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Biomedical subjects

V Lee-Own

Publications and source records attributed to V Lee-Own.

8 recordsLinked to original sources
PubMed1979

Formation of a vitamin B-12-serum complex on heating at alkaline pH.

The binding of vitamin B-12 to serum proteins during heating at alkaline pH was investigated by gel filtration of serum supplemented with cyano[57Co]-cobalamin. Heating for 5 min at 100 degrees C destroyed most of the vitamin B-12 binding activity of serum but, with further heating, the vitamin B-12 became incorporated into a complex that did not correspond in molecular size to the original vitamin B-12 binding proteins. Radioassay of vitamin B-12 in heated serum showed correspondingly first an increase then a progressive decrease in the apparent vitamin B-12 level suggesting that, on heating, vitamin B-12 was initially released then subsequently complexed by the serum. The formation of complexed vitamin B-12 was abolished by the presence of the reducing agent dithiothreitol during the heating step.

Chromatography, Gel↗
PubMed1979

Three different radioiodination methods for human spleen ferritin compared.

We compared three different methods for radioiodination of human spleen ferritin. Tracers prepared by direct oxidative iodination with use of Chloramine T or 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycoluril (Iodogen) were structurally altered by the labeling procedure, as was made apparent by gel filtration and electrophoresis on polyacrylamide gel. Tracers prepared by conjugation to radioiodinated N-succinimidyl-6-(4-hydroxyphenyl)propionate retained their structural integrity, as assessed by physicochemical methods. Such tracers bound more than 90% in antibody excess, yielded self-displacement curves parallel to unlabeled ferritin standards, and retained their immunoreactivity for nine weeks. By contrast, immunological activity and stability of tracers prepared by the oxidative methods were less satisfactory. Tracers prepared by using lodogen were unsuitable for use in the ferritin radioimmunoassay because of shallow standard-inhibition curves and poor binding in the absence of added standard. Thus, we found the conjugation procedure to be the most suitable of the three methods for preparation of spleen-ferritin tracers.

Chloramines↗
PubMed1976

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.

Amino Acids↗
PubMed1975

The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with alpha2 chains of type I collagen.

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.

Animals↗
PubMed1975

The preparation of a bacterial collagenase containing negligible non-specific protease activity.

A three stage procedure for the purification of crude bacterial collagenase is described. The three stages were ion exchange chromatography on SP-Sephadex and DEAE-cellulose, followed by molecular sieve chromatography on Sephadex G-200. The end product was eluted from Sephadex G-200 as a single peak of absorbance at 280 nm, and a single zone of activity against gelatin. The active eluate was divided into two halves, designated fraction 1 and 2 collagenase. Their activities were greater than those of commercially available collagenases when assayed viscometrically against pepsin solubilized collagen from guinea-pig skins. The non-specific protease activities in both fractions were much less than in the commercially available purified collagenases, and fraction 1 collagenase liberated only 2.6% of a [3H]-tryptophan label from a substrate of 2 mg of labelled chick embryo proteins, after an 18 hour incubation. When polymeric collagen was incubated with fraction 1 collagenase, at a final enzyme : substrate ratio of 1:160, the collagen was digested, resulting in the loss of 99.8% hydroxyproline as dialysable material.

Animals↗