Isolation and characterization of Chromatium vinosum membranes

A fractionation of Chromatium vinosum into an outer layer (cell wall) and three intracellular membrane fractions by isopycnic sucrose density centrifugation of a total membrane fraction obtained by lysis of lysozyme-EDTA spheroplasts is decribed. The three intracellular fractions (I, II, and III) have apparent densities of 1.11, 1.14, and 1.16, respectively, and contain the bulk of the photosynthetic pigments. Fraction II is enriched in bacteriochlorophyll and contains about 49% of the total membrane protein and 60% of the membrane bacteriochlorophyll. The outer membrane fraction (IV, cell wall) has a density of 1.23 and contains 5% of the membrane protein and 0.8% of the bacteriochlorophyll. Fraction I is enriched in lipids and phosphorus and has only a trace of diaminopimelate (DAP). Fractions II and III both contain a significant content of DAP. Fraction IV has no DAP, but has a fatty acid composition similar to that of the envelope fraction. Electrophoresis of the fractions on sodium dodecylsulfate-containing gels yielded from 8–13 bands of protein. Fractions I, II, and III contained the same series of unique proteins, while fraction IV contained another group of unique proteins. In fraction IV the bulk of the proteins traveled in one band with a molecular weight of 41,500. Examination of the fractions and whole spheroplasts in the electron microscope showed that fractions I, II and III were composed of large membrane structures in the form of membrane reticulum with bud-like appendages, and elongated flattened tubes. Fraction IV was composed of large ovoid structures which were seen to lie on the outer surface of the whole spheroplasts. These results suggest that the normal in vivo state of the intracellular membranes is that of an interconnected series of tubules and vesicles extending throughout the cell cytoplasm.     

Hemolytic potency and phospholipase activity of some bee and wasp venoms

1. The action of crude venoms of four aculeate species: Apis mellifera, Vespa crabro, Vespula germanica and Vespula vulgaris on human erythrocytes was investigated in order to determine the lytic and phospholipase activity of different aculeate venoms and their ability to induce red blood cell hemolysis. 2. Bee venom was the only extract to completely lyse red blood cells at the concentration of 2-3 micrograms/ml. 3. Phospholipase activity in all of the examined vespid venoms was similar and the highest value was recorded in V. germanica. 4. Vespid venoms exhibited phospholipase B activity, which is lacking in honeybee venom. 5. In all membrane phospholipids but lecithin, lysophospholipase activity of vespid venoms was 2-6 times lower than the relevant phospholipase activity. 6. The incubation of red blood cells with purified bee venom phospholipase A2 was not accompanied by lysis and, when supplemented with purified melittin, the increase of red blood cell lysis was approximately 30%.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.     

Snake venom cardiotoxins and bee venom melittin activate phospholipase C activity in primary cultures of skeletal muscle

The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.     

Purification and characterization of the anticoagulant principle of Trimeresurus mucrosqualatus venom

By means of CM-Sephadex column chromatography, Trimeresurus mucrosquamatus venom was separated into 20 fractions. Fraction XX had the marked anticoagulant action. This fraction was refractionated three times on Sephadex G-75, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single, symmetrical boundary with a value of 1.61 S was obtained by ultracentrifugation. It was a single peptide chain with a molecular weight of 11 700. The isoelectric point was higher than pH 10.The anticoagulant principle possesses phospholipase A activity and was calcium ion dependent. It did not possess proteolytic, tosyl-L-arginine methyl ester esterase, phosphodiesterase and alkaline phosphomonoesterase activities of the crude venom. The phospholipase A activity was heat-labile at pH 7.4, but was heat-stable at pH 5.6. The anticoagulant activity was more resistant to heat treatment as compared with phospholipase A activity.The anticoagulant action of the purified principle was competitively inhibited by platelet phospholipid, tissue thromboplastin and cephalin, and was neutralized by antiserum. The anticoaugulant principle inhibited platelet aggregation induced by ADP. It did not destroy fibrinogen, Factor X, prothrombin and thrombin; nor did it induce fibrinolysis nor interfere with the interaction between thrombin and fibrinogen. It is concluded that the anticoagulant action of this phospholipase A was due to the inhibition of the activations of Factors X and II through the activation of the procoagulant activity of phospholipids mediated partly by phospholipid-binding activity of this venom enzyme and partly by its enzymatic hydrolysis of phospholipids.     

Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats.

Short term administration of the venoms of the snakes Naja haje, Naja nigricollis, and Cerastes vipera and of the scorpion Leiurus quinquestriatus on the mitotic index of the duodenal mucosal cells of the white rat, Rattus rattus, has been studied. All the venoms increased the number of dividing cells of the duodenal mucosa significantly. Naja haje crude venom was fractionated into three fractions. Fraction I had no effect on the mitotic index whereas fractions II and III increased it significantly. Treatment of rats with Naja haje venom fractions II and III after blocking the histamine or the serotonin receptors did not affect the stimulatory action of the two venom fractions on the mitotic index, which it increased significantly. It was suggested that the venoms of Naja haje, Naja nigricollis, Cerastes vipera, and Leiurus quinquestriatus and Naja haje venom fractions possessed a mitogenic activity. Fraction II of Naja haje venom acted through both the muscarinic and adrenergic receptors while fraction III acted on the adrenergic ones.     

Phospholipase A of sea snake Laticauda semifasciata venom. Isolation and properties of novel forms lacking tryptophan.

The venom gland extracts of the sea snake Laticauda semifasciata contained at least four forms of phospholipase A separable on a CM-cellulose column. They were designated as phospholipases A I-IV in the order of elution from the column. Phospholipases A I, III, and IV were isolated in a homogeneous state. They were similar to one another in amino acid composition and molecular weight (14,000) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phospholipase A I contained one tryptophan residue. whereas III and IV did not. Although all these forms had the same A2-type positional specificity, they were classified into two groups (I, and III and IV) on the basis of enzymic properties. Phospholipase A I had a higher specific activity and showed normal kinetics, whereas III and IV had approximately one-tenth of the specific activity of I and showed biphasic kinetics due to their activation by the reaction products. Phospholipase A I, the major form, seems to be identical with phospholipase A reported previously (Tu, A.T., Passey, R.B., & Toom, P.M. (1970) Arch. Biochem. Biophys. 140, 96-106), whereas the other two, III and IV, are new. Phospholipase A I became more like III and IV in enzymic properties on modification with N-bromosuccinimide.     

Studies on the hemolytic and hydrolytic actions of phospholipases against mammalian erythrocyte membranes.

The hemolytic actions of three kinds of phospholipase C on horse and sheep erythrocytes were studied in relation to their hydrolytic activities on the phospholipid components of these red cells. Clostridium novyi (oedematiens) type A phospholipase C hemolyzed horse red cells by hydrolyzing phosphatidylcholine. However, the enzyme did not lyse sheep cells nor did it hydrolyze any phospholipid under the same conditions, although this enzyme hydrolyzed both sphingomyelin and phosphatidylethanolamine in the phospholipid mixture extracted from sheep red cells. Clostridium perfringens phospholipase C hemolyzed not only horse red cells by hydrolyzing phosphatidylcholine but also sheep red cells by hydrolyzing sphingomyelin. Sphingomyelin on sheep red cell membrane was hydrolyzed 10 times faster by this enzyme than that on horse red cell membrane. Pseudomonas aureofaciens phospholipase C hemolyzed horse red cells by attacking phosphatidylcholine and phosphatidylethanolamine. The enzyme did not attack sheep red cells but it did hydrolyze phosphatidylethanolamine in the extracted phospholipid mixture from sheep cells. The hemolytic activity of phospholipase C depends not only on the enzyme and the asymmetric distribution of phospholipids in the erythrocyte membrane but also on the accessibility of the enzymes to the phospholipids in the surface of the membranes. Hemolysis by phospholipase C belongs to a hot-cold type of lysis.     

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.     

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A₂ are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA₂, but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.     

Human transfer factor: fractionation and biologic activity.

Human transfer factor (TF) was fractionated by exclusion chromatography and the fractions were tested for biologic activity in vivo and in vitro. Specific TF activity in vivo was found to reside in the major UV-absorbing peak (Fraction III). Fraction III eluted at 2.7 X V(O) and transferred tuberculin, candida, or KLH-reactivity to previously negative recipients. Fraction III from nonreactive donors was ineffective. When the fractions were tested in vitro, we found that both the mitogenic activity of whole TF and the suppressive activity to mitogen activation when present in TF was found in Fraction I. Fraction III contained components responsible for augmentation of PHA and PWM responses. In addition, Fraction III contained the component responsible for antigen-dependent augmentation of lymphocyte transformation. Fraction IV was suppressive to antigen-induced lymphocyte transformation. These data suggest that TF preparations contain components which can affect immune reactions in both specific and nonspecific ways.     

Purification and properties of the main coagulant and anticoagulant principles of Vipera russellii snake venom

Vipera russellii venom was separated into thirteen fractions by means of DEAE-Sephadex A-50 column chromatography. Fraction III possessed anticoagulant and phospholipase A activities and Fraction XI possessed procoagulant and caseinolytic activities, both were further purified by gel filtration on Sephacryl S-200 column. Purified procoagulant (Component II) was a two-chain protein with molecular weight of 86 000 consisting of A-chain (Mr 66 000) and B-chain (Mr 20 000). It was a glycoprotein containing 7.8% neutral sugar and 715 amino-acid residues. The procoagulant activity was 10-times that of the crude venom. It was an acidic proteinase with isoelectric point of pH 4.2. Upon heat treatment at 60 degrees C, Component II was stable at pH 5.5 and 7.2 for 3 h, but was destroyed completely after 30 min at pH 8.9. It was devoid of esterase or amidase activity. Purified anticoagulant (Component I) was a single peptide chain with molecular weight of 16 000. It was carbohydrate free and contained 136 amino-acid residues. It was a basic protein with an isoelectric point of larger than pH 10. It was a potent phospholipase A with an enzymatic activity of 510 +/- 30 mumol/min per mg using phosphatidylcholine as substrate, and 1 microgram/ml was sufficient to cause 100% hemolysis by the indirect hemolytic method. Upon heat treatment at 90 degrees C, Component I was heat stable at pH 5.5 for more than 3 h, but was destroyed completely after 2 h at pH 7.2 and 8.9. The anticoagulant activity of Component I could be neutralized by platelet factor 3, tissue thromboplastin and cephalin.     

Composition of a Photosystem I chlorophyll protein complex from Anabaena flos-aquae

The use of Triton X-100 to solubilize membrane fragments from Anabaena flos-aquae in conjunction with DEAE cellulose chromatography allows the separation of three green fractions. Fraction 1 is detergent-solubilized chlorophyll, and Fraction 2 contains one polypeptide in the 15 kdalton area. Fraction 3, which contains most of the chlorophyll and shows P-700 and photosystem I activity, shows by SDS gel electrophoresis varying polypeptide profiles which reflect the presence of four fundamental bands as well as varying amounts of other polypeptides which appear to be aggregates containing the 15 kdalton polypeptide. The four fundamental bands are designated Band I at 120, Band II at 52, Band III at 46, and Band IV at 15 kdaltons. Band I obtained using 0.1% SDS contains chlorophyll and P-700 associated with it. When this band is cut out and rerun, the 120 kdalton band is lost, but significant increases occur in the intensities of Bands II, III, and IV as well as other polypeptides in the 20–30 kdalton range.The use of 1% Triton X-100 coupled with sucrose density gradient centrifugation allows the separation of three green bands at 10, 25 and 40% sucrose. The 10% layer contains a major polypeptide which appears to be Band IV. The 25 and 40% layers show essentially similar polypeptide profiles, resembling Fraction 3 in this regard, except that the 40% layer shows a marked decrease in Band III. Treatment of the material layering at the 40% sucrose level with a higher (4%) concentration of Triton X-100 causes a loss (disaggregation) of the polypeptides occurring in the 60–80 kdalton region and an increase in the lower molecular weight polypeptides. Thus, aggregation of the lower molecular weight polypeptides accounts for the variability seen in the electrophoresis patterns. Possible relations of the principal polypeptides to the known photochemical functions in the original membrane are discussed.     

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy-d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.     

Effects of Naturally Occurring Aquatic Organic Fractions on Am Uptake by Scenedesmus obliquus (Chlorophyceae) and Aeromonas hydrophila (Pseudomonadaceae)

Naturally occurring organics were extracted from water collected from Skinface Pond near Aiken, S.C. Organics were separated into four nominal diameter size fractions (I, >0.0183; II, 0.0183 to 0.0032; III, 0.0032 to 0.0009; IV, <0.0009 mum) by membrane ultrafiltration and introduced into Scenedesmus obliquus and Aeromonas hydrophila cultures to determine their effects on Am availability for uptake. Effects on Am uptake were determined in actively growing S. obliquus cultures after 96 h of growth and in dense cultures of nongrowing cells after 4 h. Uptake by A. hydrophila was determined after 4 and 24 h in actively growing cultures. All organic fractions stimulated S. obliquus growth, with the most pronounced effects due to larger organic fractions, whereas no apparent growth stimulation of A. hydrophila was observed for any organic fraction. For both long-term and short-term studies, cellular Am concentration (picocuries/cell) increased with increasing Am concentration for S. obliquus and A. hydrophila. Fraction IV increased Am uptake by both S. obliquus and A. hydrophila during 4-h incubations. During 96-h incubations fraction I was flocculated and cosedimented, with S. obliquus and A. hydrophila cells causing an apparent increase in Am uptake. Fractions II and III reduced apparent Am uptake by S. obliquus as a result of biological dilution caused by increased algal growth due to the organics. Fraction IV caused a reduction in Am uptake by S. obliquus not attributable to biological dilution. Organics increased Am uptake by A. hydrophila during 4- and 24-h incubations. A. hydrophila also caused flocculation of fraction I during 96-h incubations.     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A(2) are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA(2), but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.     

Purification and properties of mammalian toxins from the venom of the Brazilian scorpion Tityus serrulatus Lutz and Mello

The water-soluble part of the dried venom from the scorpion, Tityus serrulatus Lutz and Mello (range, Southeastern Brazil), showed 16 polypeptide bands on polyacrylamide gel electrophoresis. This material exhibited toxic and hyaluronidase activity but no phospholipase, phosphodiesterase, protease, or fibrinolytic activity. Fractionation on glycinamide-treated Sephadex G-50 afforded three protein fractions, which were non-toxic, equitoxic, and three times more toxic than the water-soluble venom. Subsequent separation of the toxic fractions on carboxymethyl-cellulose with phosphate buffers furnished five toxic components, which were further purified on carboxymethyl-cellulose with a salt gradient in acetate buffer. Toxin γ, the major and most basic toxin, is a 62-residue protein that, unlike other scorpion toxins, contains methionine. Automated Edman degradation showed the amino-terminal sequence to be H-Lys-Glu-Gly-Tyr-Leu-Met-Asp-His-Glu-Gly-Cys-Lys-Leu-Ser-Cys-Phe-Ile-Arg-Pro-Ser-Gly-Tyr-Cys-Gly-Arg-Glu-Cys-Gly-Ile-. Toxin γ is the first example of a fifth structural type of mammalian toxin from scorpion venom. Its amino-terminal sequence shows greater homology with toxins similar to Centruroides suffusus suffusus toxin III and Androctonus australis toxin II than with toxins similar to A. australis toxin I or Bhutus occitanus tunetanus toxin I.     

Effects of lipolytic enzymes on the photochemical activities of spinach chloroplasts.

1. Spinach class II chloroplasts were treated with purified potato lipolytic acyl-hydrolase and venom phospholipase A2, and their lipid degradations and the effects on the photochemical activities were followed. 2. Potato lipolytic enzyme hydrolyzed monogalactosyldiacylglycerol at a faster rate than phospholipids such as phosphatidylglycerol and phosphatidylcholine. The treatment caused a rapid decrease of Photosystem I activity, and a less change of Photosystem II activity. 3. Venom phospholipase A2 which preferentially hydrolyzed phosphatidylglycerol, caused a rapid decrease of Photosystem II activity and only a slight decrease of photosystem I activity. 4. Potato enzyme and phospholipase A2 degraded the membrane lipids of glutaraldehyde-fixed chloroplasts at a rather slightly higher rate than those of non-treated chloroplasts. 5. The results suggested a possible correlation between monogalactosyldiacylglycerol degradation and decay of Photosystem I activity and between phosphatidylglycerol degradation and decay of Photosystem II activity. A possible mechanism is discussed.