Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.     

Amino terminal sequence of heavy and light chains from ratfish immunoglobulin

The ratfish,Callorhinchus callorhinchus, a representative of the Holocephali, has a natural serum hemagglutinin (M _r 960 000), composed of heavy (M _r 71000), light (M _r 22 500), and J (M _r 16 000) chains. To approach the mechanisms that generate diversity at this level of evolution, the amino terminal sequence of the heavy and light chains was determined by automated microsequencing. The chains are unblocked and have modest internal sequence heterogeneity. The heavy chains show sequence similarity with the terminal region of the heavy chain from the horned shark,Heterodontus francisci, and other species. In contrast to the heavy chain, the ratfish light chains display low sequence similarity with their shark kappa counterparts. However, their similarity with the variable region of the chicken lambda light chains is about 75%.     

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzedAbz-QVVAGA-EDDnp andAbz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10⁷M^–1 sec^–1 range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10⁵ M^–1 sec^–1 range due to lowerk _cat values, with theK _m 's being identical to those with papain. OnlyAbz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending on their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based on these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN₂ was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k ₊₀= 1,100,000 M^–1 sec^–1) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN₂. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.     

Taurine Inhibits Protein Kinase C-Catalyzed Phosphorylation of Specific Proteins in a Rat Cortical P2 Fraction

Abstract: We previously reported that taurine inhibits the phosphorylation of specific proteins in a P₂ synaptosomal fraction prepared from the rat cortex. In the present study, the regulation of the phosphorylation of an ～20K M_r protein whose phosphorylation is inhibited by taurine was further investigated. The phosphorylation of the ～20K M_r protein in a hypo-osmotically shocked P₂ fraction from rat cortex was dependent on the free Ca²⁺ in the reaction medium. Depolarization induced by 30 mM K⁺ stimulated the phosphorylation of the ～20K M_r protein in an intact synaptosomal P₂ preparation by 30-fold. This stimulation was inhibited 35% by taurine, whereas guanidinoethanesulfonic acid, a taurine analogue, did not have any effect, thereby indicating the specificity of taurine. Addition of phorbol 12-myristate 13-acetate, a phorbol ester, together with phosphatidylserine, stimulated the phosphorylation of the ～20K M_r protein in the hypo-osmotically shocked P₂ synaptosomal fraction by fivefold, whereas cyclic AMP, cyclic GMP, and calmodulin did not have any effect on the phosphorylation of this particular protein. Phorbol 12-myristate 13-acetate–stimulated phosphorylation of the ～20K M_r protein is blocked 30% by taurine. Taurine also inhibited phorbol 12-myristate 13-acetate-activated phosphorylation of two other proteins that were similar in molecular weight and isoelectric point to the ～20K M_r protein on two-dimensional gels. These results suggest that taurine modulates the phosphorylation of specific proteins regulated by the signal transduction system in the brain. Thus, taurine may modulate neuroactivity by inhibiting the phosphorylation of specific proteins involved in regulatory function.     

Complexity of cellulases from Trichoderma reesei with acidic isoelectric points: A two-dimensional gel electrophoretic study using immunoblotting

Antibody specific to Trichoderma reesei cellulase (65 kDa, isoelectric point, pI, 7.7) shows immuno-cross reactivity with acidic hydrolase complexes containing other cellulases, (pI_app. 3.4–4.5) when tested under conditions of 2D-electrophoresis (1^st dim. PAGIF, 2^nd dim. SDS-PAGE) together with Western blotting. Degradation pattern of ¹⁴C(U)-labeled G₁–G₅ of the 65 kDa cellulase was followed by a 2-directional oligodextrin mapping procedure.Using preparative IEF, homologous antigen portions were detected in cellulases present within acidic hydrolase complexes showing mainly identical molar weight (M_r 65 kDa and 57 kDa) but a range of charge (pI 3.4–4.5). The pattern of acidic cellulases as found after analytical 2D-electrophoresis was reconstituted by preparative IEF (pI_app. 2.7–5.1) followed by SDS-PAGE separation. Homogeneous fractions (upon IEF) gave up to 8 different polypeptides per complex upon SDS-PAGE (M_r 70−20 kDa). Charge heterogeneity of individual acidic hydrolase complexes upon IEF is discussed as one reason for ‘multiplicity’ of acidic cellulases.     

Human immunodeficiency virus protein gp120 interferes with β-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes

Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of³²P into a soluble acidic protein of 80,000M _r , which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to³H-myristic acid indicated that the acidic 80,000M _r protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M _r MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.     

Cation binding to β-casein. a comparison of electrostatic models

The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are k_H,P^o = 3 × 10⁶, k_Ca,P^o = 120, k_Na,P^o = 1, k_H,C^o = 7 × 10⁴ and k_Ca,C^o = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k_H,P^o and k_Ca,C^o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated k^o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.     

Spontaneous Withdrawal from Long-Term Treatment with Morphine Accelerates the Turnover of α2-Adrenoceptors in the Rat Brain: Up-Regulation of Receptors Associated with Increased Receptor Appearance

Abstract: The aim of this study was to quantify and compare the turnover of brain α₂-adrenoceptors during chronic morphine treatment and after spontaneous morphine withdrawal in rats. The oral administration of increasing doses of morphine (10–90 mg/kg) for 20 days did not alter the specific binding of the agonist [³H]clonidine in the cerebral cortex. However, spontaneous opiate withdrawal (24 h) significantly increased the density of cortical α₂-adrenoceptors (B_max for [³H]clonidine was 21% greater). The recovery of [³H]clonidine binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (1.6 mg/kg) was assessed in naive, morphine-dependent, and morphine-withdrawn rats to study the process of α₂-adrenoceptor repopulation and to calculate receptor turnover parameters. The simultaneous analysis of receptor recovery curves revealed that the turnover of brain α₂-adrenoceptors in morphine-withdrawn rats was accelerated [appearance rate constant (r) = 21 fmol/mg of protein/day; disappearance rate constant (k) = 0.25 day^?1] compared with those in morphine-dependent (r = 13 fmol/mg of protein/day; k = 0.14 day^?1) and naive (r = 15 fmol/mg of protein/day; k = 0.16 day^?1) rats. Moreover, this analysis also indicated that the increased density of cortical α₂-adrenoceptors observed during morphine withdrawal was due to a significantly higher receptor appearance (Δr = 37–57%) and not to a decreased receptor disappearance, which in fact showed also an increase (Δk = 56–79%). It is proposed that the increased rate of α₂-adrenoceptor production in the brain of morphine-dependent rats during spontaneous withdrawal is most probably mediated by the overactivity of the adenylyl cyclase/cyclic AMP system induced by opiate addiction.     

In isolated human centrosomes,the associated kinases phosphorylate a specific subset of centrosomal proteins

Summary— Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34^cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230–240; Bailly et al, 1989, EMBO J 8, 3985–3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M_r 230–220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34^cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M_r 100000 and 37000) were phosphorylated only by p34^cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.     

Clonospecific structural heterogeneity in the Thy-1 molecule from mouse T lymphocytes

The Thy-1 molecule immunoprecipitated from detergent-solubilized, ¹²⁵I-labeled cell-surface proteins was shown to be processed in two distinct ways by mouse T lymphocytes: one leading to the expression by thymocytes, concanavalin A-activated spleen blasts, and six of nine T-cell clones of a molecule of 25–28 kd, and another, observed in three other T-cell clones, leading to the expression at their surface of a so far undescribed low M _r (23 kd) form of Thy-1. The results of two-dimensional gel electrophoresis and neuraminidase, endoglycosidase H, and endoglycosidase F treatment revealed that the observed heterogeneity of Thy-1 molecules from peripheral cloned T cells was due to major differences in the maturation and sialylation of their N-linked complex-type oligosaccharide residues. It was also found that a given T-cell clone could express T200, LFA.1, and transferrin receptor molecules with a low or high M _r. Furthermore, and in contrast to previously reported results, this study revealed that the differences in cell-surface glycoprotein profiles could not be correlated with the Lyt-2,3/T4 phenotypes, the specificity for allo-H-2, allo-I-A, allo-I-E, or GAT + I-A^k determinants, nor with the cytolytic or helper/amplifier potential of the various T-cell clones examined. The possible implications of these findings are discussed.     

Effect of Introducing a Lactone Group into Typhasterol and Teasterone to Promote Rice Lamina Inclination

Protein phosphorylation may be required for plant cell response to phytohormones and other extracellular signals. Protein phosphorylation and protein kinase activity in the culm of heading time of rice (Oryza sativa L.) were studied. Before heading, protein kinase activity was increased by Ca²⁺ in the membrane fraction of the panicle and culm. The protein kinases with M_r of 51,900, 49,200, and 45,500 isolated from the membrane fraction of culm increased the protein phosphorylation of M_r and pI of 40,000/7.5 and 40,000/7.6 in the culm extract. The activation of protein kinases, associated with membrane and subsequent protein phosphorylation, thus appears to be involved in the regulation of heading time in rice.     

Involvement of the antiplatelet activity of magnesium sulfate in suppression of protein kinase C and the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger

Magnesium sulfate is widely used to prevent seizures in pregnant women with hypertension. The aim of this study was to examine the inhibitory mechanisms of magnesium sulfate in platelet aggregation in vitro. In this study, magnesium sulfate concentration-dependently (0.6–3.0 mM) inhibited platelet aggregation in human platelets stimulated by agonists. Magnesium sulfate (1.5 and 3.0 mM) also concentration-dependently inhibited phosphoinositide breakdown and intracellular Ca⁺² mobilization in human platelets stimulated by thrombin. Rapid phosphorylation of a platelet protein of M_r 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12-13-dibutyrate (PDBu, 50 nM). This phosphorylation was markedly inhibited by magnesium sulfate (3.0 mM). Magnesium sulfate (1.5 and 3.0 mM) further inhibited PDBu-stimulated platelet aggregation in human platelets. The thrombin-evoked increase in pHi was markedly inhibited in the presence of magnesium sulfate (3.0 mM). In conclusion, these results indicate that the antiplatelet activity of magnesium sulfate may be involved in the following two pathways: (1) Magnesium sulfate may inhibit the activation of protein kinase C, followed by inhibition of phosphoinositide breakdown and intracellular Ca⁺² mobilization, thereby leading to inhibition of the phosphorylation of P47. (2) On the other hand, magnesium sulfate inhibits the Na⁺/H⁺ exchanger, leading to reduced intracellular Ca⁺² mobilization, and ultimately to inhibition of platelet aggregation and the ATP-release reaction.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Human pre-α-inhibitor: isolation from a by-product of industrial scale plasma fractionation and structural analysis of its H3 heavy chain

Pre-α-inhibitor (PαI) is a serine proteinase inhibitor from human plasma. It comprises bikunin (BK) responsible for antiprotease activity, covalently linked to a heavy chain H3. Here we describe its isolation from a side fraction of an industrial preparation of plasma clotting factors. By using a highly specific polyclonal antiserum prepared from rabbit immunized with a H3P polypeptide obtained in a bacterial expression system, we were able to identify the fractions containing PαI. Then, taking advantage of the differential affinity of the members of the inter-α-inhibitor family (IαI) for heparin-Sepharose and blue-Sepharose, we isolated PαI. Its specific antitryptic activity was 580 IU/g, higher than that of IαI: 420 IU/g. Its M_r, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with or without prior reduction, was 130 000. Its peptide chains were identified by N-terminal sequencing. The H3 heavy chain was isolated from PαI by alkaline dissociation and anion-exchange chromatography. Its electrophoretic mobility was compared to that of the H1 and H2 heavy chains of IαI. In reducing conditions, it was quite similar to that of H2 (M_r 85 000) but clearly different from that of H1 (M_r 78 000). Thus, the so-determined apparent M_r of H3 was overestimated since its molecular mass determined by MALDI-TOF was 74 100. This result agrees with the proposed structure for H3. Indeed, by carbohydrate analysis and PNGase F digestion, we demonstrate that the two potential N-glycosylation sites present in the core-protein (theoretical mass: 69 454) are really occupied by two N-glycans, probably of biantennary type.     

Cation binding to beta-casein. A comparison of electrostatic models

The binding of cations of β-casein at pH 6.6 was considered previously. Available for three sodium concentiations, I = 0.04, 0.08, or 0.16 M are: [1] proton releases between I and [2] for each I, as calcium activity is increased, correlated sequences of monomer net charge, proton release, site bound calcium and protein Solvation- Models for ion binding are examined. Critical considerations are the intrinsic binding constants between hydrogen[H], calcium[Ca] and sodium[Na] ions and phosphate[P] and caiboxyIate[C] sites, and the effects of electrostatic interaction between sites as influenced by spatial fixed charge distribution, ionic strength and dielectric constant [D]. Anticipated intrinsic binding constants are k_H,P^o = 3 × 10⁶, k_Ca,P^o = 120, k_Na,P^o = 1, k_H,C^o = 7 × 10⁴ and k_Ca,C^o = 5.6Distributed charge models, either surface or volume, are inadequate since any reasonable monomer size yields fixed charge densities requiring k_H,P^o and k_Ca,C^o which are too low when the maximum in D is 75. Also, with increasing calcium binding, calculated proton release is only 0.4 to 0.5 of that observed.Discrete charge models accept anticipated k^o and yield calculated sequences of calcium binding and proton release which are in good agreement with those observed provided that: (1) using the known amino acid sequence of the phosphate-containing acidic peptide portion of the molecule, pep tide fixed charge is distributed at the lowest I so as to minimize electrostatic free energy; (2) in the region of fixed charge, D is approximately 5; (3) the distances between peptide fixed charges decrease with increasing ionic strength or calcium binding and (4) while protein is in solution, the acidic peptide and the remainder of the molecule are essentially electrostatically independent.     

Phosphorylation of the α-subunit of (Na+ + K+)-ATPase by carbachol in tissue slices and the role of phosphoproteins in stimulus-secretion coupling

This study examined the changes in protein phosphorylation in response to cholinergic (muscarinic) stimulation of salivary secretion in the rat submandibular gland. Carbachol stimulation was associated with phosphorylation in a number of protein bands as detected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and autoradiography. The molecular masses (M_r) of two proteins, in which the amount of phosphorylation more than doubled in response to carbachol, were 22 000 and 96 000. The M_r 96 000 protein precipitated at 120 000 × g while most of the M_r 22 000 protein remained in the supernatant at this speed. The effect of carbachol on the phosphorylation of the M_r 22 000 and 96 000 proteins was blocked by atropine, indicating that the cholinergic receptor involved is muscarinic. The time course of phosphorylation of the M_r 22 000 protein consisted of a rapid incrase in phosphorylation within the first min of carbachol stimulation. This increased phosphorylation persisted for less than 1 min. The increased phosphoryaltion of the M_r 96 000 protein also occurred within the first min but it persisted for at least 10 min. However, removal of the muscarinic agonist, carbachol, resulted in the rapid dephosphorylation of this protein. When the plasma membranes were purified, the M_r 96 000 protein was phosphorylated by ATP in the presence of Na⁺ and Mg²⁺. It was dephosphorylated by K⁺. This proves that the M_r 96 000 dalton protein is the α-subunit of the (Na⁺ + K⁺)-ATPase.     

Architecture and polypeptide composition of HeLa cytoskeletons: Modification of cytoarchitectural polypeptides during mitosis

Substrate-attached asynchronous HeLa cells were extracted with Triton X-100 and analysed by electron microscopy and two-dimensional gel electrophoresis. Such Triton cytoskeletons showed actin filament bundles, microtubules, intermediate filaments, and actin networks in the substrate-associated lamellae, and contained around 90 polypeptides (48 basic, 42 acidic; 52% of total actin, 99% of vimentin, 41% of α-actinin and 30% of β-tubulin).Cytoskeletons produced by further extraction in high and low salt buffers (L-H-L) showed only intermediate filaments, the nucleus and residual actin, and contained a total of 19 polypeptides (13 acidic, 6 basic). Of these, 12 corresponded to abundant acidic proteins in the 47,000 to 70,000 M_r region as determined by staining with Coomassie blue and labelling with a mixture of ¹⁴C-labelled amino acids. Using L-H-L extracted cytoplasts, and employing an actin depolymerising protein from slime moulds, seven abundant acidic IEF³ polypeptides were shown to be present in these intermediate filament-enriched, substrate-attached cytoplast cytoskeletons. These polypeptides (L-H-L cytoplast polypeptides) corresponded to vimentin (IEF 26, 54,000 M_rmr) and six polypeptides (IEF 12, 68,000 M_r; IEF 24, 56,000 M_r; IEF 31, 50,000 M_r; IEF 35, 49,000 M_r; IEF 36, 48,500 M_r and IEF 46, 43,500 M_r) not previously reported as present in cytoskeletons. Peptide analysis showed that these were not related as products of modification or proteolysis.Labelling of mitotic and interphase cells with [³⁵S]methionine followed by one-dimensional peptide map analysis showed that IEF 24, 26 (vimentin), 31 and 36 are preferentially modified during mitosis. These modifications correspond to phosphorylations of IEF 26 (vimentin) and 31, and to an unknown type for IEF 24. IEF 36 is phosphorylated in interphase to yield IEF 37, and the latter is further phosphorylated in mitosis. These results suggest that modification of the L-H-L cytoplast polypeptides may be important in the reorganization of cytoskeletal elements that takes place during cell division.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.