Vitamin B12-binding Protein Research Articles

Selective Inhibition of Human Neutrophil Functional Responsiveness by Erbstatin, an Inhibitor of Tyrosine Protein Kinase

The role of tyrosine kinases in the responses of human neutrophils to chemotactic factors was examined using the recently described inhibitor erbstatin. Pre-incubation with erbstatin decreased the amount of tyrosine phosphorylation induced by the formylated oligopeptide formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) without effecting the binding of [3H]-fMet-Leu-Phe. Erbstatin also dose-dependently inhibited the production of superoxide anion induced by fMet-Leu-Phe and platelet-activating factor, but did not affect the oxidative burst induced by either the calcium ionophore A23187 or the phorbol ester phorbol 12-myristate 13-acetate. Furthermore, erbstatin diminished the cytosolic acidification elicited by fMet-Leu-Phe, platelet-activating factor, and leukotriene B4. In contrast, erbstatin was without effect on the increase in the levels of cytoplasmic free calcium and polymerized actin elicited by fMet-Leu-Phe, C5a, leukotriene B4 and platelet-activating factor, whereas the increase in cytoplasmic free calcium elicited by platelet-derived growth factor was inhibited by erbstatin. In addition, erbstatin affected neither the release of elastase stimulated by these agonists nor the release of beta-glucosaminidase, lysozyme or vitamin B12-binding protein induced by fMet-Leu-Phe. These results indicate that tyrosine protein kinases are involved in the signaling pathways employed by chemotactic factors in the stimulation of selective functional responses (and superoxide production in particular) in human neutrophils.

Selective inhibition of human neutrophil functional responsiveness by erbstatin, an inhibitor of tyrosine protein kinase

The role of tyrosine kinases in the responses of human neutrophils to chemotactic factors was examined using the recently described inhibitor erbstatin. Pre-incubation with erbstatin decreased the amount of tyrosine phosphorylation induced by the formylated oligopeptide formyl- methionyl-leucyl-phenylalanine (fMet-Leu-Phe) without effecting the binding of [3H]-fMet-Leu-Phe. Erbstatin also dose-dependently inhibited the production of superoxide anion induced by fMet-Leu-Phe and platelet- activating factor, but did not affect the oxidative burst induced by either the calcium ionophore A23187 or the phorbol ester phorbol 12- myristate 13-acetate. Furthermore, erbstatin diminished the cytosolic acidification elicited by fMet-Leu-Phe, platelet-activating factor, and leukotriene B4. In contrast, erbstatin was without effect on the increase in the levels of cytoplasmic free calcium and polymerized actin elicited by fMet-Leu-Phe, C5a, leukotriene B4 and platelet- activating factor, whereas the increase in cytoplasmic free calcium elicited by platelet-derived growth factor was inhibited by erbstatin. In addition, erbstatin affected neither the release of elastase stimulated by these agonists nor the release of beta-glucosaminidase, lysozyme or vitamin B12-binding protein induced by fMet-Leu-Phe. These results indicate that tyrosine protein kinases are involved in the signaling pathways employed by chemotactic factors in the stimulation of selective functional responses (and superoxide production in particular) in human neutrophils.

Proinftammatory Interactions of Pyocyanin and I-Hydroxyphenazine with Human Neutrophils In Vitro

The effects of the Pseudomonas aeruginosa-derived pigments, pyocyanin and 1-hydroxyphenazine (1-hp), on membrane-associated oxidative metabolism and release of lysozomal enzymes by human neutrophils were investigated in vitro. Pyocyanin, but not 1-hp, increased the generation of superoxide and the rate and duration of oxygen uptake by activated neutrophils. Both agents increased the myeloperoxidase-mediated iodinating activity of neutrophils, which in the case of 1-hp was due to stimulation of the release of myeloperoxidase by activated neutrophils. 1-hp also increased the release of lysozyme by activated neutrophils. Pyocyanin caused only slight enhancement of the release of myeloperoxidase and lysozyme by stimulated neutrophils but was more potent with respect to the release of the specific granule marker, vitamin B12-binding protein. These data indicate the existence of diverse, proinflammatory interactions of pyocyanin and 1-hp with human phagocytes, which may intensify neutrophil-mediated tissue damage during P. aeruginosa infections.

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2−) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2+-containing and Ca2+-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2− production in a dose-dependent manner. In cytochalasin B-treated fMet-Leu-Phe-induced O2− production. In Ca2+-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimntal conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.

Differential augmentation by recombinant human tumor necrosis factor-alpha of neutrophil responses to particulate zymosan and glucan.

Zymosan (Z) and its major insoluble carbohydrate component beta-linked glucan activate human neutrophils (PMN) through a trypsin-sensitive recognition mechanism. This mechanism is believed to involve the PMN CR3R. Both Z and glucan generated dose and time-dependent release of the secondary lysosomal granule marker vitamin B12 binding protein, leukotriene B4 (LTB4) and superoxide from PMN and were phagocytosed with similar dose-dependent kinetics. The PMN superoxide and LTB4 responses to glucan; however, were consistently greater than those to the same doses of Z. The phagocytosis of both particles was significantly reduced after partial digestion with beta-laminarinase but not beta-glucosidase or alpha-mannosidase suggesting a recognition mechanism dependent on intact beta-1,3-glucosidic bonds in both particles. TNF-alpha (rhTNF-alpha) promoted a time- and dose-dependent increase in the expression of PMN CR3 up to 60 min. The increased expression of CR3 was paralleled by the release of the secondary lysosomal granule marker vitamin B12-binding protein. This granule contains a population of CR3R in its boundary membrane and it is the fusion of this membrane with the plasma membrane that may represent the mechanism by which CR3 expression is increased. Preincubation of PMN with 10(-9)M rhTNF-alpha augmented phagocytosis, LTB4, and superoxide generation by PMN in response to activation by Z. In contrast, none of the responses to glucan was significantly increased after incubation with rhTNF-alpha. These differences suggest a lack of absolute homology between the recognition mechanisms for zymosan and glucan and that there is a component of the recognition mechanism for zymosan that is independent of that for glucan and is up-regulated after rhTNF-alpha pretreatment.

Activation of the inflammatory response of neutrophils by Tamm-Horsfall glycoprotein

The activation of human polymorphonuclear leukocytes (PMN) by particulate Tamm-Horsfall glycoprotein (THG) represents an interaction hitherto unrecognized. The potential pathophysiological effect of this phenomenon within the interstitium of the kidney is highlighted by the activation of the respiratory burst, as well as by comprehensive PMN degranulation. Products of the interaction are expressed in terms of phagocytosis, luminol-dependent chemiluminescence, granule marker enzyme release and arachidonic acid metabolism. Significant quantities of the primary, secondary and tertiary granule markers, myeloperoxidase, vitamin B12 binding protein and N-acetyl-beta-D-glucosaminidase, respectively, were secreted in a dose and time-dependent manner. Phagocytosis of the glycoprotein was accompanied by the generation of significant quantities of leukotriene B4. Furthermore, the ability of such a particulate ligand to activate the alternative pathway of complement clearly represents a capacity to augment the inflammatory response. Should the interaction of THG with PMN take place within the interstitium of the kidney, augmented by the deposition of complement proteins on the surface of insoluble aggregates, the resulting inflammatory response may lead to marked tissue damage and eventually result in interstitial fibrosis.

Functional and biochemical properties of ovine neutrophils

Ovine neutrophils were isolated and characterised by their morphology, biochemical and functional responses. Two major granule types were observed, peroxidase positive and peroxidase negative, which were identified as the ovine equivalent of the human azurophil and specific granules respectively. A third type of granule identified, which was present at low frequency and was peroxidase negative, was possibly the ovine equivalent of the bovine large granule. Superoxide production following stimulation with PMA, A23187, PAF, ConA and opsonized zymosan (ZC), was 20–50% less, compared to bovine and human neutrophils. Coincubation of PMA with either PAF or A23187 enhanced superoxide production by 4 to 5 fold above that of the latter stimulants alone. The amount of β-glucuronidase was similar to, while myeloperoxidase was more than twice that found in bovine neutrophils. Vitamin B12 binding protein was found in very small amounts, compared to that of bovine or human neutrophils. It was observed that coincubation of PMA with PAF, or A23187 resulted in an inhibition of β-glucuronidase secretion and an enhancement of myeloperoxidase secretion, respectively. Phagocytic capability of ovine neutrophils was found to be optimal at a neutrophil to ZC ratio of 1 : 10, and which corresponded with an enhanced myeloperoxidase secretion.

Recombinant human granulocytemacrophage colony-stimulating factor induces granule exocytosis from human polymorphonuclear neutrophils

Exposure of human polymorphonuclear neutrophils (PMNs) to recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in a time- and concentration-dependent (3-100 units/ml) extracellular release of a specific (vitamin B12-binding protein) but not azurophil granule constituent (myeloperoxidase). Negligible granule exocytosis occurred if PMNs were not preincubated with cytochalasin B prior to contact with GM-CSF. The extent of degranulation elicited with GM-CSF was reduced but not abolished when PMNs were incubated with EGTA in calcium-free medium. GM-CSF did not stimulate a rise in the cytosolic-free calcium concentration ([Ca2+]i), and it had no effect on PMN protein kinase C (PKC) activity.

Extracellular adenosine nucleotides stimulate protein kinase C activity and human neutrophil activation.

We studied the effect of adenosine nucleotides on several aspects of the functional activation of human peripheral blood polymorphonuclear leukocytes (PMN). Radiolabeled ATP bound to PMN in a manner suggesting the existence of specific binding sites because: 1) binding was reversed (92 +/- 6%) by 100-fold excess concentrations of unlabeled ATP but minimally by either ADP (43 +/- 12%) or GTP (37 +/- 8%); and 2) binding saturation was achieved (i.e., specific binding did not increase) above 250 microM ATP. Binding studies revealed that significant ATP hydrolysis occurred, even at low temperatures and in the presence of phosphatase inhibitors. Adenosine nucleotides activated signal transduction mechanisms in PMN because: 1) 1 to 100 microM ATP and 5'-adenylylimidodiphosphate (AMP-PNP) stimulated increased production of 1,2-diacylglycerols; 2) ATP (0.5 to 500 microM) and ADP (0.1 to 10 mM) induced increased insoluble protein kinase (PKC) activity in a dose-dependent manner when used at concentrations greater than 50 microM; 3) ATP (greater than or equal to 50 microM) induced a shift in the solubility of phorbol receptors from mostly soluble (89% in untreated cells) to mostly insoluble (68%), whereas ADP, GTP, and GDP were effective at higher concentrations; and 4) greater than or equal to 50 microM ATP stimulated increased phosphorylation of endogenous PMN proteins. AMP-PNP induced PKC activity and phosphoprotein changes that were qualitatively similar to those observed when PMN were treated with ATP, suggesting that extracellular ATP hydrolysis is not required for signal transduction to activate PKC. Functionally, ATP stimulated the secretion of specific (but not azurophil) granules because vitamin B12-binding protein and low levels of lysozyme, but not beta-glucuronidase, were released; qualitatively similar results were obtained by using AMP-PNP. These results suggest that certain adenosine nucleotides employed at physiologically relevant concentrations stimulate increased 1,2-diacylglycerol production, PKC activity, granule secretion, and endogenous phosphoprotein formation in a manner that is independent of extracellular ATP hydrolysis.

Measurements of Unsaturated Vitamin B12-Binding Capacity and Myeloperoxidase as Indices of Severity of Acute Inflammation in Serial Colonoscopy Biopsy Specimens from Patients with Inflammatory Bowel Disease

Measurements of tissue content of myeloperoxidase, a constituent of neutrophil azurophil granules and of unsaturated vitamin B12-binding protein from neutrophil-specific granules, have been used to assess intestinal inflammation. This paper reports results of a prospective evaluation of such measurements in serial colonoscopy biopsy specimens from patients with inflammatory bowel disease. Histologic grading of acute inflammation was based on perceived numbers of neutrophil polymorphs in sections from an immediately adjacent biopsy specimen. The mean + 2 SD range for unsaturated vitamin B12-binding protein activity in homogenates of histologically normal specimens was 62 pg mg protein-1. Values increased progressively up to 900 pg mg-1 protein in the most severely inflamed specimens. Unsaturated vitamin B12-binding protein measurements generally distinguished among histologic grades of inflammation, whereas myeloperoxidase activities failed to do this, probably because substantial myeloperoxidase activity was found in uninflamed colonic mucosa, suggesting a non-neutrophil source for this enzyme.

Staurosporine inhibits the respiratory burst and induces exocytosis in human neutrophils

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.

The opsonizing ligand on Salmonella typhimurium influences incorporation of specific, but not azurophil, granule constituents into neutrophil phagosomes.

Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.

Structure of the cDNA Encoding Transcobalamin I, a Neutrophil Granule Protein

Transcobalamin I (TCI) is a member of the R binder family of vitamin B12 binding proteins. It is a major protein constituent of secondary granules in neutrophils. We have isolated and characterized full length cDNA clones encoding TCI in order to determine whether its expression is coordinately regulated with the appearance of secondary granules and whether it is consequently a useful marker of granulocyte development. Partial amino acid sequences of human R protein were obtained from tryptic digestion fragments. Using the polymerase chain reaction, a partial TCI cDNA probe was isolated by selective amplification of a region of cDNA located between two oligonucleotides deduced from the available partial amino acid sequences. The amplified probe was then used to obtain full length clones from a granulocyte cDNA library. Identity of the clones was confirmed by matching DNA sequence to known peptide amino acid sequence. TCI is transcribed to a single 1.5-kilobase mRNA species. The predicted protein sequence is 433 amino acids long. We have compared the sequence of TCI to that of rat intrinsic factor. The two proteins have areas of extensive homology which implicate regions potentially important for vitamin B12 binding. TCI mRNA was present in late neutrophil precursors but absent from uninduced and induced HL60 cells.

Formyl peptide-induced chemotaxis of human polymorphonuclear leukocytes does not require either marked changes in cytosolic calcium or specific granule discharge. Role of formyl peptide receptor reexpression (or recycling).

We examined the role of intracellular and extracellular calcium on the ability of human polymorphonuclear leukocytes to migrate chemotactically and reexpress (or recycle) formyl peptide receptors when challenged with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP). Extracellular calcium was not required for either optimal chemotactic responses or receptor reexpression. Depletion and chelation of intracellular calcium resulted in significant diminution in the ability of polymorphonuclear leukocytes to release the specific granule constituents lactoferrin and vitamin B12-binding protein during the process of chemotaxis, but had no effect on the capability of these cells to respond chemotactically. Similarly, chelation of intracellular calcium did not affect the ability of these cells to reexpress a population of formyl peptide receptors. Inhibition of receptor reexpression, by a nonagglutinating derivative of wheat-germ agglutinin, was associated with inhibition of chemotactic responses to FMLP. Thus, it appears that large changes in cytosolic free calcium are not necessary for formyl peptide-induced polymorphonuclear leukocyte chemotaxis. In contrast, continuous reexpression (or recycling) of formyl peptide receptors is required for polymorphonuclear leukocyte chemotactic responses to FMLP, a process that appears to be independent from specific granule fusion with plasma membrane.

Effect of tumor necrosis factor on granule release and LTB4 production in adherent human polymorphonuclear leukocytes

Tumor necrosis factor (rTNF) has previously been shown to induce PMN chemotaxis, stimulate PMN adhesion to vascular endothelium and stimulate hydrogen peroxide secretion from PMNs adhered to biological surfaces. We investigated the activity of both rTNF alpha and rTNF beta on adherent and suspension cultures of human PMNs. rTNF alpha selectively stimulated the release of the specific granule in a dose dependent manner. Exocytosis of the specific granule was measured with an enzyme-immunoassay for lactoferrin and a radioassay for vitamin B12-binding protein. Adherent PMNs released up to 60% of the total lactoferrin content of the cells with no increase in myeloperoxidase (MPO) secretion when stimulated with 0.1-10 nM rTNF alpha. The PMNs in suspension cultures also selectively released the specific granule, although total release was reduced suggesting that adherence of PMNs increased their ability to respond to physiological stimuli. When PMNs in suspension cultures or adherent cells were stimulated with rTNF alpha, no LTB4 production was detectable, yet the cells retained the ability to synthesize LTB4 when stimulated with calcium ionophore A23187. Neither rTNF alpha or rTNF beta stimulated the release of the azurophilic granule, measured by the secretion of MPO and neutrophil elastase activity. These results suggest that a function of rTNF alpha and rTNF beta on PMNs is the release of the contents within the specific granule.

Affinity chromatographic identification of vitamin B12-binding proteins in egg white.

To identify vitamin B12-binding proteins in egg white, an affinity chromatographic isolation procedure was applied. A fraction tightly absorbed on vitamin B12 immobilized in agarose was eluted with 1 mM cyanocobalamin, and then separated by isoelectric focusing in sucrose density gradient. Two isolated proteins (or perhaps two forms of the same protein) of isoelectric points 6.2 and 7.2 and the same Mr 90,000 were found capable of detectable vitamin B12 binding. They are probably glycoproteins, each composed of a single polypeptide chain.

Quantitation of intracellular Mac-1 (CD11b/CD18) pools in human neutrophils.

The adhesive glycoprotein Mac-1 (CD11b/CD18) of the CD11/CD18 complex contributes to multiple neutrophil inflammatory functions. Activation of neutrophils by chemotactic stimuli results in a rapid, protein synthesis-independent increase in surface Mac-1 derived from incompletely defined intracellular compartments. Therefore, we developed a novel quantitative lectin immunoblot technique to define intracellular pools of Mac-1 in subcellular neutrophil fractions resolved on discontinuous Percoll gradients. In cavitates of unstimulated neutrophils, 30% and 26% of total Mac-1 was identified in beta [1.10 gm/ml; vitamin B12 binding protein (vit B12 B.P.)-rich] or pre-gamma (1.07 gm/ml; vit B12 B.P.-poor) granular fractions, respectively, whereas 24% was associated with the plasma membrane-rich gamma (1.06 gm/ml) fractions. N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation (10(-8) M, 15 min, 37 degrees C) significantly diminished Mac-1 in pre-gamma (-18% of total, P less than 0.05) but not beta fractions (+6% of total). Under these conditions, the content of Mac-1 in gamma fractions increased 13% in association with four- to eightfold increase in surface Mac-1 expression (OKM-1 binding). These findings suggest that chemotactic stimuli increase plasma membrane and/or surface Mac-1 on human neutrophils by mobilizing a novel intracellular granule pool.

Botulinum C2 toxin ADP-ribosylates actin and enhances O2- production and secretion but inhibits migration of activated human neutrophils.

The binary botulinum C2 toxin ADP-ribosylated the actin of human neutrophils. Treatment of human neutrophils with botulinum C2 toxin for 45 min increased FMLP-stimulated superoxide anion (O2-) production 1.5-5-fold, whereas only a minor fraction of the cellular actin pool (approximately 20%) was ADP-ribosylated. Effects of botulinum C2 toxin depended on toxin concentrations, presence of both components of the toxin, and incubation time. Cytochalasin B similarly enhanced O2- production. The effects of botulinum C2 toxin and cytochalasin B were additive at submaximally, but not maximally effective concentrations and incubation time of either toxin. Botulinum C2 toxin also enhanced stimulation of O2- production by Con A and platelet-activating factor, but not by phorbol 12-myristate 13-acetate (PMA). Botulinum C2 toxin increased FMLP-induced release of N-acetyl-glucosaminidase by 100-250%; release of vitamin B12-binding protein induced by FMLP and PMA was enhanced by approximately 150 and 50%, respectively. Botulinum C2 toxin blocked both random migration of neutrophils and migration induced by FMLP, complement C5a, leukotriene B4, and a novel monocyte-derived chemotactic agent. The data suggest that botulinum C2 toxin-catalyzed ADP-ribosylation of a minor actin pool has a pronounced effect on the activation of human neutrophils by various stimulants.

Combined Congenital Deficiencies of Intrinsic Factor and R Binder

Coexisting deficiencies of both intrinsic factor (IF) and R binder were identified in an Algerian boy who presented with severe megaloblastic anemia, growth retardation, and neurologic dysfunction with typical features of subacute combined degeneration of the spinal cord. The anemia responded completely to cyanocobalamin and folic acid. IF was absent from gastric juice, but acid secretion and gastric mucosa were normal. R binders were absent from gastric juices as well as from serum, saliva, and polymorphonuclear leukocytes. The patient's father exhibited absence of R binder in his serum with a low serum vitamin B12 level and was asymptomatic. This unique case of simultaneous IF and R binder deficiencies suggests a genetic association between these two functionally and immunologically dissimilar, but structurally close vitamin B12-binding proteins.

Kinetics and activation parameters of the reaction of cyanide with free aquocobalamin and aquocobalamin bound to a haptocorrin from chicken serum.

The kinetics of the reaction of cyanide with free aquocobalamin (H2OCbl) and with aquocobalamin bound to a vitamin B12-binding protein (haptocorrin) from chicken serum (HC-H2OCbl) have been investigated as a function of temperature and pH. The mechanism of replacement of H2O from protein-bound and free H2OCbl is apparently the same and involves attack of both CN- (k1) and HCN (k2). The reactions with HC-H2OCbl are somewhat slower than those with free H2OCbl, k1 being 22-fold smaller and k2 7-fold smaller at 25 degrees C. The relatively small effect of the protein on the rate constants supports the view that the metal in HC-H2OCbl is readily accessible to solvent. Activation parameters suggest that the transition states for ligand substitution are stabilized by nucleophilic participation (at least by CN-) and that ligand substitution on the protein-bound cobalamin proceeds through more ordered, concerted transition states. The latter effect suggests that the Co-O bond of H2OCbl is strengthened upon binding to the haptocorrin.