R-type Protein Research Articles

Radioimmunoassay for assessing exocrine pancreatic insufficiency, based on the differential enzymatic degradation of cobalamin-binding proteins.

Pancreatic proteases degrade the protein moiety of the R protein-cobalamin complex but not the intrinsic factor-cobalamin complex. Accordingly, we used these two proteins as substrates in an in vitro enzymatic assay to assess pancreatic function by incubating basal jejunal fluids with a mixture of intrinsic factor and cyano[57Co]cobalamin coupled to R-type protein and then using immunoprecipitation to determine the distribution of isotopically labeled cobalamin bound to the two proteins. With normal jejunal fluids, 91.2 (SD 6.1)% and 4.5 (SD 5.5)% of cyano[57Co]cobalamin was precipitated with antisera to intrinsic factor and anti-R protein, respectively. In the patients' jejunal fluids, the cyano[57Co]cobalamin precipitated with the respective antisera was 5.3 (SD 10.0)% and 96 (SD 6.2)%. In patients with other gastrointestinal problems, the sequestration of cobalamin was indistinguishable from that observed with the normal fluids. The clearcut discrimination this radioimmunoassay provided between abnormal and normal samples was confirmed by parallel comparative chromatographic analysis.

A derivative of R-type cyanocobalamin binding proteins in the human intestine a candidate antibacterial molecule

Human jejunal fluid contains a protein which has a molecular radius of 3.37 nm, an M r of 60 600 and a mena p I of 3.35 and binds a cobalamin with a K of 0.1 · 10 9 l/mol. This protein also couples cobalaminanalogues lacking the nucleotide moiety, cross-reacts with R-type proteins and is resistant to proteolysis in the intestine. These findings refute the hypothesis that cobalamin-analogue binders are present and effectively inhibit the bacterial uptake of analogues in the intestine.

Absorption, plasma transport, and cellular retention of cobalamin analogues in the rabbit. Evidence for the existence of multiple mechanisms that prevent the absorption and tissue dissemination of naturally occurring cobalamin analogues.

Analogues of cobalamin (Cbl; vitamin B(12)) are prevalent in nature as a result of bacterial synthesis, and are of additional interest because of their potential use as antimetabolites and chemotherapeutic agents. We have synthesized 14 Cbl analogues containing (57)Co and have compared their gastrointestinal absorption, plasma transport, and cellular retention to that of [(58)Co]Cbl in rabbits. Many of the Cbl analogues were bound with low affinity by intrinsic factor, and none of these [(57)Co]Cbl analogues were taken up by the ileum or absorbed into the body in amounts comparable to that of [(58)Co]Cbl. The Cbl analogues that were bound by intrinsic factor with high affinity were taken up by the ileum but, in many cases, they were retained there in significant amounts. Most of the Cbl analogues were bound by plasma transcobalamin II with high affinity and all of these transcobalamin II-[(57)Co]Cbl analogue complexes were taken up by a variety of tissues in a manner that was indistinguishable from that of transcobalamin II-[(58)Co]Cbl. The few analogues that were bound by transcobalamin II with low affinity were taken up by tissues in lesser amounts, and 20-70% of these analogues was rapidly excreted in the urine as occurs with native Cbl when it is present in plasma in unbound form. All of the Cbl analogues were bound by the granulocyte R-type Cbl-binding protein with high affinity and all of the R-type protein-[(57)Co]Cbl analogue complexes were cleared rapidly from plasma exclusively by hepatocytes as occurs with R-type protein-[(58)Co]Cbl. Some Cbl analogues were released back into the plasma and were disseminated among a variety of tissues via transcobalamin II as occurs with native Cbl. Other Cbl analogues were retained in the liver and eventually excreted in the feces and urine without accumulating in other tissues. These studies indicate that intrinsic factor and the ileum prevent certain Cbl analogues from entering the body and that the granulocyte R-type protein and hepatocytes prevent the dissemination of certain Cbl analogues that may gain entry such as during infections with Cbl analogue-producing bacteria. The fact that transcobalamin II binds and transports a large number of Cbl analogues indicates that these protective mechanisms can be circumvented and supports the feasibility of using Cbl analogues as antimetabolites in vivo.

Recognition of two intracellular cobalamin binding proteins and their identification as methylmalonyl-CoA mutase and methionine synthetase.

The granulocyte R-type cobalamin binding protein delivers cobalamin (Cbl) exclusively to hepatocytes, and transcobalamin II delivers Cbl to various mammalian cells. Both protein-Cbl complexes enter cells by pinocytosis, and the protein moieties are rapidly degraded in lysosomes. The liberated Cbl is subsequently bound to a high-molecular-weight intracellular cobalamin binding protein (ICB). The nature of ICB-Cbl is unknown but appears important because ICB-[57Co]Cbl is missing from cultured fibroblasts of a group of patients whose cells take up CN-[57Co]Cbl normally but do not convert it to either of its coenzyme forms. We have examined supernatants of sonicated rabbit livers and have found that 65% of the total endogenous Cbl elutes from Sephadex G-150 as ICB-Cbl and that this fraction also contains the two mammalian Cbl-dependent enzymes, methylmalonyl-CoA mutase (methylmalonyl-CoA CoA-carbonylmutase;EC 5.4.99.2) and methionine synthetase (tetrahydropteroylglutamate methyltransferase; 5-methyltetrahydropteroyl-L-glutamate:L-homocysteine-S-methyltransferase; EC 2.1.1.13). Gradient elution from DEAE-Sephadex reveals that 90--95% of the ICB--Cbl elutes with methylmalonyl-CoA mutase and 5--10% elutes with methionine synthetase. ICB--[57Co]Cbl first appears 2 hr after the intravenous injection of CN[57Co]Cbl bound to granulocyte R-type protein. This ICB-[57Co]Cbl is associated with either methylmalonyl-CoA mutase or methionine synthetase although the latter appears to be formed at a relatively faster rate. Our studies indicate that mammalian cells contain two ICBs, that these proteins are methylmalonyl-CoA mutase and methionine synthetase, and that the primary abnormality in the group of patients mentioned above lies at a step that is common to the formation of both Cbl coenzymes and that precedes the stable binding of Cbl to both methylmalonyl-CoA mutase and methionine synthetase.

Isolation and characterization of a novel vitamin B12-binding protein associated with hepatocellular carcinoma.

High levels of a novel vitamin B12-binding protein (hepatoma B12 BP) have been observed recently in plasma obtained from three adolescent patients with hepatocellular carcinoma. This protein has now been isolated in homogeneous form from the plasma and pleural fluid of two of these patients by the use of affinity chromatography with vitamin B12-Sepharose. The hepatoma B12 BP belongs to the R-type group of B12-binding proteins and is essentially indistinguishable from the recently isolated human milk and saliva R-type proteins in terms of: (a) immunologic properties based on immunodiffusion and immunoprecipitation assays; (b) amino acid composition; (c) molecular weight based on amino acid and carbohydrate content; and (d) absorption spectra. Both hepatoma B12 BPs contain more sialic acid and less fucose than the milk and saliva B12 BPs. All four proteins contain similar amounts of galactose, mannose, galactosamine, and glucosamine. Differences in sialic acid content appear to account for the differences in electrophoretic mobility that were observed among the four proteins. Differences in total carbohydrate content appear to account for the differences in apparent molecular weight that were observed with both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Tumor tissue from one of the patients contained 10 times as much R-type protein as did normal liver tissue from the same patient. This suggests, although it does not prove, that synthesis by the tumor is the cause of the high levels of R-type protein found in the plasma of certain patients with hepatocellular carcinoma. Plasma survival studies performed with rabbits indicate that the hepatoma B12 BP has a prolonged plasma survival and suggests that his parameter is also of importance.

Human plasma R-type vitamin B12-binding proteins. II. The role of transcobalamin I, transcobalamin III, and the normal granulocyte vitamin B12-binding protein in the plasma transport of vitamin B12.

The normal human granulocyte vitamin B12-binding protein, transcobalamin I, and transcobalamin III, have been labeled with 125I-labeled N-succinimidyl 3-(4-hydroxyphenyl)propionate and utilized for plasma clearance studies performed with rabbits. Both moieties of 125I-labeled granulocyte vitamin B12-binding protein-[57Co]vitamin B12 were cleared rapidly from the plasma (is less than 90% by 5 min) by the liver. After 30 min, the bulk of the 125I reappeared in the plasma in small molecular weight (less than 1000) form and was rapidly excreted in the urine. After 60 min the bulk of the [57Co]vitamin B12 reappeared in the plasma bound to rabbit transcobalamin II and was subsequently taken up by a variety of tissues. Approximately 15% of the 125I-labeled granulocyte vitamin B12-binding protein-[57Co-a1vitamin B12 was excreted intact into the bile during the period from 10 to 80 min after injection. The hepatic uptake of the protein-vitamin B12 complex was blocked by the prior injection of desialyzed fetuin but not by native fetuin. Similar results were obtained with 125I-labeled transcobalamin III-[57Co]vitamin B12. Approximately 90% of both moieties of 125I-labeled transcobalamin I-[57Co]vitamin B12 had prolonged plasma survivals similar to that of 125I-labeled bovine serum albumin. After treatment with neuraminadase, both moieties of the 125I-labeled transcobalamin I-[57Co]vitamin B12 complex were cleared rapidly from the plasma by the liver in a manner that was indistinguishable from that observed in the case of untreated granulocyte vitamin B12-binding protein and transcobalamin III. These observations indicate that desialyzed transcobalamin I and the native forms of the granulocyte vitamin B12-binding protein and transcobalamin III are cleared from plasma by the mechanism elucidated by Ashwell and Morell (Ashwell, G., and Morell A. G. (1974) Adv. Enzymol. 41, 99-128) that is capable of clearing a wide variety of asialoglycoproteins. These observations have implications concerning the function of the human R-type vitamin B12-binding proteins, the nature of the enterohepatic circulation of vitamin B12, the biological significance of the mechanism described by Ashwell and Morell, and the etiology of the increased plasma concentration of human R-type protein that occurs frequently in chronic myelogenous leukemia and occasionally in hepatocellular carcinoma and other solid tumors.