Kenneth D. Gibson

Supports for reverse-phase high-performance liquid chromatography of large proteins

Abstract Reverse-phase high-performance liquid chromatography supports have been developed for use in separating proteins up to 300,000 M r . They are based on silica supports to which octyl, cyanopropyl, or diphenyl groups are covalently bonded. Their effectiveness in rapidly separating several standard proteins is demonstrated. Applications presented include the separation of the α 1 and α 2 chains of chick Type I collagen within 1 h and the separation of the α and β components of human Type I collagen.

Separation of the major species of interstitial collagen by reverse-phase high-performance liquid chromatography

Abstract Results presented here demonstrate a further application of reverse-phase high-performance liquid chromatography to the separation of large proteins. At a pH near 4.5 with a high pyridine concentration, we have completely separated three major types of human collagen (Types I, II, and III) from mixtures. We illustrate the application of this technique to the preparation of Types I and II collagen from lathyritic chick cartilage extracts.

Some species of human leukocyte interferon are glycosylated.

The carbohydrate content of all of the species of human leukocyte interferon (IFN-alpha) which have been derived from patients with chronic myelogeneous leukemia (CML) and purified to homogeneity has now been determined. Amino sugar content was measured by high-performance liquid chromatography and fluorescamine detection of acid hydrolysates of each sample. Two species showed significant amounts of glucosamine. Most of the purified species of leukocyte interferon from a myeloblast cell line were also tested, and two species were found to contain sugar residues. These forms also differed from the CML interferons in that they revealed the presence of greater amounts of galactosamine. The apparent lack of carbohydrate in some of the higher-molecular-weight species of interferon implicated factors other than glycosylation in the molecular weight differences. The results indicate that some species of IFN-alpha are glycosylated to various degrees.

Membranes of Rhodopseudomonas spheroides: II. Precursor-product relations in anaerobically growing cells

Abstract Rhodopseudomonas spheroides, growing exponentially under anaerobic conditions in the light, was exposed to a 3-min pulse of labeled leucine or phenylalanine, followed by a chase of several hours. Subcellular fractions were isolated from cells harvested at various times during the chase, and their specific radioactivities determined. The results confirmed that chromatophores are formed from a precursor structure. From the decay of their specific activity, it was concluded that isolated small membrane fragments are precursors of chromatophores. Examination of the labeling of individual protein bands separated by gel electrophoresis showed that all the chromatophore protein bands except one acquire label at the same rate, and also confirmed that the proteins of the small membrane fragments have the behavior expected of precursors.

Characterization of two circular satellite species of deoxyribonucleic acid in Rhodopseudomonas spheroides

Abstract DNA, isolated from a strain of Rhodopseudomonas spheroides , showed two components when banded in CsCl, with buoyant densities of 1.719 and 1.724 g/ml. The lighter band comprised about 8% of the total. Using a mild lysis procedure, it was possible to extract DNA of which 15–20% was circular. Circular DNA, isolated by dye-buoyant density centrifugation, contained two components with buoyant densities 1.718 and 1.724 g/ml. Sedimentation of the circular DNA gave two peaks, with S 20,w values of 78.2 S and 52.7 S ; sedimentation in alkali, and in neutral solution after denaturation, showed that the first peak was closed circular and the second peak open circular DNA. Band-buoyant density sedimentation of the circular DNA in alkaline CsCl initially showed two peaks; after a time, each peak split into two. The alkaline buoyant densities of the four peaks were consistent with open and closed circles with neutral buoyant densities 1.718 and 1.724 g/ml. It is concluded that Rps. spheroides contains two circular satellite DNA species, with the same molecular weight of 70–75 × 10 6 daltons, but with slightly different base compositions.

Is adrenal proenkephalin glcosylated

Abstract Adrenal proenkephalin contains the sequence AsnSerSer that is a typical site for the attachment of asparagine-linked carbohydrate. The 5300- and 18,200-Da bovine adrenal proteins derived from proenkephalin contain this recognition sequence and were therefore analyzed for the presence of both amino and neutral sugars. Less than 0.05 mol of amino sugar and less than 0.1 mol of neutral sugar were found per mole of each protein. No amino sugar was detected in other high-molecular-weight adrenal [Met]enicephalin-containing proteins. Together these findings indicate that bovine adrenal proenkephalin does not contain asparagine-linked carbohydrate.

Effect of RNA synthesis inhibitors on stimulation of sulfation by L-3,5,3′-triiodothyronine

Abstract Stimulation of sulfation by T3 1 in chick embryo cartilage was blocked when actinomycin D or camptothecin was added to the incubation medium together with the T3. When either inhibitor was added 2 hr after the T3, the stimulation was not affected. The results suggest that there is an early step in the action of T3 that involves synthesis of a stable species of RNA that is larger than 5S. α-Amanitin had no effect on the stimulation of sulfation by T3, although it inhibited synthesis of poly(A)-rich RNA almost completely. This suggests that the action of T3 is not associated with increased synthesis of RNA by RNA polymerase II. We conclude that the stimulation of sulfation by T3 involves synthesis of a species of RNA that is larger than 5S but is not a messenger.

Characterization of an α-glucan from chick embryo sternum whose synthesis is stimulated by l-3,5,3′-triiodothyronine and human serum

Abstract Incorporation of [3H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l -3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 n m triiodothyronine and 5–20% ( v v ) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [3H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [32P]phosphate, [35S]sulfate, [3H]uridine, or [3H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.