Absorption and retention of free and milk protein-bound cyano- and hydroxocobalamins. An experimental study in rats.

INTRODUCTION: Cobalamin/Vitamin B12 (Cbl) is an essential vitamin, supplied mainly as hydroxocobalamin (OHCbl) by animal products, including cows' milk. Cyanocobalamin (CNCbl) is the usual form in vitamin pills. The aim was to explore absorption and tissue accumulation of two Cbl forms, administered alone or bound to milk protein. MATERIALS AND METHODS: We synthesized labeled OH[(57)Co]Cbl from commercially available CN[(57)Co]Cbl. Recombinant bovine transcobalamin (rbTC) was produced in yeast and skimmed milk obtained off the shelf. Male Wistar rats (250-300 g) received labeled Cbl by gastric gavage. First, we administered CN[(57)Co]Cbl, free or rbTC-bound (n = 15 in each group). Rats were sacrificed after two, 24, and 48 h. In the following studies, rats were sacrificed after 24 h. We compared absorption of free or rbTC-bound CN[(57)Co]Cbl added to cows' milk and analogous absorption of OH[(57)Co]Cbl, free or rbTC-bound, to absorption of free CN[(57)Co]Cbl, (n = 10 in each group). Blood, tissues, 24-h urine and feces were collected. Labeled Cbl was measured using a gamma counter. Results are expressed as percentage of administered dose. RESULTS: Absorptions of CNCbl and OHCbl were neither influenced by rbTC-binding nor administration in milk. Absorption increased in the first 24 h with no further tissue accumulation during the subsequent 24 h. Accumulation of free CNCbl and (OHCbl) was 1.4, (4.1) (liver); 20.2, (16.4) (kidney); and 0.05, (0.02) (plasma)% 24 h after administration. Total organ accumulations were 21.6, (20.5)%. While total accumulations of CNCbl and OHCbl were equal, distributions between liver, kidney, and plasma showed significant differences (p < 0.0001; p = 0.01; p < 0.0001). CONCLUSIONS: Cbl added to milk (spiked with rbTC) has high bioavailability matching that of free Cbl. OHCbl and CNCbl are absorbed equally well, but much more OHCbl accumulated in the liver. Benefits of oral supplementation with OHCbl compared to CNCbl should be investigated.

An amphiphilic, catalytically active, vitamin B12 derivative.

We performed the reaction of vitamin B12 with N,N-dimethylformamide dimethyl acetal for primary amide activation, and added MeOH as a nucleophile, to afford cobalester, the first amphiphilic cobalamin derivative. The unique combination of redox properties and solubility represents an asset for its use as a catalyst in C-C bond forming reactions.

Crystallization and preliminary X-ray diffraction analysis of human transcobalamin, a vitamin B12-transporting protein.

Transcobalamin is a cobalamin-binding protein in mammalian plasma that facilitates the cellular uptake of vitamin B(12). Human transcobalamin was crystallized using polyethylene glycol and ethanol as precipitants. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.04, b = 145.27, c = 164.96 A. A complete data set to 3.2 A resolution was collected from a single crystal using synchrotron radiation. Estimation of the crystal packing (V(M) = 3.2 A(3) Da(-1)) and self-rotation function analysis suggest the presence of two molecules in the asymmetric unit related by non-crystallographic twofold symmetry.

Measurement of transcobalamin by ELISA.

BACKGROUND: Transcobalamin is essential for the cellular internalization of cobalamin. Methods to quantify the unsaturated protein are available, but few attempts have been made to develop methods to quantify the sum of unsaturated and cobalamin saturated transcobalamin. METHODS: gamma-Globulins from two polyclonal rabbit antibodies against recombinant human transcobalamin were used as capture and detection antibodies, and recombinant human transcobalamin was used as calibrator in an ELISA design. RESULTS: The ELISA is specific for transcobalamin and has a detection limit of <1.6 pmol/L. The imprecision (CV) is 4-6% for mean concentrations of 13-70 pmol/L. The central 95% interval for serum from healthy blood donors (n = 77) was approximately 600-1500 pmol/L and showed limited variation with age and sex. No correlation was observed between the marker of acute phase reaction, C-reactive protein, and transcobalamin in plasma. CONCLUSIONS: The ELISA measures total transcobalamin in serum and thus can be used for measurement of transcobalamin in patients treated with cobalamin.

Kinetic and structural characterization of a two-domain streptokinase: dissection of domain functionality.

The mammalian protease plasminogen can be activated by bacterial activators, the three-domain (alpha, beta, gamma) streptokinases and the one-domain (alpha) staphylokinases. These activators act as plasmin(ogen) cofactors, and the resulting complexes initiate proteolytic activity of host plasminogen which facilitates bacterial colonization of the host organism. We have investigated the kinetic mechanism of the plasminogen activation mediated by a novel two-domain (alpha, beta) streptokinase isolated from Streptococcus uberis (Sk(U)) with specificity toward bovine plasminogen. The interaction between Sk(U) and plasminogen occurred in two steps: (1) rapid association of the proteins and (2) slow transition to the active complex Sk(U)-PgA. The complex Sk(U)-PgA converted plasminogen to plasmin with the following parameters: K(m) < or = 1.5 microM and k(cat) = 0.55 s(-)(1). The ability of proteolytic fragments of Sk(U) to activate plasminogen was investigated. Only two C-terminal segments (97-261 and 123-261), which both contain the beta-domain (126-261), were shown to be active. They initiated plasminogen activation in complex with plasmin, but not with plasminogen, and thereby exhibited functional similarity to the staphylokinase. The fusion protein His(6)-Sk(U) (i.e., Sk(U) with a small N-terminal tag) acted exclusively in complex with plasmin as well. These observations demonstrate that (1) the N-terminal alpha-domain, including a native N-terminus, was necessary for "virgin" activation of the associated plasminogen in the Sk(U)-PgA complex and (2) the C-terminal beta-domain of Sk(U) is important for recognition of the substrate in the Sk(U)-PgA complex.

Functional analyses of two cellular binding domains of bovine lactadherin.

The glycoprotein bovine lactadherin (formerly known as PAS-6/7) comprises two EGF-like domains and two C-like domains found in blood clotting factors V and VIII. Bovine lactadherin binds to alpha(v)beta(5) integrin in an RGD-dependent manner and also to phospholipids, especially phosphatidyl serine. To define and characterize these bindings the interactions between lactadherin and different mammalian cell types were investigated. Using recombinant forms of bovine lactadherin, the human breast carcinomas MCF-7 cells expressing the alpha(v)beta(5) integrin receptor were shown to bind specifically to RGD containing lactadherin but not to a mutated RGE lactadherin. A monoclonal antibody against the alpha(v)beta(5) integrin receptor and a synthetic RGD-containing peptide inhibited the adhesion of MCF-7 cells to lactadherin. Green monkey kidney MA-104 cells, also expressing the alpha(v)beta(3) together with the alpha(v)beta(5) integrin, showed binding to bovine lactadherin via both integrins. To investigate the interaction of lipid with lactadherin two fragments were expressed corresponding to the C1C2 domains and the C2 domain. Both fragments bound to phosphatidyl serine in a concentration-dependent manner with an affinity similar to native lactadherin (K(d) = 1.8 nM). A peptide corresponding to the C-terminal part of the C2 domain inhibited the binding of lactadherin to phospholipid in a concentration-dependent manner, and finally it was shown that lactadherin mediates binding between artificial phosphatidyl serine membranes and MCF-7 cells. Taken together these results show that lactadherin can act as link between two surfaces by binding to integrin receptors through its N-terminus and to phospholipids through its C-terminus.

Conformational changes of transcobalamin induced by aquocobalamin binding. Mechanism of substitution of the cobalt-coordinated group in the bound ligand.

Binding of aquo-, cyano-, or azidocobalamin (Cbl.OH(2), Cbl.CN, and Cbl.N(3), respectively) to the recombinant human transcobalamin (TC) and haptocorrin from human plasma was investigated via stopped-flow spectroscopy. Association of cobalamins with haptocorrin always proceeded in one step. TC, however, displayed a certain selectivity for the ligands: Cbl.CN or Cbl.N(3) bound in one step with k(+1) = 1 x 10(8) M(-1) s(-1) (20 degrees C), whereas binding of Cbl.OH(2) under the same conditions occurred in two steps with k(+1) = 3 x 10( 7) M(-1) s(-1) (E(a) = 30 kJ/mol) and k(+2) = 0.02 s(-1) (E(a) = 120 kJ/mol). The second step of Cbl.OH(2) binding was interpreted as a transformation of the initial "open" intermediate TC.Cbl.OH(2) to the "closed" conformation TC(Cbl) with displaced water. The backward transition from the closed to the open conformation was the reason for the identical rate-limiting steps during substitution of H(2)O in TC.Cbl.OH(2) for cyanide or azide according to the reaction TC(Cbl) --> TC.Cbl.OH(2) + CN(-)/N(3)(-). The cyano and azido forms of holo-TC which were produced behaved as the open proteins. Different conformations of holo-TC, determined by the nature of the active group in the bound Cbl, may direct transportation of cobalamins in the organism.

Sequence, S-S bridges, and spectra of bovine transcobalamin expressed in Pichia pastoris.

Transcobalamin (TC) -encoding cDNA was isolated from a bovine mammary gland cDNA library. Hybridization of the cloned bovine TC-cDNA to RNA samples from bovine tissues showed that the most intensive synthesis of a TC positive 1.9-kilobase mRNA occurred in kidney, lymphatic nodes, and liver. Bovine TC was expressed in yeast Pichia pastoris, and the isolated recombinant protein showed cobalamin (Cbl) and receptor binding properties similar to TCs from other sources. Alignment of the related Cbl carriers (haptocorrins and intrinsic factors from other species) with bovine TC (414 residues) revealed four conservative clusters in the sequence (85-98, 137-147, 178-190, and 268-288), which may be responsible for Cbl binding. Three S-S bonds connected Cys residues 3-252, 98-294, and 147-190. Treatment with an S-S reducing agent caused liberation of Cbl from TC-Cbl. A significant change was observed in the TC-Cbl absorbance spectrum upon substitution of Co(2+)-coordinated H(2)O by azide. The reaction developed several orders of magnitude slower, and the spectral distortions were much stronger than those in free Cbl. This may be caused by significant deformation of the Cbl molecule and/or by its shielding when bound to TC.

A refined kinetic analysis of plasminogen activation by recombinant bovine tissue-type plasminogen activator indicates two interconvertible activator forms.

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.

Transcobalamin from cow milk: isolation and physico-chemical properties.

The concentration of endogenous cobalamin (Cbl) in cow milk was 3.3 nM while the Cbl-binding capacity was 0.05 nM. Both endogenous and newly added Cbl showed similar quantitative distribution between a 280 kDa protein complex (45%) and a 43 kDa Cbl-binder (55%). Long time incubation, as well as urea treatment, was accompanied by a slow release of the 43 kDa Cbl-binder from the 280 kDa fraction. No other Cbl-binding proteins appeared after these procedures. The 43 kDa binder from cow milk, depleted of the ligand by urea treatment, reacted with Cbl even in the presence of a B12-analogue cobinamide (Cbi) at the ratio Cbl:Cbi = 1:40. The stokes radius of the binder changed from 2.7 nm for the Cbl-free protein to 2.5 nm for the Cbl-saturated form and the Cbl-saturated binder was able to displace human transcobalamin (TC) from the TC-receptor. The interaction between the protein and Cbl was significantly suppressed at pH 2.0. The N-terminal sequence of the purified 43 kDa Cbl-binder revealed homology with TC from human and rabbit plasma. In conclusion we have shown that TC is the main Cbl-binding protein in cow milk. This is surprising, since previous studies on human and rat milk have shown another Cbl-binder, apo-haptocorrin, to be the dominating Cbl-binding protein.

Binding of cobalamin and cobinamide to transcobalamin from bovine milk.

We have studied the interaction between transcobalamin (TC) and the ligands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Unsaturated TC was adsorbed on CM-Sepharose in order to ensure fast separation of the matrix-bound protein from the reaction medium. The forward reaction TC+Cbl-->TC-Cbl (rate constant k+Cbl) and the backward reaction TC-Cbl-->TC+Cbl (k-Cbl) were followed in time. A single-step binding model (with no intermediate protein-ligand complex) was sufficient to fit the data. The calculated rate constants were k+Cbl = 0.6 nM-1 min-1 and k-Cbl = 1.3 x 10(-4) min-1, which corresponded to the TC-Cbl dissociation constant KDCbl = 0.2 pM. Reaction between TC and Cbl developed against electrostatic forces, and the effective charges of the interacting species were estimated as both +1 or both -1. The competition between Cbl and Cbi for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k+Cbi = 0.03 nM-1 min-1, k-Cbi = 0.03 min-1, and KDCbi = 1 nM. Slow dissociation of TC-Cbl guarantees its stability in plasma for 5-10 h, while Cbi bound to TC would be transferred to haptocorrin in less than 1 h.

Creatine-creatine phosphate shuttle modeled as two-compartment system at different levels of creatine kinase activity.

In order to characterize ADP-ATP and creatine-creatine phosphate (Cr-CrP) shuttles a minimal mathematical model with two compartments and cyclic turnover of matter was designed. The 'mitochondrial' compartment contained 'ATP-synthase' and 'mitochondrial creatine kinase' (mitCK). The 'cytoplasmic' compartment consisted of 'ATPase', 'cytoplasmic creatine kinase' (cytCK) and an 'ADP-binding structure'. The exchange of metabolites between these compartments was limited. Different levels of cytCK and mitCK expression as well as different exchange rate constants between the compartments were assigned to obtain several different modes. Every steady state obtained in the presence of low ATPase activity ('resting' conditions) was then disturbed by a steep activation of ATPase ('muscle performance') and afterwards the transition to a new steady state was followed in time. The ATP-buffering capacity of the system initially acquired by cytCK expression significantly increased after additional mitCK supplement. Nevertheless, even the complete Cr-CrP shuttle failed to maintain a high [ATP]/[ADP] ratio during long term 'muscle performance' due to the rate limiting CK-transphosphorylation in the mitochondria. The facilitated diffusion of Cr and CrP was not critical, and the model worked with the same efficiency even at equal permeabilities for nucleotides and guanidines. Under 'resting conditions' the main flux of matter went through the Cr-CrP shuttle, resulting in 'pumping' of CrP. This ensured a 40 s delay in the [ATP] decrease at 'work'. The partial systems without mitCK were not as effective, and this delay was 0-10 s. However, the ADP-ATP shuttle was of more importance at the steady state achieved under 'working' conditions.

A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength.

The quantitative aspects of mitochondrial creatine kinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the enzyme adsorption. An analysis of our own data as well as of the data from the literature is consistent with the adsorption site of the octameric mitCK being composed of 4 amino acid residues with pK = 8.8 in the free enzyme. The pK value changes to 9.8 upon binding of the protein to the membrane. Lysine is suggested as the main candidate to form the adsorption site of mitCK. Deprotonated octameric mitCK easily dissociated from the membrane (Ka = 0.39 mM at ionic strength I = 7.5 mM and 5 degrees C); after protonation its affinity increased many times (Kah = 39 nM). Determination of mitCK adsorption capacity by another method at pH 7.4, when the enzyme is almost protonated, gave Kah = 15 nM. The effect of ionic strength on mitCK adsorption may be described in terms of Debye-Hückel's theory for activity coefficients assuming the charges of the interacting species to be +4 and -4. The dissociation constant for the mitCK-membrane complex at pH 7.4 and I = 0 was evaluated by different approaches as approx. 1 nM. Extramitochondrial ATP (or ADP) shifted greatly the equilibrium between the adsorbed and the free mitCK towards the solubilized state, since in the adsorbed protein the external ligands had access to four binding sites and in the free protein to eight sites.

[The effect of temperature and pH on ATP hydrolysis of Na,K-ATPase in duck salt glands]. / Vliianie temperatury i pH na gidroliz ATP Na,K-ATPazoi solevykh zhelez utki.

The shape of the substrate curve for duck salt gland Na,K-ATPase during ATP hydrolysis depends on incubation conditions. Under optimal conditions (pH 7.4, 37 degrees C) this curve has an intermediate plateau at 0.7-0.9 mM ATP. Both the acidification of the medium and the decrease in the incubation temperature below 20 degrees C transforms this dependence into a simple hyperbole which is characteristic of the hydrolysis of other substrates (GTP or UTP) under optimal conditions. Recently it has been suggested that the deviation from the Michaelis-Menten kinetics during Na,K-ATPase operation is due to the formation of short-living oligomers in the course of the ATP hydrolyzing cycle. The results obtained are interpreted in terms of possible effects of temperature and pH on the interprotomer interaction in the oligomeric complexes of Na,K-ATPase.

[Radiologic diagnosis of non-Hodgkin's lymphoma in abdominal and retroperitoneal sites]. / Luchevaia diagnostika nekhodzhkinskikh limfom abdominal'noi i retroperitoneal'noi lokalizatsii.

A total of 112 patients with non-Hodgkin's lymphomas of abdominal and retroperitoneal localization were examined by the ultrasonic and x-ray methods, in 83 of them x-ray computer-aided tomography was used. Angiographic examinations and puncture biopsy monitored by US or x-ray CAT were carried out if indicated. The authors discuss the problems of ultrasonic and x-ray semeiotics of the lymphomatous involvement of the lymph nodes, spleen, gastrointestinal tract. They claim that USE, permitting the specification of further plan of examination of each patient, and x-ray examination as a method to assess the gastrointestinal status are the optimal methods for the primary diagnosis of non-Hodgkin's lymphomas. US data may prompt further application of x-ray CAT, whose results will supplement the US findings. Angiographic examinations should be carried out only in the most difficult diagnostic situations, bearing in mind the invasive nature of the method and its high price.

[Study of myocardial morphology in chronic obstructive bronchitis complicated by pulmonary hypertension based on the results of endomyocardial biopsy]. / Morfologiia miokarda pri khronicheskom obstruktivnom bronkhite, oslozhnennom legochnoi gipertenziei, po rezul'tatam éndomiokardial'noi biopsii.

Endomyocardial biopsy along with right heart catheterization was first performed in 12 patients with chronic obstructive bronchitis complicated by transient or sustained pulmonary hypertension. Light and electron microscopic findings showed signs of severe myocardial dystrophy in 7 patients and prevalent hypertrophy in the remaining patients. Endomyocardial biopsy provided valuable information on the state of the myocardium, yielding evidence for the heretogeneous nature of right ventricular myocardial changes. Two morphological variants of cor pulmonale--hypertrophic and dystrophic--could be identified with regard to the findings.

The structural features of beef heart mitochondrial creatine kinase.

Two forms of mitochondrial creatine kinase (Mi-CK) having Mr 320 kDa and 240 kDa as determined by gel-filtration on Sephacryl S-300 in 0.1 M Tris-HCl pH 7.4 were investigated. The sedimentation coefficient values for these two forms were found to be identical and equal to 12.3 S. When studied by electron microscopy the main type of images for the 320 kDa and 240 kDa Mi-CK appeared as annular particles, 12-14 nm in diameter, with a well-detected subunit structure and a central hollow, 3-4nm in diameter filled with the dye. The results of the averaging of the main type of individual Mi-CK images and particles of the two-dimensional crystal layer point to the overall geometry of the Mi-CK molecule structure as containing eight subunits arranged by a 4-fold symmetry around the central hollow. It may be that the eight identical subunits of crystalline Mi-CK are arranged with a P422 symmetry. However in both cases the averaged main images do not show a mirror symmetry. The multiplicity of the observed projections close to annular one provides additional evidence in favour of the great lability and structural mobility of the Mi-CK subunits. It allows to assume that two forms (320 kDa and 240 kDa) are not the different oligomers but they are two functionally distinct conformational states of octameric molecule of Mi-CK.