Purification of bovine somatomedin.

A procedure for the purification of bovine somatomedin (SM⁴) is presented. The purification scheme utilizes ultrafiltration through membranes of nominal mol. wt. cutoffs, molecular sieve chromatography and finally iso-electric focusing. Two peaks of SM activity, measured by the $\text{in vitro}$ stimulation of ³⁵S-Na₂SO₄ and ³H-thymidine uptake by costal cartilage, were present after focusing; an acidic component having a pI of 6.0 – 6.7 and a basic component having a pI in the range of 7.8 – 8.3. The acidic component comprised 2% of the initial activity and was 120,000-fold purified: the basic component comprised 10% of the initial activity and was 350,000-fold purified relative to the starting material. These components are similar in molecular size and pI to SM-A and SM-C isolated from human plasma.     

Measurement of urinary somatomedin C by a chemiluminescent method

We present a method for the measurement of the total Somatomedin C (SmC) content in human early morning urine samples after dialysis, extraction, and concentration. We modified a chemiluminescence immunoassay, previously developed for SmC determination in serum, for analysis of SmC in urine. Appropriate sensitivity was obtained by the preparation of a new chemiluminescent tracer (AEEI-COOH-SmC) and the optimization of a competitive non-equilibrium immunoassay system which had a detection limit of 0.24 fmol SmC per tube.     

A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column

Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.     

A substance resembling somatomedin C in the American cockroach

Material antigenically resembling somatomedin C (type I insulin-like growth factor, IGF-I) is demonstrated in the American cockroach Periplaneta americana by means of a monoclonal antibody immunoperoxidase technique. It was localized histochemically in neuronal cell somata and axonal fibers (probably interneurons) of the central nervous/neuroendocrine system and in 'endocrine-type' cells lining the midgut epithelium. The IGF-I-like substance is different from vertebrate insulin and also distinct from materials immunostained by different insulin antibodies in the brain and neurohaemal complex of this insect species. These findings are viewed in the light of recent reports on the presence and action of insulin-like chemicals in insects, and with respect to the existence of an insect brain-midgut system similar to the mammalian brain-gastroenteropancreatic system.     

A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens.

This paper describes a simple and rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, together with analysis of the kinetic behaviour and some properties of the enzyme. The purification consisted of two steps, 2',5'-ADP-Sepharose 4B affinity chromatography and DEAE Sepharose Fast Flow ion exchange chromatography in procedure which took two working days. The enzyme was obtained with a yield of 13.7% and had a specific activity of 2.64 U/mg protein. The overall purification was about 19,700-fold. The molecular weight of the enzyme was found to be 62 +/- 3 kDa by Sephadex G-200 gel filtration chromatography. A protein band corresponding to a molecular weight of 69.2 +/- 3.2 kDa was obtained on SDS polyacrylamide slab gel electrophoresis. On chromatofocusing, lens glucose-6-phosphate dehydrogenase gave a single peak at pI 5.14. The activation energy of the reaction catalyzed by the enzyme was calculated from Arrhenius plot as Ea = 5.88 kcal/mol. The pH versus velocity curve had two peaks at pH 7.7 and 9.6. By the double-reciprocal plots and the product inhibition studies, it was shown that the enzyme follows 'Ordered Bi Bi' sequential kinetics. From the graphical and statistical analyses, KmNADP+, KmG-6-P, KiNADPH, Ki6-PGA were estimated to be 0.008 +/- 0.002, 0.035 +/- 0.013, 0.173 +/- 0.007 and 1.771 +/- 0.160 mM, respectively. The observed kinetic behaviour of glucose-6-phosphate dehydrogenase from bovine lens was in accordance with the enzyme from other sources.     

Partial purification of somatomedin from human plasma.

Fractional precipitation of human plasma using ethanol, followed by chromatography on S.P. Sephadex, yielded a somatomedin-enriched fraction freed from substantial amounts of inhibitory substances. Heat coagulation of the proteins present in this fraction allowed the recovery of appreciable amounts of active components which were then chromatographed on Sephadex G-75 and S.P. Sephadex. A major part of the activity was associated with components less than 4,000 daltons, suggesting that somatomedin, or an active fragment thereof, had been dissociated from a carrier protein by the heat treatment. The range of pH employed throughout was 5.3-9.8. Recoveries of about 30% of biological activity with fold-purification up to 38, as measured by radioactive sulphate uptake in the chick pelvic cartilage assay, were higher than those obtained using acid-ethanol extraction.     

Simultaneous purification and polymerization method for bovine serum albumin preparation

Bovine serum albumin (BSA) has various applications in blood group serology and different research purposes. In this study purification of BSA has been compared with human serum albumin (HSA) using modified ethanol precipitation method based on the method of Cohn. The purification process was carried out under controlled conditions, particularly of ethanol concentration, pH, ionic strength and temperature. It was revealed that the produced BSA and HSA have purity more than 95%. It is obvious that HSA can be used, as a drug when the amount of its polymers is less than 5% whereas polymer generation is required in order to enhance the potentiating properties of BSA in agglutination of red cells. We propose here a simple and rapid two-step method for simultaneously purification and polymerization of BSA. By this method simply BSA with desired amount of polymers was obtained by 40% ethanol concentration.     

An improved method for the purification of kinesin from bovine adrenal medulla.

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 micrograms of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca++ ATPase of 0.4 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.     

Mitogenic activity of hydroxyapatite: requirement for somatomedin C

Synovial hyperplasia is a feature of the chronic synovitis associated with basic calcium phosphate crystals [hydroxyapatite (HA), octacalcium phosphate, tricalcium phosphate] and calcium pyrophosphate. Each of these crystals stimulated mitosis of cultured human skin fibroblasts or canine synovial fibroblasts in a concentration-dependent fashion. We examined the effect of pure somatomedin C (Sm-C) on HA crystal induced mitogenesis. Confluent cultures of human fibroblasts were rendered quiescent by incubation in the presence of 1% platelet-poor-Sm-C free plasma (PPSCFP) for 24 hours. HA crystals stimulated thymidine incorporation 2.3-fold over control value. Addition of Sm-C significantly augmented the effect of HA crystals (P less than 0.01). Nearly identical effects were observed in the presence of 100 micrograms/ml HA crystals or 15 ng/ml PDGF. Monoclonal antibodies against Sm-C had little effect on the basal 3H thymidine uptake by control cells incubated in 1% PPSCFP but blocked over 50% of the HA crystal or PDGF-induced 3H thymidine incorporation both in the presence or absence of Sm-C. The incomplete blocking suggested either the presence of other "progression" factors, such as insulin-like growth factor II in the conditioned media or the possibility that HA or PDGF in high enough dosage enabled cells to escape their dependence on Sm-C for DNA synthesis.     

A simple method for the purification of myxobacteria

Pure cultures of many myxobacteria could be quickly obtained by treating fruiting bodies with highly dosed mixtures of antibiotics     

Chemical synthesis of a gene for somatomedin C.

A synthetic gene for somatomedin C, a human growth factor, has been assembled by a single ligation of 23 oligodeoxyribonucleotides, which were chemically synthesized by an improved solid phase phosphotriester method.