Structural and Functional Analysis of Chloroplast Membrane Using Trypsin and Chymotrypsin as Structural Modifiers

(1) Similar results were obtained after controlled digestion of spinach chloroplasts with trypsin and chymotrypsin, but the specificity of digestion of chymotrypsin differed from that of trypsin. Trypsin weakly uncoupled photosynthetic electron transport but chymotrypsin did not. (2) Both changes of DCIP and Fecy reduction activity and the recovery of CCCP inhibition by electron donors of PSⅡ during proteolytic enzyme digestion showed that trypsin not only affected oxidizing side and reducing side of PSⅡ, but also partially inactivated the reaction center of PSⅡ. (3) The effects of CCCP on photosynthetic electron transport in chloroplasts digested with trypsin and chymotrypsin indicated the probable presence of "channel" in PSⅡ. These results support the interpretation that there is a fine structure in PSⅡ membrane. Modification of the protein components of PSⅡ in the membrane might alter their function.     

Thylakoid membrane protein topography. Location of the termini of the chloroplast cytochrome b6 on the stromal side of the membrane.

The orientation of cytochrome b6 in the thylakoid membrane and the question of whether the number of membrane spanning helices is an even or odd number was tested through the relative trypsin susceptibility of epitopes (Asp-5 to Gln-14) and (Ile-205 to Leu-214) at the NH2 and COOH termini, respectively, of the 214-residue cytochrome b6 polypeptide. A structure of the cytochrome with an even number of helices and the NH2 and COOH termini on the stromal side of the membrane was inferred from the following: 1) cleavage of cytochrome b6 by trypsin added to thylakoids occurs by removal of both of the exposed NH2- and COOH-terminal epitopes. The epitopes at the termini were more sensitive to trypsin after prior treatment of thylakoids with carboxypeptidase A, indicating that these epitopes are shielded on the stromal side of the membrane by the COOH termini of other proteins. 2) Both epitopes were more trypsin-sensitive in thylakoid membranes than was cytochrome f that is only sensitive to trypsin acting on the lumen side of the membrane. 3) The NH2- and COOH-terminal epitopes of cytochrome b6 were also more sensitive to trypsin added to thylakoid membranes than were the oxygen-evolving complex 16- and 33-kDa proteins that are completely located on the lumen side. 4) The order of trypsin susceptibility was reversed in inside-out membranes, where the cytochrome NH2- and COOH-terminal epitopes were less sensitive than the 16- and 33-kDa proteins. The decreased relative sensitivity of the cytochrome b6 epitopes occurs in spite of a greater absolute sensitivity of these epitopes to trypsin in inside-out membranes. 5) The greater absolute sensitivity can be explained by a 4-helix model that includes trypsin-sensitive sites on the lumen side.     

Effect of Bovine Serum Albumin Incubation on Lettuce Chloroplasts Digested by Trypsin

1. Bovine serum albumin stimulates the DCIP photoreduction activity of lettuce chloroplasts which has been treated with trypsin. When these chloroplast preparations were washed by tricine buffer such "reversible action" can still be obtained. It is possible that bovine serum albumin may be incorporated into trypsin destroyed site of the membrane. 2. Trypsin-induced CCCP inhibitory effect on DCIP photoreduction activity is reversed by bovine serum albumin. 3. Bovine serum albumin partially reverses the trypsin-induced unstacking of lettuce chloroplast membranes. 4. After trypsin digestion, there are absorbance decreases around 500–640 nm. Bovine serum albumin has no effect on these absorbance decreases. It is concluded that the membrane-bound proteins responsible for different functions of chloroplast are heterogeneous. The results also show that there are gate and channel near the position of PSⅡ on chloroplast membrane.     

Enzyme immobilization on fibrin.

The following conclusions can be drawn concerning the utilization of fibrin to immobilized enzyme systems. Fibrin can be used both as a powder or membrane, to covalently immobilize trypsin with retention of activity. Carbon-14 labeled trypsin can be used to estimate the amount of immobilized enzyme on a proteinaceous support. Significant amounts of noncovalently coupled (adsorbed) enzyme are present on the surface of the support. Esterase activity of the immobilized labeled trypsin was inversely proportional to the amount of attached enzyme. Optimum TAME hydrolysis occurred at pH 8-8.4. The storage stability of trypsin was enhanced. Inhibition of trypsin esterase activity occurred at substrate concentrations greater than 30mM.     

Sidedness of ceramide-phosphoethanolamine synthesis on rat liver plasma membrane

Phosphatidylethanolamine:ceramide-ethanolaminephosphotransferase catalyzes the synthesis of ceramide-ethanolamine, a sphingomyelin analogue. Its transverse localization in rat liver plasma membrane was studied by treating intact and deoxycholate- or Triton X-100-disrupted membrane vesicles with trypsin or bacterial protease. The latency of ATPase was preserved during protease treatment; its value was 80% in the membrane vesicles obtained by sucrose gradient procedure alone and 91.2% in the vesicles isolated after sucrose gradient plus two-phase partitioning. This suggested that membrane integrity was not altered and that 90% of the vesicles were right-side out. When the sucrose gradient was followed by the two-phase procedure, 62% of phosphatidylethanolamine:ceramide-ethanolamine-phosphotransferase was accessible to the protease action, but only 45% in vesicles obtained by sucrose gradient alone. Our results suggest that at least a sizable portion of the active center of the enzyme responsible of biosynthesis of ceramide-phosphoethanolamine is located on the external side of liver plasma membrane and that the other is embedded in the membrane interior and is not accessible to trypsin, even in the presence of detergent.     

Isolation of Hemagglutinin and Neuraminidase Subunits of Hemagglutinating Virus of Japan

When purified hemagglutinating virus of Japan (HVJ) was treated with trypsin, two major surface antigens were released from the virus. The "hemagglutinin" subunits obtained by this method were reactive with homologous hemagglutination-inhibition antibody and could be detected by an antibody-blocking test. They adsorbed to but did not agglutinate red cells and thus appeared to be "monovalent." The neuraminidase subunits were obtained in fully active form and did not adsorb to red cells. This finding suggests that these two activities of HVJ are associated with different subunits of the virus particle. The hemagglutinin and neuraminidase subunits could be partially separated by zonal rate centrifugation or gel filtration on Sephadex G-200. The molecular weights estimated for these subunits were approximately 124,000 and 114,000, respectively. After treatment with trypsin, virus-associated hemagglutinin and neuraminidase activities were both reduced significantly. The electron micrographs of such trypsinized virus particles showed complete or partial loss of surface projections. These results suggested that the subunits obtained by this method seemed to be those projections liberated from the virus by the action of trypsin.     

Oriented adsorption of purple membrane to cationic surfaces

We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine- treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze- fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.     

Integration of the Serratia marcescens haemolysin into human erythrocyte membranes

The haemolytic activity of Serratia marcescens is determined by two proteins, ShlA and ShlB. ShlA integrates into the erythrocyte membrane and causes osmotic lysis through channel formation. The conformation of ShlA and its interaction with erythrocyte membranes were studied by determining the cleavage of ShlA by added trypsin. Our results suggest that the conformation of inactive ShlA (from an ShlB- strain) differs from the active ShlA, and that in a hydrophobic environment (detergent or membrane) active ShlA assumes a conformation distinct from that in buffer. Only active haemolysin adsorbed to erythrocytes. ShlA was firmly integrated into the erythrocyte membrane since it was only released under conditions which also dissolved the integral erythrocyte membrane proteins. Moreover, ShlA integrated into 'ghosts' remained there and was not haemolytic when incubated with erythrocytes. From the trypsin cleavage pattern obtained with haemolysin and C-terminally truncated, but still active, haemolysin derivatives integrated into erythrocytes, and sealed and unsealed erythrocyte 'ghosts', we conclude that ShlA is preferentially cleaved by trypsin at a few sites but only from the inside of the erythrocyte. Haemolysin in the erythrocyte membrane forms a water-filled channel and is resistant to trypsin and other proteases.     

Trypsin-enabled construction of anti-fouling and self-cleaning polyethersulfone membrane

Constructing anti-fouling and self-cleaning membrane surfaces based on covalent attachment of trypsin on poly(methacrylic acid)-graft-polyethersulfone (PMAA-g-PES) membrane was reported. The carboxylic acid groups enriched on asymmetric PMAA-g-PES membrane surface were activated with 1-ethyl-(3-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) and employed as chemical anchors for the conjugation with amino groups of trypsin. Activity assays showed that such chemically immobilized trypsin was much more active and stable than that of the physically adsorbed counterpart. Trypsin covalently attached on membrane surface could substantially resist protein fouling in dynamic flow process. The considerable enhancement of protein solution permeation flux was observed as a consequence of rapid enzymatic degradation of protein deposited onto membrane surface. The permeation flux of the membrane could be recovered upon simple hydraulic flush after protein filtration, suggesting superior self-cleaning property. After multi-cycle BSA filtration over 15-day period, the active self-cleaning membrane maintained more than 95.0% of its initial flux.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: translocation of regions outside the channel-forming domain

Summary C-terminal fragments of colicin E1, ranging in mol wt from 14.5 to 20kD, form channels with voltage dependence and ion selectivity qualitatively similar to those of whole E1, placing an upper limit on the channel-forming domain. Under certain conditions, however, the gating kinetics and ion selectivity of channels formed by these different E1 peptides can be distinguished. The differences in channel behavior appear to be correlated with peptide length. Enzymatic digestion with trypsin of membrane-bound E1 peptides converts channel behavior of longer peptides to that characteristic of channels formed by shorter fragments. Apparently trypsin removes segments of protein N-terminal to the channel-forming region, since gating behavior of the shortest fragment is little affected by the enzyme. The success of this conversion depends on the side of the membrane to which trypsin is added and on the state, open or closed, of the channel. Trypsin modifies only closed channels from thecis side (the side to which protein has been added) and only open channels from thetrans side. These results suggest that regions outside the channel-forming domain affect ion selectivity and gating, and they also provide evidence that large protein segments outside the channel-forming domain are translocated across the membrane with channel gating.     

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Topological investigations. Study of the trypsin sensitivity of the N-acetylglucosaminyl and mannosyl-transferase activities located in the outer mitochondrial membrane

The trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyl-transferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.     

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.     

Enzymic solubilization of the human intestinal brush border membrane enzymes.

The releases of proteins, maltase, lactase, sucrase, trehalase, alkaline phosphatase, gamma-glutamyltransferase and leucylnaphthylamide-hydrolyzing activity from human intestinal brush bborder membrane vesicles by various enzymes (especially pancreatic proteases) have been studied. The brush border membrane enzymes are not solubilized by digestion with trypsin and chymotrypsin but are largely released after treatment with papain or elastase. Most of the enzymes are fully active after the proteolytic treatment. All proteins released by papain and elastase have been identified by electrophoresis to already known intestinal hydrolases. Electron microscopy of brush border membrane vesicles demonstrates "knob-like" structures (particles) attached to the external side of the membrane. During papain treatment, enzyme removal runs parallel with the disappearance of the particles. During elastase treatment it is not possible to correlate the release of the enzymic activities with the removal of the particles. The results indicate that most of the intestinal hydrolases are surface components attached to the external side of the membrane. They are in accord with the concept that the brush border membrane enzymes are organized within the membrane in a mosaic-like pattern.     

The role of inner membrane in the realization of cAMP-dependent activation of mitochondrial enzymes

It was shown that the increase in the activities of transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase after incubation of mitochondria with cAMP is due to the stimulating effect of cAMP on mitochondria, but not to the increased stability of mitochondria to the incubation procedure. Treatment of mitochondria with trypsin prevents the action of cAMP on the both enzymes. The integrity of the inner mitochondrial membrane is necessary for the manifestation of cAMP effect. Pretreatment of mitochondria with the local anesthetic, lidocaine, prevents the activation of NAD(P)(+)-transhydrogenase and NAD(+)-dependent isocitrate dehydrogenase during subsequent incubation of mitochondria with cAMP. It is concluded that the role of the inner mitochondrial membrane consists in the reception of the cAMP signal for the internal compartment of mitochondria, i.e. for mitoplasts. Peripheral protein(s) on the external side of the inner mitochondrial membrane seems to play a role in cAMP reception.     

"Nonclassical" secretion of annexin A2 to the lumenal side of the enterocyte brush border membrane

Annexin A2 is a member of the annexin family of Ca(2+)-dependent lipid binding proteins and believed to be engaged in membrane transport processes in a number of cell types. In small intestinal enterocytes, we localized annexin A2 to the brush border region, where it was found mainly on the lumenal side of the microvilli, showing an apical secretion by a "nonclassical" mechanism. In addition, annexin A2 was associated with surface-connected, deep apical tubules in the apical terminal web region and with an underlying pleiomorphic, tubulo-vesicular compartment (subapical compartment/multivesicular bodies). By subcellular fractionation, the 36 kDa full-length form of annexin A2 was approximately equally distributed between the Mg(2+)-precipitated fraction (containing intracellular and basolateral membranes) and the microvillar membrane fraction. In addition, a 33 kDa molecular form of annexin A2 was seen in the latter fraction that could be generated from the full-length annexin A2 by digestion with trypsin. Taken together, the results suggest that annexin A2 acts in exocytic apical membrane trafficking and is proteolytically cleaved in situ by pancreatic proteinases once it has become externalized to the lumenal side of the brush border membrane. On the basis of its well-known membrane fusogenic properties, we propose a model for the nonclassical membrane translocation of annexin A2.     

Adsorption of commercially prepared trypsin using a membrane support material

Summary The ability of a recently developed affinity membrane to adsorb commercially prepared trypsin was investigated. Several buffered solutions of trypsin which varied in their initial concentrations from 62.5 mg/l to 1,000 mg/l, were passed through a stacked bed of seven membranes; dry wt 350 mgs. The adsorbed protein was eluted using acetic acid; 2.2 mgs to 5.3 mgs of trypsin was desorbed. The adsorption capacity tended to a maximum of 16 mg /g dry wt when the initial feed concentration of trypsin was 1 g/l. There was no loss in enzyme activity after desorption; 11,500 IU ± 500 IU.     

Effects of a supernatant protein activator on microsomal squalene-2,3-oxide-lanosterol cyclase.

A soluble protein termed "supernatant protein factor" (SPF) that stimulates microsomal squalene epoxidase has been isolated in this laboratory (Ferguson, J.B., and Bloch, K. (1977) J. Biol. Chem. 252, 5381-5385). We now show that the purified protein also stimulates microsomal squalene-2,3-oxide leads to lanosterol cyclase but has no effect on the subsequent conversion of lanosterol to cholesterol. Phospholipid, specifically phosphatidylglycerol or phosphatidylethanolamine, is required for maximal stimulation of the cyclase by purified SPF. The response of microsomal squalene epoxide-lanosterol cyclase to SPF was abolished by pretreatment of the membranes with phospholipase A2 or by low concentrations of deoxycholate, indicating that an intact membrane system is required. Digestion of intact microsomes with trypsin had no effect on the SPF-stimulated cyclase activity. However, in the presence of 0.4% deoxycholate, trypsin completely inhibited microsomal squalene epoxide-lanosterol cyclase. We conclude that the cyclase is located on the luminal side of the microsomal membrane. SPF also significantly enhances the formation of lanosterol from squalene-2,3-oxide already bound to microsomes. This finding is constant with the proposal that SPF influences intramembrane events.     

The beta subunit controls the gating and dihydropyridine sensitivity of the skeletal muscle Ca2+ channel.

The skeletal muscle (SKM) L-type Ca2+ channel is composed of a central subunit designated alpha 1, which contains the pore and the dihydropyridine (DHP) binding domains and three associated subunits, alpha 2/delta, beta, and gamma, which influence the activity of the SKM alpha 1. Coexpression of SKM alpha 1 and SKM beta in stably transfected mouse L cells results in a dramatic increase in DHP binding accompanied by fast gated Ba2+ currents. We report here that this "SKM alpha 1 beta-related phenotype" can be converted upon intracellular trypsin treatment into a slowly inactivating, DHP sensitive "SKM alpha 1 phenotype." These observations indicate that current amplitude, fast inactivation, and DHP sensitivity are modulated by an interaction of SKM alpha 1 and SKM beta on the internal side of the membrane.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.