₈Ac₆c has been investigated in chloroform and acetonitrile solutions by 2D NMR techniques. Only one set of sharp signals is observed in both solvents. This evidence strongly supports the hypothesis that only one conformational state exists in the chosen solvents. The interpretation of the experimental data points to the existence for C₈-Ac₆c of a very rigid structure stabilized by intramolecular hydrogen bonds. The measured NOE effects allow the calculation of internuclear distances, which have been used as restraints in molecular dynamic calculations. The proposed conformation of the molecule shows that the Pro-Pro-Phe segment retains the conformation observed in natural CLA both in solution and in the solid state and that the Ac₆c residue indeed reinforces the ring rigidity not permitting the formation of any appropriate cavity in which inorganic cations could be complexed.     

Sodium gradient dependence of proline and glycine uptake in rat renal brush-border membrane vesicles

The sodium-dependent entry of proline and glycine into rat renal brushborder membrane vesicles was examined. The high K_m system for proline shows no sodium dependence. The low K_m system for glycine entry is strictly dependent on a Na⁺ gradient but shows no evidence of the carrier system having any affinity for Na⁺. The low K_m system for proline and high K_m system for glycine transport appear to be shared. Both systems are stimulated by a Na⁺ gradient and appear to have an affinity for the Na⁺. The effect of decreasing the Na⁺ concentration in the ionic gradient is to alter the K_m for amino acid entry and, at low Na⁺ concentrations, to inhibit the V for glycine entry.     

Altered glycine decarboxylation inhibition in isonicotinic Acid hydrazide-resistant mutant callus lines and in regenerated plants and seed progeny

Isonicotinic acid hydrazide (INH), an inhibitor of the photorespiratory pathway blocking the conversion of glycine to serine and CO₂, has been used as a selective agent to obtain INH-resistant tobacco (Nicotiana tabacum) callus cells. Of 22 cell lines that were INH-resistant, none were different from wild-type cells in their ability to take up [³H]INH or to oxidize INH to isonicotinic acid. In 7 of the 22 cell lines, INH resistance was associated with decreased inhibition of NAD-dependent glycine decarboxylation activity in isolated mitochondrial preparations. In the cell line that was most extensively investigated (I 24), this biochemical phenotype (exhibiting a 3-fold higher K_i with INH) was observed in leaf mitochondria of regenerated plants and of plants produced from them by self-fertilization. After crosses between resistant and sensitive plants, the decreased inhibition of glycine decarboxylation was observed among F₂ and backcross progeny only in those plants previously identified as INH-resistant by callus growth tests. In contrast, in siblings identified as INH-sensitive, glycine decarboxylation was inhibited by INH at the wild-type level. This demonstration of the transfer of an altered enzyme property from callus to regenerated plants and through seed progeny fulfills an important requirement for the use of somatic cell genetics to produce biochemical mutants of higher plants.     

The synthesis of phosvitin in vitro by slices of liver from the laying hen

1. Slices of liver from laying hens incorporated Na₂¹⁴CO₃ and NaH₂³²PO₄ into phosvitin. Slices of liver from immature birds did not do so to any appreciable extent. The ³²P was incorporated into O-phosphorylserine in the phosvitin molecule. 2. Kidney, spleen, muscle, large and small intestine, ovary and oviduct from laying birds did not incorporate Na₂¹⁴CO₃ into phosvitin. 3. Slices of liver from laying hens carried out a net synthesis of phosphoprotein under the standard conditions of incubation. Slices from the livers of immature pullets did not do so. 4. Liver from the laying hen incorporated [2-¹⁴C]glycine, [3-¹⁴C]serine and [2-¹⁴C]glutamic acid into phosvitin. Part of the glycine was shown to be present as serine in the final product. 5. Slices of liver from immature birds treated with oestradiol synthesized phosvitin from [2-¹⁴C]glycine, but the addition of oestrogens in vitro to slices from untreated immature birds did not promote synthesis during a 3 hr. incubation period.     

Nucleotide sequence determination of bacteriophage T4 glycine transfer ribonucleic acid

The nucleotide sequence of a T4 tRNA with an anticodon for glycine has been determined using ³²P-labeled material from T4-infected cultures of Escherichia coli. The sequence is: pGCGGAUAUCGUAUAAUGmGDAUUACCUCAGACUUCCAAψCUGAUGAUGUGAGTψCGAUUCUCAUUAUCCGCUCCA-OH. The 74 nucleotide sequence can be arranged in the classic cloverleaf pattern for tRNAs. The anticodon of T4 tRNA^Gly is UCC with a possible modification of the U. The tRNA molecule would thus be expected to recognize the glycine codons GGG and GGA. Comparative analysis of tRNAs^Gly from T2 and T6 indicate that their sequences are identical with that from T4.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.     

Adult human CD133/1 kidney cells isolated from papilla integrate into developing kidney tubules

Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin⁺ and CD133/1⁺ cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1⁺ cells. Isolated CD133/1⁺ papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1⁺ progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Capillary electrophoresis of 99mtechnetium radiopharmaceuticals

Diagnostically used ^99mTc kit radiopharmaceuticals were analyzed using capillary zone electrophoresis with radioactivity detection: ^99mTc-bis(bis(2-ethyloxyethyl)phosphino)ethane (^99mTc-Myoview, ^99mTc-Tetrofosmin), ^99mTc-trans(1,2-bis(dehydro-2,2,5,5,-tetramethyl-3-furanone-4-methylene-amino)ethane)-tris(3-methoxy-1-propyl)phosphine) (^99mTc-Technescan Q12, ^99mTc-Furifosmin), ^99mTc-methoxyisobutylisonitrile (^99mTc-MIBI), ^99mTc-l,l-ethylenecysteine diethylester dimer (^99mTc-ECD), ^99mTc-d,l-hexamethylene propyleneamine oxime (^99mTc-HMPAO), ^99mTc-diethylenetriaminepentaacetic acid (^99mTc-DTPA), ^99mTc-ethylene hepatobiliary iminodiacetic acid (^99mTc-EHIDA), ^99mTc-l,l-ethylenecysteine dimer (^99mTc-EC), ^99mTc-mercaptoacetylglycylglycylglycine (^99mTc-MAG₃), ^99mTc-dimercaptosuccinic acid (^99mTc-DMSA), ^99mTc-methylene diphosphonate (^99mTc-MDP) and ^99mNaTcO₄. A pressure-driven capillary zone electrophoresis was employed to detect small anions of high electrophoretic mobility and cations within one run. Effective ^99mTc complex charges could be determined by a neutral internal standard. All complexes showed the expected electrophoretic behaviours in view of their charges. Pure products were obtained for the majority of the studied complexes. In the case of ^99mTc-Q12, ^99mTc-EHIDA and ^99mTc-MDP, complex mixtures were detected. The high potential of CE for the analysis of ^99mTc radiopharmaceuticals could be shown.     

Functional Interaction Between Release Factor One and P-site Peptidyl-tRNA on the Ribosome

Translation termination at UAG is influenced by the nature of the 5′ flanking codon inEscherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA⁺) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA₃^ProversustRNA₂^Pro, tRNA₄^ThrversustRNA_1,3^Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA₂^Gly) and GGG (tRNA_2,3^Gly) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High α-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.     

N- and [C]NMR determination of utilization of glycine for synthesis of storage protein in the presence of glutamine in developing cotyledons of soybean

Solid-state ¹⁵N- and [¹³C] NMR have been used to measure quantitatively the utilization of glycine in the presence of glutamine for the synthesis of storage protein in immature cotyledons of soybean (Glycine max L. cv. Elf) in culture. The presence of an equal molar amount of glycine in the medium causes a decrease in the use of glutamine-amide nitrogen. Glycine nitrogen is incorporated extensively into peptide bonds (in amounts greater than what would be expected if it appeared solely in glycine residues), but is used sparingly for synthesis of histidine ring residues, guanidino nitrogen residues of arginine, and lysine residues. The modest use of glycine carbon in protein synthesis does not parallel the use of glycine nitrogen.     

Stimulation of CDP-choline biosynthesis by enantiomeric lysophosphatidylcholines in rat intestinal mucosa

The phosphorylcholine cytidyltransferase activity of the cytosol fractions of rat intestinal crypt and villus cells was observed to be stimulated by addition of 3-sn-lysophosphatidylcholine. A 15- to 20-fold increase in activity was seen following a prior removal of phospholipids with acetone and butanol. 3-sn-Lysophosphatidylcholines of C¹⁶ and C¹⁸ carbon fatty acids were three to four times more effective than those with fatty chains of shorter length. The 3-sn-phosphatidyl and 3-sn-lysophosphatidyl ethanolamine, inositol, and serine were ineffective. 3-sn-Lysophosphatidic acid and 3-sn-glycerophosphorylcholine did not stimulate the transferase activity indicating that both fatty acyl and phosphorylcholine moieties of the 3-sn-lysophosphatidylcholine molecule were required for the observed effect. Addition of the enantiomeric 1-sn-lysophosphatidylcholine had an effect comparable to that of 3-sn-lysophosphatidylcholine on the phosphorylcholine cytidyltransferase activity when assayed in rat intestinal mucosa and in pigeon liver. It is suggested that the stimulatory effect of lysophosphatidylcholine may be related to a specific detergent property dependent upon the peculiar balance of hydrophilic and hydrophobic components in the molecule.     

Identification of an ATP-Driven, Osmoregulated Glycine Betaine Transport System in Listeria monocytogenes

The ability of the gram-positive, food-borne pathogen Listeria monocytogenes to tolerate environments of elevated osmolarity and reduced temperature is due in part to the transport and accumulation of the osmolyte glycine betaine. Previously we showed that glycine betaine transport was the result of Na⁺-glycine betaine symport. In this report, we identify a second glycine betaine transporter from L. monocytogenes which is osmotically activated but does not require a high concentration of Na⁺ for activity. By using a pool of Tn917-LTV3 mutants, a salt- and chill-sensitive mutant which was also found to be impaired in its ability to transport glycine betaine was isolated. DNA sequence analysis of the region flanking the site of transposon insertion revealed three open reading frames homologous to opuA from Bacillus subtilis and proU from Escherichia coli, both of which encode glycine betaine transport systems that belong to the superfamily of ATP-dependent transporters. The three open reading frames are closely spaced, suggesting that they are arranged in an operon. Moreover, a region upstream from the first reading frame was found to be homologous to the promoter regions of both opuA and proU. One unusual feature not shared with these other two systems is that the start codons for two of the open reading frames in L. monocytogenes appear to be TTG. That glycine betaine uptake is nearly eliminated in the mutant strain when it is assayed in the absence of Na⁺ is an indication that only the ATP-dependent transporter and the Na⁺-glycine betaine symporter occur in L. monocytogenes.     

The natriuretic mechanism of Gamma-Melanocyte-Stimulating Hormone

Gamma-Melanocyte Stimulating Hormone (Gamma-MSH) regulates sodium (Na⁺) balance and blood pressure through activation of the melanocortin receptor 3 (MC3-R). The mechanism of the natriuretic effect is proposed to involve binding of MC3-R either in the kidney to directly inhibit tubular Na⁺ transport or in the brain to inhibit central neural pathways that control renal tubular Na⁺ absorption. This study aimed to clarify the mechanism involved in the natriuretic effect of Gamma-MSH on MC3-R in kidney cells. In Ussing chamber studies, we observed no effects of Gamma-MSH on NaCl transport in the mouse inner medullary collecting duct cell line (mIMCD-K2). We also found that neither MC3-R protein nor mRNA was expressed in mouse kidney, suggesting that renal Gamma-MSH action may not be mediated through direct effects on tubular Na⁺ transport but rather through effects on central neural pathways that innervate the kidney.     

Glycine as a precursor of bacterial purines

The synthesis of purines in Aerobacter aerogenes was studied by the use of labeled glycine [CH₂(N¹⁵H₂)C¹³OOH]. Adenine and guanine were isolated from the lyophilized cell material. The isotopes were found in the bases. Evidence for the incorporation of the whole glycine molecule is presented.     

Structure and biological activity of hagfish insulin

An isomorphously phased electron density map of hagfish (Myxine glutinosa) insulin has been calculated at a resolution of 3·1 Å spacing. The molecule crystallises with one molecule per asymmetric unit but is organised as a symmetric dimer lying on a 2-fold crystal axis. The structure of the hagfish insulin monomer is much more similar to that of pig insulin molecule 2 than molecule 1 of the dimer that constitutes one third of the 2 Zn insulin hexamer. There are different conformations however at the N and C termini of the B-chain. At the C terminus the two final residues on hagfish insulin partially obscure the A1 glycine residue, which in pig insulin is exposed. This structural difference has been shown, however, not to be responsible for the reduced activity of the hagfish insulin.