Purification and characterization of the polycation-stimulated protein phosphatase catalytic subunit from porcine renal cortex

The predominant form of phosphorylase phosphatase activity in porcine renal cortical extracts was a polycation-stimulated protein phosphatase. This activity was present in extracts in a high-molecular-weight form which could be converted to a free catalytic subunit by treatment with ethanol, urea, or freezing and thawing in the presence of beta-mercaptoethanol. The catalytic subunit of the polycation-stimulated phosphatase was purified by chromatography on DEAE-Sephacel, heparin-Sepharose, and Sephadex G-75. The phosphatase appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis. The enzyme had an apparent Mr of 35 000 on gel filtration and SDS-polyacrylamide gel electrophoresis. The purified phosphatase could be stimulated by histone H1, protamine, poly(D-lysine), poly(L-lysine) or polybrene utilizing phosphorylase a as the substrate. It preferentially dephosphorylated the alpha-subunit of phosphorylase kinase. The phosphatase was highly sensitive to inhibition by ATP. These results suggest that the renal polycation-stimulated phosphatase catalytic subunit is very similar to or identical with the skeletal muscle phosphatase form which has been previously designated phosphatase-2Ac.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

Chemical and physical characterization of rabbit sperm acrosome stabilizing factor

Rabbit Acrosome Stabilizing Factor (ASF) is an epididymal product that reversibly inhibits the process of sperm capacitation. The native molecular weights of the monomer and dimer ASF were determined from sedimentation and diffusion data at 129,000 and 259,000 Mr. The monomer is composed of 92,000 and 38,000 Mr subunits according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with size heterogeneity demonstrated for the latter. The stoichiometry of the subunits appears to be one-to-one by gel scanning. Amino acid and carbohydrate compositions are characteristic of a globular glycoprotein, which is high in cysteine content and is 8.3% carbohydrate by weight. The sugar composition suggests the presence of both high mannose and complex N-linked oligosaccharides with the unusual feature of appreciable amounts of glucose. The isoelectric character of ASF spans a range from 5.3 to 7.0.     

An immunopathologic study of a 330-kD protein defined by monoclonal antibodies and reactive with anti-RTE alpha 5 antibodies and kidney eluates from active Heymann nephritis

There is evidence indicating that the glomerular Ig deposits of Heymann's nephritis (HN)--a model of epimembranous glomerulonephritis--may be formed at least in part in situ by binding of free circulating antibody with brush border (BB) antigen expressed by glomerular epithelial cells. In this work, we provide evidence that a 330-kD protein defined by seven monoclonal antibodies is responsible for HN. 1) Ig eluted from glomeruli of rats with HN induced classically with crude BB preparation bind specifically the 330-kD antigen; 2) passive immunization with monoclonal antibodies induces epimembranous glomerular Ig deposits; 3) active immunization with the 330-kD antigen induces proteinuric glomerulonephritis; 4) the 330-kD antigen was present in the nephritogenic preparation purified by Edgington, Glassock, and Dixon, because it was identified by the corresponding heterologous antisera. These results, obtained by a completely different approach, confirm and extend those of Kerjaschki and Farquhar and provide a link with the classical studies on HN.     

Differential Proteolysis of Glycinin and beta-Conglycinin Polypeptides during Soybean Germination and Seedling Growth

The degradation of the major seed storage globulins of the soybean (Glycine max [L.] Merrill) was examined during the first 12 days of germination and seedling growth. The appearance of glycinin and β-conglycinin degradation products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cotyledon extracts followed by electroblotting to nitrocellulose and immunostaining using glycinin and β-conglycinin specific antibodies. The three subunits of β-conglycinin were preferentially metabolized. Of the three subunits of β-conglycinin, the larger α and α′ subunits are rapidly degraded, generating new β-conglycinin cross-reactive polypeptides of 51,200 molecular weight soon after imbibition of the seed. After 6 days of growth the β-subunit is also hydrolyzed. At least six polypeptides, ranging from 33,100 to 24,000 molecular weight, appear as apparent degradation products of β-conglycinin. The metabolism of the glycinin acidic chains begins early in growth. The glycinin acidic chains present at day 3 have already been altered from the native form in the ungerminated seed, as evidenced by their higher mobility in an alkaline-urea polyacrylamide gel electrophoresis system. However, no change in the molecular weight of these chains is detectable by sodium dodecyl sulfate-polyarylamide gel electrophoresis. Examination of the glycinin polypeptide amino-termini by dansylation suggests that this initial modification of the acidic chains involves limited proteolysis at the carboxyl-termini, deamidation, or both. After 3 days of growth the acidic chains are rapidly hydrolyzed to a smaller (21,900 molecular weight) form. The basic polypeptides of glycinin appear to be unaltered during the first 8 days of growth, but are rapidly degraded thereafter to unidentified products. All of the original glycinin basic chains have been destroyed by day 10 of growth.     

Imino 1H- and 31P-NMR analysis of the interaction of propidium and ethidium with DNA

At low temperature and low salt concentration, both imino proton and ³¹p-nmr spectra of DNA complexes with the intercalators ethidium and propidium are in the slow-exchange region. Increasing temperature and/or increasing salt concentration results in an increase in the site exchange rate. Ring-current effects from the intercalated phenanthridinium ring of ethidium and propidium cause upfield shifts of the imino protons of A · T and G · C base pairs, which are quite similar for the two intercalators. The limiting induced chemical shifts for propidium and ethidium at saturation of DNA binding sites are approximately 0.9 ppm for A · T and 1.1 ppm for G · C base pairs. The similarity of the shifts for ethidium and propidium, in both the slow- and fast-exchange regions over the entire titration of DNA, shows that a binding model for propidium with neighbor-exclusion binding and negative ligand cooperativity is correct. The fact that a unique chemical shift is obtained for imino protons at intercalated sites over the entire titration and that no unshifted imino proton peaks remain at saturation binding of ethidium and propidium supports a neighbor-exclusion binding model with intercalators bound at alternating sites rather than in clusters on the double helix. Addition of ethidium and propidium to DNA results in downfield shifts in ³¹P-nmr spectra. At saturation ratios of intercalator to DNA base pairs in the titration, a downfield shoulder (approximately ?2.7 ppm) is apparent, which accounts for approximately 15% of the spectral area. The main peak is at ?3.9 to ?4.0 ppm relative to ?4.35 in uncomplexed DNA. The simplest neighbor-binding model predicts a downfield peak with approximately 50% of the spectral area and an upfield peak, near the chemical shift for uncomplexed DNA, with 50% of the area. This is definitely not the case with these intercalators. The observed chemical shifts and areas for the DNA complexes can be explained by models, for example, that involve spreading the intercalation-induced unwinding of the double helix over several base pairs and/or a DNA sequence- and conformation-dependent heterogeneity in intercalation-induced chemical shifts and resulting exchange rates.     

N2-(1-carboxyethyl)methionine. A ''pseudo-opine'' in octopine-type crown-gall tumours.

A novel methionine-containing plasmid-determined compound, N2-(1-carboxyethyl)methionine (NCEM) has been identified in crown-gall tumours induced by octopine-type strains of Agrobacterium tumefaciens. NCEM is probably synthesized by octopine synthase. Cell-free preparations from octopine-type strains of A. tumefaciens can degrade NCEM; however, the bacterium cannot transport the compound into the cell, although these strains can take up and degrade the octopine family of opines.     

Some thoughts on the evolutionary basis for the prominent role of ATP and ADP in cellular energy metabolism

The predominance of the adenosine triphosphate/adenosine diphosphate (ATP/ADP) couple in cellular phosphorylation reactions, including those that form the basis for cellular energy metabolism, cannot be explained on thermodynamic grounds since a variety of "high energy phosphate" compounds (including ADP itself) found in the cell would, based on thermodynamic considerations, be at least as effective as ATP in serving as a phosphoryl donor. How then did present-day organisms come to rely on the ATP/ADP couple as the principal mediator of phosphorylation reactions? The early appearance of adenine compounds in the prebiotic environment is suggested by experiments indicating that, relative to other purine or pyridimine compounds, adenine derivatives are preferentially synthesized under simulated prebiotic conditions (Ponnamperuma et al., 1963). In addition to the roles of adenine nucleotides in phosphorylation reactions, other adenine derivatives (e.g. Coenzyme A, flavin adenine dinucleotide, puridine nucleotides) are employed in a variety of metabolic roles. The principal function of the adenine moiety in these latter cases is in the binding of these derivatives to the relevant enzyme. The capability for binding of the adenine moiety appears to have arisen early in evolution and been exploited in a multitude of contexts, a suggestion consistent with observed similarities between the binding sites of several enzymes employing adenine derivatives as substrate. The early availability of suitable adenine compounds in the biosphere and development of complementary binding sites on cellular proteins, coupled with the expected advantages in having a limited number of metabolites as central mediators of endergonic and exergonic metabolism could readily have led to the observed pre-eminence of adenine nucleotides in cellular energy metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular and intracellular acid-base status following strenuous activity in the sea raven (Hemitripterus americanus)

Summary Exhausting activity in the sea raven resulted in a pronounced extracellular acidosis, which consisted of a large, short-lived respiratory component and a small, longer-lived metabolic component. Thi disturbance had been corrected by 12 h. White muscle experienced a pronounced intracellular acidosis of chiefly metabolic origin, with pH_i dropping from a resting value of 7.51 to a low of 7.10 immediately post-activity. The recovery of pH_i was associated with a reduction in muscle lactate. Despite the large increase in , cardiac muscle pH_i remained constant postactivity, actually showing an alkalosis at 30 min into recovery. Maintenance of cardiac muscle pH_i was achieved by an accumulation of HCO ₃ ^– intracellularly.