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In cooperative aggregation of bovine platelets induced by simultaneous addition of PAF and collagen at subthreshold concentrations the following observations were made. (i) Formation of phosphatidic acid and arachidonic acid metabolites, which characterize PAF and collagen alone-induced aggregation, respectively, was observed very obviously. (ii) A thromboxane antagonist did not completely block the cooperative aggregation. The extent of residual aggregation activity was dependent on concentration of collagen used in the simultaneous administration with PAF. These results suggest that both signal transduction pathways activated by PAF and collagen alone at high concentrations are attained by simultaneous addition of both agonists at subthreshold concentrations through unknown mechanisms.

Serum albumin is the most abundant protein in blood. This protein is unique in that it does not have single specific function but a number of physiological roles. They include regulation of osmotic pressure, transportation of hydrophobic substances such as fatty acids, and an enzyme-like activity [l-3]. It is believed from biochemical and physico-chemical studies that serum albumin has a limited number of binding sites for fatty acids and that the binding of fatty acids induces conformational changes of the protein [4-71. In spite of intensive studies, however, the relationship between a function and the structure has never been thoroughly understood. We have also demonstrated one other biological function of bovine serum albumin which is the specific aggregation of washed platelets [8,9]. Here, we show that there might be two domains in serum albumin; one is responsible for binding with platelets (binding site) and the other is for actual aggregation (aggregating site). The former is not affected by the fatty acids but fatty acids are necessary for the latter to function.

Modified asparaginase, in which 4 tryptophan residues were modified with 2-hydroxy-5-nitrobenzyl bromide, had little enzymic activity and retained immunoreactivity [(1976) FEBS Lett. 65, 11-15]. Addition of IgG or its Fab towards asparaginase to the modified asparaginase gave rise to marked enhancement of the enzymic activity. Native asparaginase (4 subunits) lost the enzymic activity due to dissociation into subunits by dilution of the enzyme solution. However, in the presence of Fab, asparaginase did not lose enzymic activity on dilution, probably due to no dissociation into subunits occurring. Asparaginase 2-Hydroxy-5-nitrobenzyl bromide Anit-asparaginase IgG

Chymotrypsin was modified in the zymogen form with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2), followed by activation with trypsin. The modified enzyme was soluble in benzene and retained its enzymic activity. Acid-amide bond formation by the modified enzyme proceeded efficiently in benzene: N-benzoyltyrosine butylamide was made from N-benzoyl-L-tyrosine ethyl ester and n-butyl-amine, and benzoyltyrosine(oligo)phenylalanine ethyl esters were formed from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine ethyl ester.

1. Introduction During the course of investigations of the mechanism of energy-transducing system in living cells, various ATPase inhibitors have been found in the ATPase molecule; chloroplast ATPase inhibitor (CFi-I) [l], mitochondrial ATPase inhibitor (Fi-I) [2], bacterial ATPase inhibitor (BFi-I) [3] and one of the troponin components in muscle (TN-I) [4]. They were found as one component of the subunits in each ATPase molecule and play an important role in regulating the ATPase activity. It has been shown in our laboratory that a troponin component, TN-I, inhibits non-competitively mitochondrial, chloroplast or bacterial ATPase, and that the Fi -inhibitor inhibits strongly actomyosin or chloroplast ATPase [5-91. These experiments deal with the isolation of a protein from a commercial aprotinin preparation, which dramatically inhibits chloroplast and mito- chondrial ATPase. 2. Materials and methods An aprotinin preparation, Trasylol (lot no. 43 13p, 500 000 KIE/70-100 mg) [lO,ll], was supplied from Bayer AC. The chloroplast coupling factor 1 (CF,) was prepared from spinach by the method in [ 121 and was activated by heat at 60°C for 4 min. The mitochondrial coupling factor l(F i), AS-particles, was

It was reported that on denaturation ovalbumin loses to a considerable extent the ability to react with antibodies against native ovalbumin [ 1,2]. Although many investigators tried to isolate peptides with antigenicity from ovalbumin by enzymic and chemical degradations, there was not any evidence of production of fragments with immunoreactivity [3,4]. These facts imply that the conformation of ovalbumin is responsible to its antigenicity. It is known that native ovalbumin is not digested with trypsin or chymotrypsin at all, owing to the rigid conformation of ovalbumin in native state [4]. In [5], the significance of tyrosine residues in ovalbumin was pointed out as a specific amino acid responsible for antigenic determinants by chemical modification study. We demonstrate here a novel approach for rendering ovalbumin susceptible to hydrolysis with trypsin and were able to obtain two peptides with immunoreactivity. Peptides B and A thus obtained were single polypeptides stretching from N-terminal AcGlyl to Lys 189 with Mr 23 000 and from L.eu 135 to C-terminal Pro,,, containing polysaccharide with Mr 34 000. Both peptides had an ability to bind with anti-ovalbumin serum.

and myofibrils [4] have been extensively studied to clarify the mech- anism of energy transducing systems. In our laboratory, we demonstrated that troponin component TN-I, ‘which is an inhibitor of actomyosin ATPase, strongly inhibits the mitochondrial and chloroplast ATPase activities [S-7]. Mitochondrial ATPase inhibitor (Fr- inhibitor) also inhibited the chloroplast and acto- myosin ATPase activities [8,9] as well as the mito- chondrial ATPase activity. These results showed the similarity of the action of ATPase inhibitors on ATPases obtained from different sources with a system of heterogeneous combination. The actomyosin ATPase activity was reported [lo] to be inhibited by basic proteins such as salmine, lysozyme and cytochrome c. It is demon- strated here that the chloroplast ATPase activity is inhibited by basic proteins and peptides and that the inhibitory action is due to ammo groups in the molecule of inhibitor proteins.