Differences in the cholinergic proteolipid isolated by affinity chromatography from normal and denervated rat leg muscle

The cholinergic proteolipid from rat leg muscle, isolated by Sephadex LH-20 column chromatography, was purified about 5 fold further by affinity chromatography in organic solvents. The affinity column consisted of p-phenyltrimethylammonium as the active group and a pulse of acetylcholine or acid was used to desorb the specific fraction. From normal controls 36 μg of specific protein per g fresh tissue were obtained. In the denervated muscle there was no increase in this protein after three days, but it increased by 120% after six days. Binding studies carried out with ¹⁴C-acetylcholine, ³H-α-bungarotoxin and ¹⁴C-d-tubocurarine showed that only the specific fraction was able to bind the ligands. Three days after denervation the specific activity (nmoles/mg protein) for ¹⁴C-acetylcholine increased 400% and for ³H-α-bungarotoxin 100% over the control; on the contrary, there was no change in the binding of ¹⁴C-d-tubocurarine. These results are discussed in relation to the different pharmacological properties of the functional and extrajunctional receptors in skeletal muscle.     

Isolation of a chymotrypsin inhibitor from Culex pipiens by affinity chromatography

A chymotrypsin inhibitor was isolated from adult mosquitoes of Culex pipiens by affinity chromatography. Sepharose with covalently coupled chymotrypsin from Culex larvae was used as the fractionation matrix. The isolated inhibitor migrates as a heavy major and a faint minor band on 7.5% polyacrylamide gel and has a molecular weight of 6,800. At higher concentrations it exhibits a 82% inhibition of the mosquito enzyme. Its inhibitory activity is not affected by pH above 5 and appears stable against heat denaturation.     

Isolation of ADP-ribosyltransferase by affinity chromatography

An affinity adsorbent for ADP-ribosyltransferase (EC 2.4.2.30) has been synthesized by coupling 3-aminobenzamide to Sepharose 4B. Using this material, ADP-ribosyltransferase from human placenta has been purified from crude extract to homogeneity within a few hours. The enzyme has an apparent Km for NAD+ of 52 microM. Its molecular mass is 115,000 as determined by gel electrophoresis. The enzyme is DNA dependent and stimulated by histone, its temperature optimum is at 25 degrees C, and its pH optimum is around pH 9. alpha-NAD+, thymidine, caffeine, theophylline, theobromine, 3-methoxybenzamide, and nicotinamide inhibit the enzyme. Purification of ADP-ribosyltransferases from horse, rat, and chicken liver was also achieved with the method described.     

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Isolation of brain tubulin by affinity chromatography

An affinity chromatographic procedure for the isolation of tubulin from brain is described. The yield is good and the method rapid and gentle. Criteria of purity are colchicine binding, SDS acrylamide gel electrophoresis, and velocity sedimentation.     

Isolation of S-100 binding proteins from brain by affinity chromatography

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.     

Isolation of tyrosinase-mRNA by affinity chromatography of polysomes

We present a method whereby some mRNAs which code for enzymes can be isolated by affinity chromatography of newly synthesized polypeptides bound to their polysome complexes. Using this method we have isolated tyrosinase-mRNA from Xenopus laevis oocytes and have analysed the translation products from the RNAs thus obtained. In vitro translation reveals the presence of two mRNAs coding for polypeptides of molecular weights of 20 000 and 32 000, respectively. The larger molecule corresponds to the molecular weight of nascent tyrosinase. Furthermore, microinjection of the mRNA into Xenopus oocytes results in the synthesis of active tyrosinase. Since this isolation method is dependent on the ability of nascent enzymes to bind to their substrate analogue, it is thought that this approach may be appropriate for obtaining mRNAs coding for other enzymes.     

Isolation of lipid-free C-reactive protein by affinity chromatography

Human C-reactive protein purification has been hampered by its association with lipids. Isolation of pure lipid-free C-reactive protein was obtained by a three step procedure. First, partially lipid-free C-reactive protein was obtained by affinity chromatography from ascitic fluids; second, lipid-bound proteins were eliminated by calcium-dependent precipitation; and third, lipid-free pure C-reactive protein was obtained by affinity re-chromatography of the supernatant. A 46-50% yield of lipid-free C-reactive protein was obtained compared with the 14.7% obtained by the old method of extraction with lipid solvents.