Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific β-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney γ-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or ⁶⁰Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Phosphorylation of a 16-kDa protein by diacylglycerol-activated protein kinase C in vitro and by vasopressin in intact hepatocytes

A replication-defective Simian virus 40 genome, with a deletion of about 120 nucleotides in the region encoding the N-terminal fourth of the large T antigen, has been isolated from the DNA of Simian cells transformed by SV40. Both the original transformants, and the murine transformants obtained by transfection with this cloned mutant DNA, produced a large T antigen displaying in immunofluorescence an exclusively cytoplasmic localization. The protein apparent molecular mass (83 kDa) was about 6% smaller than that of normal karyophilic large T. Restriction analysis showed that the deletion eliminated two close HinfI sites, at nucleotides 4459 and 4376 (map unit 0.50).     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.     

Coordinated synthesis of the nuclear protein cyclin and DNA in serum-stimulated quiescent 3T3 cells

Quantitative two-dimensional gel electrophoretic analysis (IEF) of the nuclear polypeptide cyclin together with autoradiographic studies have revealed a coordinate synthesis of cyclin and DNA after serum stimulation of quiescent 3T3 cells. These results strengthen the notion that cyclin may be a central component of the pathway(s) that regulate cell proliferation.     

A spin-labeled derivative of Procion brilliant blue MX-R. Synthesis and interaction with pig heart nucleoside diphosphate kinase

The ¹H-NMR spectrum of cucumber basic blue protein (CBP) has been recorded. Examination of the spectrum of the reduced protein suggests that one or more sidechains exist in conformations which interconvert slowly at ambient temperatures. His 39, His 84 and Met 89 are identified as copper ligands by redox titration and by amino acid sequence homology with plastocyanin and azurin. The importance of a Phe sidechain close to the Met ligand in the potential blue copper site is confirmed. Broadening of His ligand resonances at elevated temperatures reveals an exchange process at the reduced copper centre.     

Stimulation of phospholipase A2 activity by high osmotic pressure on cholesterol-containing phospholipid vesicles

Bovine serum albumin conjugates of guanosine prepared by the periodate method was used as immunogen to elicit guanosine antibodies in rabbits. The specificities of the antibodies were studied by the inhibition of their binding to [3H]Gox-red, [32P]DNA and [3H]RNA by related non-radioactive compounds. A population of antibodies is specific to Gox-red with an average association constant of around 10(7) M-1 at 4 degrees C. There are a population of antibodies which bind to [32P]ssDNA and [3H]RNA specifically at guanosine residues. RNA binding antibodies were separated into two populations by affinity chromatography.     

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of beta-lactamase through genetic recombination. This region comprising amino acid residues 70-335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of beta-lactamase.     

Sodium butyrate inhibits the synthesis of the transformation related protein p 53 in 3T6 mouse fibroblasts

Sodium butyrate, which blocks the cell cycle of many cell types in the G1 phase, strongly inhibits the synthesis of the transformation related, 53 kDa protein in 3T6 fibroblasts but much less so in SV 40 transformed mouse cells. By several criteria, this effect of the fatty acid appears to be indirect; p 53 synthesis takes place several hours after the butyrate-sensitive step in G1. The results are discussed in the light of a putative role of p 53 in growth control.