Topography of protein kinase C substrates analyzed by membrane splitting

We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [32 P]Pi into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [32P]Pi-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.     

Glucokinase in bird liver: a membrane bound enzyme

There have been numerous reports that liver of birds contain only isoenzymes of the low K_M hexokinases, but lack the high K_M glucokinase. We describe here the presence of glucokinase in livers of chicken and Japanese quail. The enzyme is membrane bound and is solubilized by vigorous mechanical disruption of the tissue. With gentle homogenization the glucokinase is recovered upon centrifugation in the 1000g pellet, from which it may be liberated by prolonged sonication. It appears to be localized in the cell plasma membrane. The activities of hexokinase and glucokinase appear to be about equal in liver parenchyma of fed chicken, but in that of Japanese quail the activity of glucokinase exceeds greatly that of hexokinase.     

Evaluation by steady-state enzyme kinetics of the role of tightly bound nucleotides during photophosphorylation

The ATP synthetase of chloroplast membranes binds ADP and ATP with high affinity, and the binding becomes quasi-irreversible under certain conditions. One explanation of the function of these nucleotides is that they are transiently tightly bound during ATP synthesis as part of the catalytic process, and that the release of tightly bound ATP from one catalytic site is promoted when ADP and P(i) bind to a second catalytic site on the enzyme. Alternatively, it is possible that the tightly bound nucleotides are not catalytic, but instead have some regulatory function. We developed steady-state rate equations for both these models for photophosphorylation and tested them with experiments where two alternative substrates, ADP and GDP, were phosphorylated simultaneously. It was impossible to fit the results to the equations that assumed a catalytic role for tightly bound nucleotides, whether we assumed that both ADP and GDP, or only ADP, are phosphorylated by a mechanism involving substrate-induced release of product from another catalytic site. On the other hand, the equations derived from the regulatory-site model that we tested were able to fit all the results relatively well and in an internally consistent manner. We therefore conclude that the tightly bound nucleotides most likely do not derive from catalytic intermediates of ATP synthesis, but that substrate (and possibly also product) probably bind both to catalytic sites and to noncatalytic sites. The latter may modulate the transition of the ATP-synthesizing enzyme complex between its active and inactive states.     

Bacterial membrane potential analyzed by spectrofluorocytometry

Cells ofMycobacterium smegmatis 607 in their late exponential growth were stained with rhodamine 123. The stained bacterial populations were analyzed by fluorocytometry and spectrofluorometry. Responses of the bacterial membrane potential to agents such as valinomycin, graimicidin, nigericin, and sodium azide were analyzed with the two techniques mentioned above. The results have shown that the flow cytometric technique allowed detection of changes in bacterial membrane potential and allowed population response analysis to ionophore activity. The applications of this technique are promising for the rapid detection of membrane potential mutants and for the estimation of the viability of a bacterial population.     

Molecular cloning of a membrane bound tyrosine-specific protein kinase from rat spleen

Tyrosine-specific protein phosphorylation is believed to play an important (though poorly understood) role in various cellular functions in many normal and malignant cells. In order to understand the function of tyrosine-specific protein kinases in normal cells, it is necessary, as an initial step, to identify genes (and proteins) for these enzymes. For this purpose cDNA libraries were constructed in plasmid vector pGEM-3Z and lambda gt11 using mRNA from rat spleen. From these cDNA libraries, cDNA clones coding for a src-related tyrosine-specific protein kinase were isolated. The largest clone (L115) was 1.94 kb in size. Various restriction fragments of this clone were subcloned in plasmid vector for sequencing. The complete nucleotide sequence of the largest clone showed an open reading frame coding for a protein of 503 amino acids. The presence of a glycine at position 2 and an arginine at position 7 indicated that this protein is likely to be acylated at glycine 2 and therefore associated with plasma membrane. This gene showed high homology to human and mouse hck and hence it is perhaps the rat homologue of hck. Moderate level of expression of this gene was observed only in the adult rat spleen and not in other tissues. These results suggest that this kinase gene is expressed in a tissue specific manner.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

An optical study of the exchange kinetics of membrane bound molecules

The kinetics of molecular exchange between lipid bilayers are studied using a special fluoresence technique. Pyrene and pyrene decanoic acid are chosen as typical examples of an apolar and amphiphilic molecule. Their property of forming dimers in the excited state (excimer) is exploited. The time dependencies of monomer and excimer intensities after rapid mixing of vesicles doped with fluorescent probe with undoped ones are studied by stopped-flow technique. The transient curves reveal the information on the exchange kinetics. A theoretical analysis shows that the molecular exchange follows a first order kinetics. Surprisingly short half life-times t_ex for this exchange process are obtained (for dipalmitoyl phosphatidylcholine t_ex = 3.3 s for T = 23 °C, t_ex = 0.5 s for T = 68 °C). Multilamellar systems (onion like structure) show much slower exchange rates. The exchange rates are nearly equal for polar and unpolar molecules. Addition of cholesterol has a strong reducing effect on this rate. Charging of dipalmitoyl phosphatidylcholine vesicle surfaces by the addition of (a) EuCl₃ to the aqueous phase and (b) dipalmitoyl phosphatidic acid to the lipid phase reduces the exchange rate by about an order of magnitude above the phase transition.In a separate experiment it is shown that the lipid exchange or fusion for two different lipids is a much slower process compared to the label exchange. In fact vesicles kept below the phase transition temperature T_tr for both lipids, do not fuse even after 70 h. Noticeable fusion occurs after 10 h when the mixture stays above T_tr. Experiment shows that the fusion of pure lipid vesicles is not very much affected by the presence of a charged lipid.Change in concentration of the monovalent ions in the aqueous solution by two orders of magnitude does not have an appreciable effect on the exchange rate of phospholipids.     

Thermostability of irreversible unfolding alpha-amylases analyzed by unfolding kinetics

For most multidomain proteins the thermal unfolding transitions are accompanied by an irreversible step, often related to aggregation at elevated temperatures. As a consequence the analysis of thermostabilities in terms of equilibrium thermodynamics is not applicable, at least not if the irreversible process is fast with respect the structural unfolding transition. In a comparative study we investigated aggregation effects and unfolding kinetics for five homologous alpha-amylases, all from mesophilic sources but with rather different thermostabilities. The results indicate that for all enzymes the irreversible process is fast and the precedent unfolding transition is the rate-limiting step. In this case the kinetic barrier toward unfolding, as measured by unfolding rates as function of temperature, is the key feature in thermostability. The investigated enzymes exhibit activation energies (E(a)) between 208 and 364 kJmol(-1) and pronounced differences in the corresponding unfolding rates. The most thermostable alpha-amylase from Bacillus licheniformis (apparent transition temperature, T(1/2) approximately 100 degrees C) shows an unfolding rate which is four orders of magnitude smaller as compared with the alpha-amylase from pig pancreas (T(1/2) approximately 65 degrees C). Even with respect to two other alpha-amylases from Bacillus species (T(1/2) approximately 86 degrees C) the difference in unfolding rates is still two orders of magnitude.     

Stopped flow kinetics of carbamylcholine binding to membrane bound acetylcholine receptor

Conformational changes upon binding of carbamylcholine to acetylcholine receptor-enriched membrane fragments have been observed by stopped-flow methods using the fluorescent probe ethidium bromide. A model consistent with both equilibrium and kinetic experiments is proposed in which the receptor binds two molecules of carbamylcholine with high affinity in a non-cooperative manner followed by binding of a third and possibly a fourth molecule with increasingly lower affinity. The receptor ligand precomplexes isomerize to different non-interconvertible complexes depending on the number of ligands bound. This kinetic model fits the data for carbamylcholine interactions with receptor prepared initially either in a low or high affinity form for ligands.     

Chlorpromazine and other psychoactive drug induced alterations of a membrane bound enzyme in rat brain

Psychoactive drugs like chlorpromazine (CPZ), imipramine, lithium and amphetamine in one way or another affect behaviour. The drug responses are presumably mediated by inducing a change in the activity of membrane bound enzymes. CPZ is very potent in inhibiting the alkaline phosphatase activity in rat brain. The combined effect of CPZ with other drugs shows that CPZ and imipramine together inhibit the enzyme activity significantly greater than the individual inhibition either by CPZ or by imipramine alone. Effective inhibition of the alkaline phosphatase activity with a single drug or combined drugs may lead to a change in neuronal permeability through glucocorticoids thereby affecting mood.     

Esolation and partial characterization of a membrane bound protein kinase from mitochondria of Saccharomyces cerevisiae.

$\text{Saccharomyces cerevisiae}$ mitochondria, isolated by enzymatic lysis of the cell wall and purified by gradient centrifugation are able to phosphorylate serine residues of exogenous phosphoproteins in the presence of added [γ³²P] ATP. Most of the protein kinase activity is bound to the mitochondrial membranes from which it can be partially solubilized by 0.7 M NaCl. The solubilized protein kinase, whose M.W. is approximately 30,000, has been partially purified by Phosphocellulose chromatography: it displays its activity toward “acidic” phosphoproteins (α_s2-casein>α_s1-casein = phosvitin > β-casein) while it does not phosphorylate histones even in the presence of cAMP. The enzyme requires Mg²⁺, which cannot be replaced by Mn²⁺, and is strongly inhibited by inorganic Phosphate.     

Automatic computation of enzyme kinetics by HPLC

Enzyme activity can be easily measured by HPLC using tracesof the product itself as an internal standard. Our procedureinvolved the development of an equation using the experimentaldata obtained in the kinetic assay. The entire procedure canthus be automated and a computer program is presented here forfacilitating the assay and saving time. The determination ofthe activity of NAD kinase is reported as an example. Received on March 15, 1990; accepted on May 17, 1990     

Co-identity of brain angiotensin converting enzyme with a membrane bound dipeptidyl carboxypeptidase inactivating Met - enkephalin.

The distribution in rat brain of angiotensin converting enzyme (EC3.4.15.1) using hippuryl-His-Leu as substrate was identical to a dipeptidyl carboxypeptidase present in membranes assayed with Met-enkephalin as substrate. Highest activity occurred in pituitary, followed by cerebellum, corpus striatum, midbrain, pons-medulla, hypothalamus, cerebral cortex and spinal cord. The ratio of products His-Leu/Tyr-Gly-Gly was identical for all regions but differed from His-Leu/Tyr. Angiotensin converting enzyme purified by immunoaffinity chromatography gave a K_m for hippuryl-His-Leu of 0.5mM and for Met-enkephalin of 0.1 mM. In the presence of the specific inhibitor of angiotensin converting enzyme, SQ 14,225, the Ki value was 10^?7M. Present data point to the co-identity of brain angiotensin converting enzyme with the dipeptidyl carboxypeptidase inactivating enkephalin.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Identification of protease IV of E.coli: An outer membrane bound enzyme

The proteolytic activity of $\text{E.coli}$ measured using ¹²⁵I-labelled αS1 casein as substrate, is mainly localised in the outer membrane and is due to an intrinsic outer membrane protein which can be solubilized by deoxycholate. This enzyme exhibits maximum activity at pH 7,5 in Tris-HCl buffer, is resistant to thermal denaturation with a half-life of 28 min. at 90°C in deoxycholate-NaCl buffer and is inhibited by ethylene-diamine tetraacetate, high concentrations of p-aminobenzamidine, tosyl-L-lysine chloromethyl ketone, tosyl-L-phenylalaninechloromethyl ketone and by two inhibitors of the processing of the secreted protein precursors, procaine and phenehylalcohol. Whole cells do not exhibit proteolytic activity, nevertheless, some is unmasked when the outer membrane is permeabilized by Tris or ethylenediamine tetraacetate or when vesicles are sonicated. This suggests that the protease is on the inner side of the outer membrane. Because the protease is different from the soluble proteases described in $\text{E.coli}$, and especially from proteases I,II and III, it has been called protease IV.