Meta-analysis on anxiety and depression in adult celiac disease.

OBJECTIVE: We used meta-analysis to test hypotheses concerning whether adult celiac disease is reliably linked with anxiety and/or depression. METHOD: We examined published reports on anxiety and depression in adult celiac disease. RESULTS: Eighteen studies on depression and eleven studies on anxiety in adult celiac disease met selection criteria. They show that depression is reliably more common and/or more severe in adults with celiac disease than in healthy adults (overall meta-analysis effect size: 0.97). The fail-safe margin of unpublished reports that would be required to negate the finding exceeds 8000. Adults with celiac disease do not, however, differ reliably in terms of depression from adults with other physical illnesses, nor do they differ reliably from healthy adults or adults with other physical illnesses in terms of anxiety. CONCLUSION: Depression is common in adult celiac disease and resembles the condition in other physical illnesses. We view the findings as support for the notion that non-specific mechanisms mediate emotional disorders in adult celiac disease.

Characterization of annexin A1 glycan binding reveals binding to highly sulfated glycans with preference for highly sulfated heparan sulfate and heparin.

Annexin A1 is a multifunctional, calcium-dependent phospholipid binding protein involved in a host of processes including inflammation, regulation of neuroendocrine signaling, apoptosis, and membrane trafficking. Binding of annexin A1 to glycans has been implicated in cell attachment and modulation of annexin A1 function. A detailed characterization of the glycan binding preferences of annexin A1 using carbohydrate microarrays and surface plasmon resonance served as a starting point to understand the role of glycan binding in annexin A1 function. Glycan array analysis identified annexin A1 binding to a series of sulfated oligosaccharides and revealed for the first time that annexin A1 binds to sulfated non-glycosaminoglycan carbohydrates. Using heparin/heparan sulfate microarrays, highly sulfated heparan sulfate/heparin were identified as preferred ligands of annexin A1. Binding of annexin A1 to heparin/heparan sulfate is calcium- but not magnesium-dependent. An in-depth structure-activity relationship of annexin A1-heparan sulfate interactions was established using chemically defined sugars. For the first time, a calcium-dependent heparin binding protein was characterized with such an approach. N-Sulfation and 2-O-sulfation were identified as particularly important for binding.

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells.

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.

Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.

P-selectin (CD62, GMP-140, PADGEM), a Ca(2+)-dependent lectin on activated platelets and endothelium, functions as a receptor for myeloid cells by interacting with sialylated, fucosylated lactosaminoglycans. P-selectin binds to a limited number of protease-sensitive sites on myeloid cells, but the protein(s) that carry the glycans recognized by P-selectin are unknown. Blotting of neutrophil or HL-60 cell membrane extracts with [125I]P-selectin and affinity chromatography of [3H]glucosamine-labeled HL-60 cell extracts were used to identify P-selectin ligands. A major ligand was identified with an approximately 250,000 M(r) under nonreducing conditions and approximately 120,000 under reducing conditions. Binding of P-selectin to the ligand was Ca2+ dependent and was blocked by mAbs to P-selectin. Brief sialidase digestion of the ligand increased its apparent molecular weight; however, prolonged digestion abolished binding of P-selectin. Peptide:N-glycosidase F treatment reduced the apparent molecular weight of the ligand by approximately 3,000 but did not affect P-selectin binding. Western blot and immunodepletion experiments indicated that the ligand was not lamp-1, lamp-2, or L-selectin, which carry sialyl Le(x), nor was it leukosialin, a heavily sialylated glycoprotein of similar molecular weight. The preferential interaction of the ligand with P-selectin suggests that it may play a role in adhesion of myeloid cells to activated platelets and endothelial cells.

"Antidotal thirst": a response to intoxication.

Albino rats increased their intake of water soon after they were given a load of 0.12 molar lithium chloride in the stomach. Alterations in blood volume and tonicity could not account for the magnitude of the thirst observed, which served to facilitate the renal excretion of the toxic lithium ions.

A monosialoganglioside is a monoclonal antibody-defined antigen of colon carcinoma.

The antigen of a monoclonal antibody that is specific for cells of human carcinoma of the colon is a monosialoganglioside as determined by the direct binding of antibody to thin-layer chromatograms of total lipid extracts of tissues. Binding of antibody to chromatograms is detected by autoradiography after the application of iodine-125-labeled F(ab')2 of rabbit immunoglobulin G antibodies to mouse immunoglobulins.

Transgenic Leishmania and the immune response to infection.

Genetic manipulation of single-celled organisms such as the Leishmania parasite enables in depth analysis of the consequences of genotypic change on biological function. In probing the immune responses to infection, use of transgenic Leishmania has the potential to unravel both the contribution of the parasite to the infection process and the cellular interactions and mechanisms that characterize the innate and adaptive immune responses of the host. Here, we briefly review recent technical advances in parasite genetics and explore how these methods are being used to investigate parasite virulence factors, elucidate immune regulatory mechanisms and contribute to the development of novel therapeutics for the leishmaniases. Recent developments in imaging technology, such as bioluminescence and intravital imaging, combined with parasite transfection with fluorescent or enzyme-encoding marker genes, provides a rich opportunity for novel assessment of intimate, real-time host-parasite interactions at a previously unexplored level. Further advances in transgenic technology, such as the introduction of robust inducible gene cassettes for expression in intracellular parasite stages or the development of RNA interference methods for down-regulation of parasite gene expression in the host, will further advance our ability to probe host-parasite interactions and unravel disease-promoting mechanisms in the leishmaniases.

A randomised controlled trial examining the longer-term outcomes of standard versus new antiepileptic drugs. The SANAD trial.

OBJECTIVES: To compare clinicians' choice of one of the standard epilepsy drug treatments (carbamazepine or valproate) versus appropriate comparator new drugs. DESIGN: A clinical trial comprising two arms, one comparing new drugs in carbamazepine and the other with valproate. SETTING: A multicentre study recruiting patients with epilepsy from hospital outpatient clinics. PARTICIPANTS: Patients with an adequately documented history of two or more clinically definite unprovoked epileptic seizures within the last year for whom treatment with a single antiepileptic drug represented the best therapeutic option. INTERVENTIONS: Arm A was carbamazepine (CBZ) versus gabapentin (GBP) versus lamotrigine (LTG) versus oxcarbazepine (OXC) versus topiramate (TPM). Arm B valproate (VPS) versus LTG versus TPM. MAIN OUTCOME MEASURES: Time to treatment failure (withdrawal of the randomised drug for reasons of unacceptable adverse events or inadequate seizure control or a combination of the two) and time to achieve a 12-month remission of seizures. Time from randomisation to first seizure, 24-month remission of seizures, incidence of clinically important adverse events, quality of life (QoL) outcomes and health economic outcomes were also considered. RESULTS: Arm A recruited 1721 patients (88% with symptomatic or cryptogenic partial epilepsy and 10% with unclassified epilepsy). Arm B recruited 716 patients (63% with idiopathic generalised epilepsy and 25% with unclassified epilepsy). In Arm A LTG had the lowest incidence of treatment failure and was statistically superior to all drugs for this outcome with the exception of OXC. Some 12% and 8% fewer patients experienced treatment failure on LTG than CBZ, the standard drug, at 1 and 2 years after randomisation, respectively. The superiority of LTG over CBZ was due to its better tolerability but there is satisfactory evidence indicating that LTG is not clinically inferior to CBZ for measures of its efficacy. No consistent differences in QoL outcomes were found between treatment groups. Health economic analysis supported LTG being preferred to CBZ for both cost per seizure avoided and cost per quality-adjusted life-year gained. In Arm B for time to treatment failure, VPS, the standard drug, was preferred to both TPM and LTG, as it was the drug least likely to be associated with treatment failure for inadequate seizure control and was the preferred drug for time to achieving a 12-month remission. QoL assessments did not show any between-treatment differences. The health economic assessment supported the conclusion that VPS should remain the drug of first choice for idiopathic generalised or unclassified epilepsy, although there is a suggestion that TPM is a cost-effective alternative to VPS. CONCLUSIONS: The evidence suggests that LTG may be a clinical and cost-effective alternative to the existing standard drug treatment, CBZ, for patients diagnosed as having partial seizures. For patients with idiopathic generalised epilepsy or difficult to classify epilepsy, VPS remains the clinically most effective drug, although TPM may be a cost-effective alternative for some patients. Three new antiepileptic drugs have recently been licensed in the UK for the treatment of epilepsy (levetiracetam, zonisamide and pregabalin), therefore these drugs should be compared in a similarly designed trial.

Mutation of Hip's carboxy-terminal region inhibits a transitional stage of progesterone receptor assembly.

Steroid receptor complexes are assembled through an ordered, multistep pathway involving multiple components of the cytoplasmic chaperone machinery. Two of these components are Hsp70-binding proteins, Hip and Hop, that have some limited homology in their C-terminal regions, outside the sequences mapped for Hsp70 binding. Within this region of Hip is a DPEV sequence that occurs twice; in Hop, one DPEV sequence plus a partial second sequence occurs. In an effort to better understand Hip function as it relates to assembly of progesterone receptor complexes, the DPEV region of Hip was targeted for mutations. Each DPEV sequence was mutated to an APAV sequence, singly or in combination. The combined mutation, APAV2, was further combined with a deletion of Hip's tetratricopeptide repeat region that is required for Hsp70 binding or with a deletion of Hip's GGMP repeat. An additional mutant was prepared by truncation of Hip's DPEV-containing C terminus. By comparing interactions of various Hip forms with Hsp70, it was determined that mutation of the DPEV sequences created a dominant inhibitory form of Hip. The mutant Hip-Hsp70 complex was not prevented from interacting with progesterone receptor, but the mutant caused a dose-dependent inhibition of receptor assembly with Hsp90. The behavior of the Hip mutant is consistent with a model in which Hip and Hop are required to facilitate the transition from an early receptor complex with Hsp70 into later complexes containing Hsp90.

Mutational analysis of the hsp70-interacting protein Hip.

The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes. During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions. In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements. Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR). Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes. Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat. Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes. Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes. This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain. In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences.

Progesterone receptor structure and function altered by geldanamycin, an hsp90-binding agent.

The assembly of progesterone receptor (PR) heterocomplexes in vitro involves at least eight components of the molecular chaperone machinery, and as earlier reports have shown, these proteins exhibit complex, dynamic, but ordered, interactions with one another and PR. Using the selective hsp90 binding agent geldanamycin (GA), we have found that PR assembly in vitro can be arrested at a previously observed intermediate assembly step. Like mature PR complexes, the intermediate complexes contain hsp90, but they differ from mature complexes by the presence of hsp70, p60, and p48 and the absence of immunophilins and p23. Arrest of PR assembly is likely due to GA's ability to directly block binding of p23 to hsp90. An important functional consequence of GA-mediated assembly arrest in vitro is the inability of the resulting PR complexes to bind progesterone, despite the presence of hsp90 in the receptor complexes. The biological significance of the in vitro observations is demonstrated by GA's ability to (i) rapidly block PR's hormone binding capacity in intact cells and (ii) alter the composition of COS cell PR complexes in a manner similar to that observed during in vitro reconstitutions. An updated model for the cyclic assembly pathway of PR complexes that incorporates the present findings with earlier results is presented.

Binding of heat shock proteins to the avian progesterone receptor.

The protein composition of the avian progesterone receptor was analyzed by immune isolation of receptor complexes and gel electrophoresis of the isolated proteins. Nonactivated cytosol receptor was isolated in association with the 90-kilodalton (kDa) heat shock protein, hsp90, as has been described previously. A 70-kDa protein was also observed and was shown by Western immunoblotting to react with an antibody specific to the 70-kDa heat shock protein. Thus, two progesterone receptor-associated proteins are identical, or closely related, to heat shock proteins. When the two progesterone receptor species, A and B, were isolated separately in the absence of hormone, both were obtained in association with hsp90 and the 70-kDa protein. However, activated receptor isolated from oviduct nuclear extracts was associated with the 70-kDa protein, but not with hsp90. A hormone-dependent dissociation of hsp90 from the cytosolic form of the receptor complex was observed within the first hour of in vivo progesterone treatment, which could explain the lack of hsp90 in nuclear receptor complexes. In a cell-free system, hsp90 binding to receptor was stabilized by molybdate but disrupted by high salt. These treatments, however, did not alter the binding of the 70-kDa protein to receptor. Association of the 70-kDa protein with the receptor could be disrupted by the addition of ATP at elevated temperatures (23 degrees C). The receptor-associated 70-kDa protein is an ATP-binding protein, as demonstrated by its affinity labeling with azido[32P]ATP. These results indicate that the two receptor-associated proteins interact with the progesterone receptor by different mechanisms and that they are likely to affect the structure or function of the receptor in different ways.

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.

Molecular cloning of human FKBP51 and comparisons of immunophilin interactions with Hsp90 and progesterone receptor.

A cDNA for human FKBP51 has been cloned and sequenced, and protein products have been expressed in both in vitro and bacterial systems. The deduced amino acid sequence for human FKBP51 is 90% identical to sequences of recently described murine proteins and is 55% identical to the sequence of human FKBP52. Human FKBP51 mRNA is expressed in a wide range of tissues, and the protein has peptidylprolyl isomerase activity that is inhibited by FK506 but not cyclosporine. FKBP51 is the same as a previously described progesterone receptor-associated immunophilin that, similar to FKBP52 and cyclophilin 40, is an Hsp90-binding protein and appears in functionally mature steroid receptor complexes along with Hsp90 and p23. Each of the three receptor-associated immunophilins displays interactions with progesterone receptor that are more dynamic than Hsp90-receptor interactions. Whereas FKBP52 and FKBP51 compete about equally well for binding to Hsp90 in a purified system, FKBP51 accumulates preferentially in progesterone receptor complexes assembled in a cell-free system. This observation provides a precedent for differential interactions between Hsp90-associated immunophilins and target proteins such as steroid receptors.

Short-term changes in maternal and neonatal urinary iodine excretion.

Investigation of maternal urinary iodine (UI) excretion in the immediate antenatal and early postpartum periods showed a precipitous fall in median values from 93 microg/L antenatally to 36 microg/L at delivery subsequently rising to 49 microg/L and 63 microg/L at days 3 and 10 postpartum respectively. The fate of ingested iodine not appearing in the maternal urine is unknown but measurement of UI in babies born to nursing mothers suggested transfer from the mother with median neonatal values of 117 and 159 microg/L being recorded at days 3 and 10. While maternal UI seemed to relatively unaffected by breast feeding, median UI from breast feeding babies (148 microg/L) was significantly greater than in those bottle feeding (50 microg/L). This was also reflected by the finding that no breast feeding baby had a UI values < 50 microg/L in comparison to 50% of bottle feeders. The depressed values in mothers and relatively high values in their infants could present a false picture and suggest the need to defer any investigations of iodine status at this time. The findings do however suggest a need for further investigations aimed at determining the fate of iodine ingested perinatally and its possible physiological significance in maintaining thyroid status in the mother and neonate.

Prevention of osteoporosis in glucocorticoid-treated neurology patients.

OBJECTIVES: Iatrogenic, including corticosteroid-induced osteoporosis is preventable with administration of osteoprotective biphosphonates. The best medical practice is published in the National Guidelines: UK Osteoporosis Consensus Group (1998, update 2002). We conducted an audit in prednisolone-treated general neurology patients, to assess compliance to national guidelines, raise awareness of osteoporosis prevention, and improve clinical practice in a tertiary neurology referral centre. METHODS AND RESULTS: Preintervention: Of the 48 cases (21 male) identified twenty-nine (61%) received osteoporosis prophylaxis. Nineteen (40%) were given biphosphonates, while 10 (21%) hormone replacement therapy or calcium and Vitamin D. INTERVENTION: Results were presented to the consultant body. Postintervention: Data were collected prospectively on 48 patients (30 male) in year 2001. Thirty-eight (79%) received prophylaxis: 35 (73%) were started on biphosphonates, while 3 (6%) on calcium and Vitamin D. This process was repeated 2 years later to assess sustainability. Of the 48 patients, 44 (92%) received prophylaxis: 41 (86%) were taking biphosphonates, while 3 (6%) calcium and Vitamin D. CONCLUSION: We present an original and complete audit on osteoporosis prophylaxis in a typical population of neurology patients. Though initial results were similar to previous reports, our audit led to significant improvement in clinical practice. National guidelines could not be followed meticulously, as our centre has no regular access to bone densitometry. Our patient population had other risk factors for osteoporosis apart from steroid use. Therefore, we recommend that neurologists in this setting use osteoporosis prophylaxis for all their patients on long-term corticosteroids.

Poly(A)-binding protein I of Leishmania: functional analysis and localisation in trypanosomatid parasites.

Regulation of gene expression in trypanosomatid parasites is predominantly post-transcriptional. Primary transcripts are trans-spliced and polyadenylated to generate mature mRNAs and transcript stability is a major factor controlling stage-specific gene expression. Degenerate PCR has been used to clone the gene encoding the Leishmania homologue of poly(A)-binding protein (Lm PAB1), as an approach to the identification of trans-acting factors involved in this atypical mode of eukaryotic gene expression. lmpab1 is a single copy gene encoding a 63 kDa protein which shares major structural features but only 35-40% amino acid identity with other PAB1 sequences, including those of other trypanosomatids. Lm PAB1 is expressed at constant levels during parasite differentiation and is phosphorylated in vivo. It is localised predominantly in the cytoplasm but inhibition of transcription with actinomycin D also reveals diffuse localisation in the nucleus. Lm PAB1 binds poly(A) with high specificity and affinity but fails to complement a null mutation in Saccharomyces cerevisiae. These properties are indicative of functional divergence in vivo.

Iodine intake in pregnancy in Ireland--a cause for concern?

BACKGROUND: Adequate dietary iodine intake is necessary to maintain maternal thyroid function at a level permitting normal neuropsychological development of the foetus. AIMS AND METHODS: To determine dietary iodine status by measuring urinary iodine excretion (UIE), proportional to dietary intake, in Irish mothers during the first trimester of pregnancy. RESULTS: Median UIE showed seasonal variations, being lower in summer than in winter. The median values in pregnant women were, summer 45microg/l, winter 68microg/l. Equivalent values for controls were 43 and 91microg/l respectively. UIE required to achieve WHO recommended daily iodine intakes would be 120-180microg/l. In the Irish subjects UIE values suggestive of iodine deficiency (<50microg/l) were observed in 55% of pregnant women tested in summer and 23% in winter. Dairy milk iodine, a major dietary iodine source, showed similar variation. CONCLUSIONS: While there is as yet no available evidence of widespread thyroid hypofunction in the Irish obstetric population, the findings are a cause of concern, which if confirmed by a more comprehensive investigation, may indicate the need for iodine prophylaxis.

Micronucleated erythrocytes as an index of cytogenetic damage in humans: demographic and dietary factors associated with micronucleated erythrocytes in splenectomized subjects.

Erythrocytes containing micronuclei serve as an indicator of genotoxic exposure in splenectomized individuals. Micronucleated erythrocytes, derived from cytogenetically damaged RBC precursors, are not selectively removed from peripheral blood in individuals who lack splenic function. The relationship between micronucleated cell frequencies and demographic, environmental, and dietary factors was examined in 44 subjects with previous splenectomy due to trauma. Their micronucleated cell counts fit a log-normal distribution, with geometric means of 3.3 micronucleus-containing cells/1000 reticulocytes and 2.7/1000 normochromatic erythrocytes. A multiple regression analysis showed that drinking five cups of coffee or tea/day (relative to none) was associated with an approximately 2-fold higher frequency of micronucleated cells. Weaker statistical associations were also noted with micronucleus frequency and the consumption of calcium supplements (associated with a higher frequency) and vitamins A, C, or E (lower frequency). An apparent trend of higher micronucleus counts with age was attenuated when other factors were considered in the regression. Cigarette smoking and decaffeinated coffee consumption were among the factors not associated with elevated micronucleated cell frequencies. Because the occurrence of micronuclei in reticulocytes reflects cytotoxic exposures within the past 3-8 days, it may be possible to test directly the relationship of these factors to micronucleus formation through intervention studies.

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.