Philip Siekevitz

Gel electrophoresis of partially purified cytochromes P450 from liver microsomes of variously-treated rats

Liver microsomes and partially purified cytochromes P450 prepared from untreated animals or those injected with phenobarbital, or 3-methylcholanthrene, or polychlorinated biphenyls, were subjected to slab-gel SDS-electrophoresis. There were observed marked differences, after these treatments, in the gel-electrophoresis patterns of the induced cytochromes P450 in the microsomes and partially purified preparations.

Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain.

Abstract: Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L‐[3H]glutamate binding sites. It was found that there was a concentration of L‐glutamate binding sites in CTX‐PSD and CL‐PSD over the respective membrane fractions, and the Bmax value of CL‐PSD (92.0 pmol/mg protein) was about three times that of CTX‐PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL‐PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L‐glutamate binding to canine CTX‐SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl− increased the number of L‐glutamate binding sites and the effect was enhanced by Ca2+; Ca2+ alone had no significant effect; (b) the Cl−/Ca2+ ‐sensitive binding sites were abolished by 2‐amino‐4‐phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na+ ion was biphasic: low concentration of Na+ (< 5 mM) decreased Cl−7Ca2+ ‐de‐pendent L‐glutamate binding sites, whereas at higher concentrations of Na+ the binding of glutamate was found to increase either in the presence or absence of Ca2+ and Cl−. In addition, the K+ ion (50 mM) was found to decrease the Na+‐independent and Cl−/Ca2‐‐independent binding of L‐glutamate to fresh CTX‐SPM by 18%, but it decreased the Na−‐dependent and Cl−/Ca2+‐independent L‐glutamate binding by 93%; in the presence of Cl, −/Ca2+, the K+ ion decreased the Na+‐dependent binding by 78%. Freezing and thawing of CTX‐SPM resulted in a 50% loss of the Na+‐dependent L‐glutamate binding sites assayed in the absence of Ca2+ and Cl−. The CL‐SPM fraction showed similar ion dependency of L‐glutamate binding except for the absence of Na−‐dependent glutamate binding sites. The CTX‐PSD fraction contained neither Na+‐dependent nor APB (or Cl−/Ca2+)‐sensitive L‐glutamate binding sites and its L‐glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L‐Glutamate binding to CTX‐SPM or CTX‐PSD fraction was not affected by pretreatment with 10 mM L‐glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX‐SPM or CTX‐PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L‐glutamate. Furthermore, binding of L‐glutamate to CTX‐SPM or CTX‐PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX‐PSD fraction with 0.5% deoxycholate, 1.0% N‐lauroyl sarcosinate, 4 M guanidine‐HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L‐glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L‐glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ‐aminobutyric acid and flunitrazepam binding sites, of the Ca2+‐dependent K+ channel, and of the voltage‐dependent Ca2+ channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.

Synthesis of microsomal membranes and their enzymic constituents in developing rat liver

Abstract The limiting membrane of the endoplasmic reticulum (ER) has a variety of enzymatic activities which are ascribed to some of its constitutive proteins ( Siekevitz, 1963 ). The location of the enzymes in the membrane is related to the function of the ER system; moreover, enzyme activity is influenced by the position and relations of the enzyme within the membrane structure ( Ernster et al ., 1962 ). The question arises whether during biogenesis of the ER membrane, the synthesis of the membrane-bound enzymes is closely connected with, and perhaps dependent on, the synthesis of other membrane components, such as phospholipids and probably non-enzymic protein. The developing rat liver appears to be a suitable system for answering this question. Available data indicate that the fetal hepatic cell has a poorly developed ER (Howatson and Ham, 1954; Peters et al ., 1963 ) and is deficient in some enzymes ( Nemeth, 1954 ; Strittmatter, 1963 ). This paper describes the differential rates of synthesis of membrane-bound enzymes in the ER of the newborn rat.

Phosphoproteins in Postsynaptic Densities

Publisher Summary The postsynaptic density (PSD) is a dense submembranous filamentous array located behind and in intimate contact with the postsynaptic membrane. PSDs have been isolated from synaptosomal plasma membranes and synaptosomes using various detergents. Canine cerebral cortex and midbrain PSDs have identical protein patterns with SDS-polyacrylamide gel electrophoresis (SDS PAGE) and contain about 10 major and at least 20 minor proteins. No membranes are seen by electron microscopy and the phospholipid content is 1% or less. No nucleic acid has been found either so that the structure is composed almost exclusively of protein. Enzymatic marker studies and radioactive mixing experiments reveal 0.1–6% contamination of the PSD fraction by mitochondrial, synaptic vesicle, plasma membrane and myelin membrane proteins. Myelin basic protein and intermediate filament protein (have been identified as contaminants. There are no Mg 2+ or Ca2+-ATPase nor adenylate cyclase activities present in the fraction. A CAMP-dependent protein kinase and the two substrates for this kinase, proteins Ia and Ib are present. The presence of actin has been identified by amino acid analysis, immunology, and by gel mobility.

The binding interaction between alpha-latrotoxin from black widow spider venom and a dog cerebral cortex synaptosomal membrane preparation.

—The major toxin of black widow spider venom, α‐latrotoxin, can be iodinated with 125I with hardly any loss in biological activity. The radioactive toxin could bind specifically to a dog cerebral cortex synaptosomal membrane preparation but not to a dog liver plasma membrane preparation. The bound protein could be recovered from the neuronal membrane preparation in an unchanged form. Non‐specific binding was only 6–10% of the total binding. The protein nature of the presumed receptor was indicated by the complete inhibition of the binding by either heating the membrane preparation at 70°C or treating the membrane with trypsin. Pre‐incubation with 2%β‐mercaptoethanol also completely inhibited the binding, while 70% inhibition was observed after pre‐treatment with 10m M‐EDTA or EGTA. From plots of the equilibrium binding data, it could be ascertained that the binding is non‐cooperative, with an apparent equilibrium dissociation constant, K1, of 1.0 nM. Kinetic data gave an apparent association rate constant of 8.2 × 105 M−1 s−1. Dissociation followed a biphasic exponential with rate constants of 1.4 × 10−3 and 5.2 × 10−5s−1 corresponding to half‐lives of 8.2 min and 3.7 h. Possible schemes for the binding interaction were proposed. Based on the present results and on previous results which indicated that α‐latrotoxin causes the release of all neurotransmitters and a depletion of the synaptic vesicle population in vertebrate synapses, a hypothetical mechanism of the action for the toxin was proposed, involving the binding of the toxin to a membrane protein receptor which interacts with filamentous proteins linking the synaptic vesicles to the axolemma.

The turnover of proteins and the usage of information.

Abstract This article is in the nature of a review with theoretical comments on protein turnover in cells, concentrating on possible modes of control of synthesis and degradation rates. While our knowledge of protein synthesis is well-developed, that of protein degradation is rudimentary. Thus, the review covers data on protein degradation with regards the influence of substrate, of the environment in which the protein finds itself, of the size and amino acid composition of protein, and on the proteolytic enzymes involved. It is stressed that a linkage must exist between synthesis and degradation, to explain the steady-state concentration of specific proteins in cells, and it explores ideas as to the nature of this linkage. Possible explanations are given of the apparent necessity of having proteins being constantly degraded; these include the need for a basic mechanism which can be easily modulated in order that concentrations of specific proteins may change, the need for the correction of possible errors arising from the transcription and translation processes, and the apparent need to make the most efficient usage, in terms of energy input, of the information machinery of the cell. Finally, ideas are suggested of the possible relevance of protein turnover to the informational molecules and complexes in the cell.

Postmortem Accumulation of Tubulin in Postsynaptic Density Preparations

Abstract: Postsynaptic density (PSD) preparations isolated from canine cerebral cortex that had been left at 0–37°C for various times were found to become enriched in two bands in a time‐ but not temperature‐dependent manner. The two bands were identified as tubulin subunits by gel mobility and immunology. Of all the isolated synaptic structures the increase in tubulin occurred primarily in the PSD fraction. The increase of tubulin also occurred in PSD preparations isolated from canine cerebellum and rat forebrain. Results obtained when PSD fractions were isolated from canine brain obtained as rapidly as possible after the death of the animal indicate that the maximum amount of tubulin in the PSD preparations is 2.5% of total Coomassie blue‐stained protein as determined by scanning of gel electrophoretograms. These results imply that tubulin is probably not a major structural protein of the PSD as it exists in situ.

The purification and some properties of ribulosebisphosphate carboxylase and of its subunits from the green alga Chlamydomonas reinhardtii

Abstract A description is presented of a rapid and efficient method for large-scale preparation of ribulosebiphosphate carboxylase (EC 4.1.1.39) from the green alga, Chlamydomonas reinhardtii, and of the separation into two purified subunits. The purification of the enzyme was accomplished essentially by (NH4)2SO4 fractionation and sucrose-density gradient centrifugation, while the separation into subunits has been done by chromatography on sodium dodecylsulfate-hydroxyapatite or by gel filtration on Sephadex G-150 in the presence of guanidine-HCl. The molecular weights of the subunits, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, were 55.0·103 and 16.5·103. The pI of the enzyme was found to be 6.25, while that of the subunits was in the range 6.0–7.0. No amino-terminal amino acid could be detected in either of the subunits. Amino acid composition of the whole enzyme and of the separated subunits were determined and compared to those from other plant species.

Characteristics of a Ca2+/calmodulin-dependent binding of the Ca2+ channel antagonist, nitrendipine, to a postsynaptic density fraction isolated from canine cerebral cortex

Synaptic membrane (SM) and postsynaptic density (PSD) fractions isolated from the cerebral cortex (CTX) and cerebellum (CL) of the canine brain were found to contain one class of specific nitrendipine binding sites. The specific binding constants were: CTX-SM, Kd = 110 pM (Bmax = 126 fmol/mg protein); CTX-PSD, Kd = 207 pM (Bmax = 196 fmol/mg); CL-SM, Kd = 100 pM (Bmax = 65 fmol/mg); CL-PSD, Kd = 189 pM (Bmax = 80 fmol/mg). Treatment of the CTX-SM and CTX-PSD fractions with 0.5% deoxycholate and 1.0% N-lauroyl sarcosinate removed 88-91% and 42-51% of the nitrendipine binding, respectively, indicating that the major nitrendipine binding present in the SM fractions are of non-synaptic origin. Moreover, the percentages of total protein and specific nitrendipine binding removed from PSDs by these detergents were similar, indicating no preferential dissociation of the latter, and suggesting that the receptor protein is firmly bound and is probably an intrinsic component of the PSD fraction. Both Ca2+ and calmodulin were found to be important for the binding of nitrendipine to the CTX-SM and CTX-PSD fractions since: R24571, a calmodulin antagonist, was found to inhibit nitrendipine binding to the CTX-SM and CTX-PSD fractions with IC50 values of 1.1 microM and 0.9 microM, respectively; removal of Ca2+ from the CTX-SM and CTX-PSD fractions with 0.2 mM EGTA resulted in losses of specific nitrendipine binding of 80 and 90%, respectively; Ca2+ alone restored nitrendipine binding to EGTA-pretreated CTX-SM fractions and not to CTX-PSD fractions, with the latter needing both Ca2+ and calmodulin to restore nitrendipine binding; EGTA treatment removed 14-16% and 89-91% of nitrendipine bound to the CTX-SM and CTX-PSD fractions, respectively, suggesting that calmodulin (but not Ca2+) is needed to maintain the nitrendipine-nitrendipine receptor-calmodulin complex; Ca2+-reconstituted EGTA-pretreated CTX-SM fractions and the Ca2+ plus calmodulin-reconstituted EGTA-pretreated CTX-SM and CTX-PSD fractions were found to have similar binding constants to those for the corresponding native, untreated fractions; and the Ca2+/calmodulin dependency on nitrendipine binding was similar to the well-known Ca2+/calmodulin dependency on phosphorylation in EGTA-pretreated PSD fractions. It needed much less Ca2+ to saturate Ca2+/calmodulin-dependent phosphorylation of the pretreated CTX-PSD fractions than the nitrendipine binding. Yet, less calmodulin was needed to saturate nitrendipine binding than the phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

SDS-polyacrylamide gel electrophoresis of membrane proteins: effect of phospholipids.

Abstract Under the conditions described in this report, it was found that the occurrence of phospholipids in membrane samples has no artifactual impact on the subsequent separation and visualization of membrane proteins in SDS-polyacrylamide gels stained with Coomassie Blue.