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The cytosolic fraction of goat cauda epididymis possesses a protein kinase (PKx) activity which is stimulated by a number of unsaturated fatty acids of which arachidonic acid is the best activator in absence of cAMP or Ca(2+). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and diacylglycerol have no effect either alone or in combination. The membrane fraction does not show any appreciable kinase activity even after detergent treatment. PKx migrates as a single band of apparent molecular mass of 116 kDa on 10% SDS-PAGE after sequential chromatographic separation on DEAE-cellulose, phenyl-Sepharose, high-Q anion exchange and protamine-agarose affinity column. PKx phosphorylates histone H1, histone IIIs and protamine sulfate, but not casein. However, the best phosphorylation was obtained with a substrate based on PKC pseudosubstrate sequence (RFARKGSLRQKNV). The kinase phosphorylates two endogenous cytosolic proteins of 60 and 68 kDa. Ser residues are primarily phosphorylated although a low level of phosphorylation is observed on Thr residues also. Ca(2+) and Mn(2+) inhibit PKx activity in the micromolar range. Staurosporine is found to inhibit the PKx activity to a significant level at sub-nanomolar concentration. Lyso-phosphatidylcholine and certain detergents at very low concentrations (<0.05%) stimulate enzyme activity to some extent. The immuno-crossreactivity study with antibody against different PKC isotypes suggests that the protein kinase under study is not related to any known PKC family. Even the antibody against PKN (a related protein kinase reported in rat testis found to be activated by arachidonic acid) does not cross-react with this protein kinase. Hence we believe that the protein kinase (PKx) reported here is different even from the PKN of rat testis. The phosphorylation of endogenous proteins by the protein kinase may be involved in cell regulation including fertility regulation and signal transduction.

Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced.

1. Alcohol dehydrogenase (EC 1.1.1.1.) has been immobilised to aminoethyl-cellulose by glutaraldehyde, to DEAE-cellulose by an s-triazine derivative and to agarose using CNBr. Lactate dehydrogenase has been immobilised to the latter two supports. 2. Their use for affinity chromatography of NAD was compared and alcohol dehydrogenase immobilised to CNBr-activated agarose chosen for detailed study due to the efficient coupling of applied enzyme and the specific nature of binding. 3. The efficiency of coupling of alcohol dehydrogenase dropped from 94.5 to 72.2% when the applied load was increased from 18 to 54 mg/g activated agarose. Activity relative to free enzyme fell from 21 to 11%. The binding of NAD was maximal between pH 5.5 and 6. With the lowest loading of enzyme, NAD binding fell from 450 to 320 mug/g support when the linear flow rate was increased from 0.84 to 3.95 cm/min. 4. NAD was completely separated from a mixture with ATP, ADP and AMP. Separation from NMN and hydrolysed RNA and DNA was evidently possible. Immobilised alcohol dehydrogenase used for 34 binding experiments over a period of weeks maintained 60% of its original enzyme activity. 5. The method was applied to yeast NAD following mechanical disruption of yeast, clarification and either ultrafiltration or hollow-fibre dialysis to permit separate purification of macromolecules and nucleotides.

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.

Fibrinogen has been purified from human platelets. Platelet fibrinogen exhibits a characteristic pattern in agar gel immunoelectrophoresis different from that of plasma fibrinogen. Stepwise plasmin degradation has been used in further elucidation of the molecular properties of the platelet protein. Examination of comparative digests by immunologic and gel electrophoretic methods has revealed that (1) the platelet protein is more resistant to plasmin degradation, (2) the plasmin-produced fragments of platelet fibrinogen differ consistently from those of its plasma counterpart, and (3) platelet fibrinogen is different from fragment X of plasma fibrinogen. It is suggested that platelet fibrinogen may contribute to the stability of the thrombus.