A new form of renin in normal human plasma: “big renin” is a mixture of inactive prorenin and the new active high molecular weight renin

A new form of active renin was separated from inactive prorenin in normal human plasma by a new affinity chromatographic method on a column of Cibacron Blue F3GA-agarose. This active renin has a molecular weight of 54,000, considerably higher than the hitherto recognized active renin of 40,000 dalton in human plasma. The molecular weight of inactive prorenin was 56,000±2,000. Active renin produced from the inactive prorenin by trypsin or pepsin digestion or by acid treatment in $\text{in vitro}$ experiments showed a molecular weight of 54,000±2,000. Active renin with a molecular weight of 40,000 was not found in 6 samples of untreated plasma of normal human subjects nor was it formed by treatment with trypsin, pepsin, or acid pH. It is concluded that a large form of active renin (54,000 dalton) exists in normal human plasma which is distinct from a smaller form and that the activatable “big renin” is a mixture of this active renin and totally inactive prorenin. This explains the absence of molecular weight change during the activation of “big renin”.     

Na+,K+-ATPase: on the nature of the "cross-linked" subunits obtained in the presence of o-phenanthroline and cupric ion.

In previous studies on the quaternary structure of Na⁺,K⁺-ATPase, cupric-phenanthroline complex (CP) has been used for the cross-linking of the enzyme subunits. Here we show that the same products obtained in the presence of CP (α,α-dimer, α,β-dimer, and products of higher molecular weight) are also obtained when the enzyme is exposed to Cu²⁺ without o-phenanthroline. The α,β-dimer (but not the α,α-dimer) is dissociated in the presence of EDTA; indicating that this product is not the result of the covalent cross-linking of the subunits through a disulfide bond. The nature of the α,α-dimer remains to be determined. The findings suggest that the results of “cross-linking” experiments with CP should be interpreted with caution until the products are more clearly identified.     

High molecular weight forms of human placental lactogen: synthesis in vitro and binding to membrane receptors for lactogenic hormones.

Translation in wheat germ extracts of poly(A)-containing RNA isolated from human term placentas resulted in the synthesis of immunoreactive forms of human placental lactogen (hPL) capable of specific binding to lactogenic receptors. The minor component coelectrophoresed on sodium dodecyl sulfate-polyacrylamide gels with authentic hPL while the major component migrated with an apparent molecular weight about 3000 larger. In addition to this precursor-like molecule, even higher molecular weight forms of hPL were observed under certain conditions: (i) when the cell-free translation products were purified by precipitation with anti-hPL serum followed by dissociation of the immunoprecipitate in guanidine hydrochloride and chromatography of the solubilized material on Sephadex G-150 in the same denaturing buffer, and (ii) when the cell-free reaction mixture was analyzed by direct chromatography on Sephadex G-150 in nondenaturing buffers. Under both sets of conditions 50–75% of the radioactivity was eluted in the column void volume, suggesting it had a molecular weight of 150,000 or more. When the high molecular weight translated product was analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the radioactive components were identical to authentic hPL and the precursorlike form, suggesting the large forms are aggregates of the smaller forms. Both the very high molecular weight forms, composed primarily of the precursor-like molecule, and the less aggregated products bound to specific lactogenic hormone receptors in rat liver membrane preparations, although the larger forms exchanged less readily with unlabeled hPL than did the monomeric form of the hormone. The aggregated, receptor-bindable cell-free translation product may be similar to high molecular weight lactogens previously described in vivo.     

On the controversy over "fast" and "slow" helix-coil transition rates in polypeptides

The controversy over “fast” and “slow” helix-coil transition rates in polypeptides is discussed. The “slow” results are derived from the assumption that multiple NMR spectra of α-CH and NH groups arise from chemical exchange. In this paper it is shown that such spectra may be obtained without invoking chemical exchange. The multiplicity arises from the difference in helicity of amino acid residues near the ends of the chain by comparison with amino acid residues nearer the middle, and from a polydispersity in molecular weight. As a consequence of this analysis, support is given to the “fast” transition rates.     

Accumulation of a large component related to thyrotropin subunits in the pituitary of thyroidectomized rats.

Rat pituitary thyrotropin-β subunit immunoreactivity is demonstrated to increase with time after thyroidectomy. This increase is due to free β subunits and a high molecular weight form. The latter is also immunoreactive in α subunit and native thyrotropin assays. Its immunological properties in these three types of assays respectively, suggest that it represents a “bid β subunit” and a “big α subunit” excluded together in Sephadex G-100 chromatography, rather than a “big thyrotropin”. These larger species could be precursor forms of the subunits. This would be in agreement with the concept of separate biosynthesis of the subunits of pituitary glycoproteic hormones.     

A unique proenkephalin-converting enzyme purified from bovine adrenal chromaffin granules

A unique proenkephalin converting enzyme specifically generating enkephalin was partially purified from lysates of adrenal chromaffin granules. The enzyme, whose molecular weight is estimated as ca. 220,000, is thiol-dependent protease, with optimal pH at around 5.5. The enzyme converts proenkephalin to enkephalins by cleaving specifically at the sites of consecutive basic amino acid residues. The enzyme also converts BAM-12P, an adrenal “big” Met-enkephalin, to Met-enkephalin in a similar manner. During the enzyme reaction, formation of [Arg⁶]-Met-enkephalin was not observed. Additionally, [Arg⁶]-enkephalins were not converted to enkephalins by the enzyme. Consequently, the enzyme was proved to be a unique converting enzyme distinct from either trypsin-like or carboxypeptidase B-like proteases.     

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro

Background aimsMultipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown.MethodsMSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to “rescue” the proliferative capacity of MSCs.ResultshPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS.ConclusionshPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity.     

Involvement of a low molecular weight component(s) in the mechanism of action of the glucocorticoid receptor.

[³H]Dexamethasone-receptor complexes from rat liver cytosol preincubated at 0° bind poorly to DNA-cellulose. However, if the steroid-receptor complex is subjected to gel filtration at 0–4° separating it from the low molecular weight components of cytosol, the steroid-receptor complex becomes “activated” enabling its binding to DNA-cellulose. This activation can be prevented if the gel filtration column is first equilibrated with the low molecular weight components of cytosol. In addition, if adrenalectomized rat liver cytosol, in the absence of exogeneous steroid, is subjected to gel filtration the macromolecular fractions separated from the “small molecules” of that cytosol have much reduced binding activity towards [³H]dexamethasone. These results suggest that rat liver cytosol contains a low molecular weight component(s) which maintains the glucocorticoid receptor in a conformational state that allows the binding of dexamethasone. Furthermore, this component must be removed from the steroid-receptor complex before binding to DNA can occur.     

The molecular forms of immunoreactive glucagon secreted by the isolated,perfused dog pancreas

The immunoreactive glucagon (IRG) in the plasma-free effluent of the arginine stimulated isolated dog pancreas was purified by immunoaffinity chromatography and characterized with respect to molecular weight. Only a 3500 dalton component was secreted from the pancreas during the first four minutes of stimulation but immunoreactive material having a molecular weight of between 150,000 and 200,000 was isolated from the secretions after prolonged stimulation. This component (which corresponds in size to the incompletely characterized “big plasma glucagon”) was dissociated to a 3500 dalton component and nonimmunoreactive material by 6 M guanadinium chloride. Components of molecular weight 9000 and 2000, which are found in plasma, and components with the immunological properties of gut GLI, were not identified in the pancreatic secretions.     

A macromolecule which gives rise to thyrotropin releasing-hormone.

Chemical and enzymatic treatment of a high-molecular weight fraction from a frog brain extract resulted in formation of a “TRH-Like material” (TRH-i). Sequential treatment with trypsin and carboxypeptidases A and B, acetic acid and then chemical amidation generated a quantity of TRH-i equivalent to 25% of the endogenous TRH. TRH-i was similar to TRH (pGlu·His·ProNH₂) as assessed by molecular weight estimations, radioimmunoassay and susceptibility to serum inactivation. TRH and TRH-i also competed with [³H]-TRH for binding to TRH receptors, stimulated prolactin synthesis and uridine uptake, and “down-regulated” TRH receptors in pituitary cells. These results suggest the possibility that TRH may be processed from a macromolecular precursor.     

Evidence for a pro-calcitonin

The biosynthesis of calcitonin was studied using radioimmunochemical methods and suspensions of calcitonin-producing cells derived from trout ultimobranchial glands. [¹⁴C]leucine was incorporated into cell proteins in a linear fashion for up to 36 hrs. Acid-extracted cellular radioactivity could be precipitated by trichloroacetic acid and calcitonin antiserum. Chromatography of the cell extracts revealed two distinct peaks of radio-immunoassayable and immunoprecipitable calcitonin activity. One peak coeluted with radioiodinated calcitonin, the other as a higher molecular weight species. The relative incorporation of [¹⁴C]leucine into the higher and lower molecular weight peaks during “pulse-chase” experiments was consistent with a precursor-product relationship between them.     

Protein recovery from enzyme‐assisted aqueous extraction of soybean

Enzyme‐assisted aqueous oil extraction from soybean is a “green” alternative to hexane extraction that must realize potential revenues from a value‐added protein co‐product. Three technologies were investigated to recover protein from the skim fraction of an aqueous extraction process. Ultrafiltration achieved overall protein yields between 60% and 64%, with solids protein content of 70%, and was effective in reducing stachyose content, with fluxes between 4 and 10 L/m² hr. Protein content was limited because of high retention of lipids and the loss of polypeptides below 13.6 kDa. Isoelectric precipitation was effective in recovering the minimally hydrolyzed proteins of skim, with a protein content of 70%, again limited by lipid content. However, protein recovery was only 30% because of the greater solubility of the hydrolyzed proteins. Recovery by the alternative of protein capture on dextran‐grafted agarose quaternary‐amine expanded bed adsorption resins decreased with decreasing polypeptide molecular weight. Proteins with molecular mass greater than 30 kDa exhibited slow adsorption rates. Expanded bed adsorption was most effective for recovery of proteins with molecular weight between 30 and 12 kDa. Overall, adsorption protein yields were between 14% and 17%. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Early alterations induced by carbon tetrachloride in the lipids of the membranes of the endoplasmic reticulum of the liver cell I. Separation and partial characterization of altered lipids

Alterations induced by carbon tetrachloride poisoning in fatty acids of liver microsomal lipids were studied. Thin layer chromatography of fatty acid methyl esters prepared from liver microsomal lipids, revealed, in the CCl₄-treated rats, the presence of a component (the “D” spot) with an R_f value lower than that of the methyl esters. The lipids recovered from this component showed a marked diene conjugation absorption when examined spectrophotometrically over the UV range, while the lipids recovered from the spot of the methyl esters showed no absorption of conjugated dienes.Studies carried out with labelled carbon tetrachloride indicated that compounds present in the “D” spot contained 28% of ¹⁴C applied to the chromatoplate. The spot of the methyl esters (the “M” spot) contained 42% of ¹⁴C applied to the chromatoplate. However, specific activity of the “D” spot was about 1000 times greater than specific activity of the “M” spot.The lipids recovered from either the “D” spot or the spot of the methyl esters were analyzed separately by gas-liquid chromatography (GLC) with an electron capture detector (ECD). It was found that the lipids recovered from the “D” spot showed no response, while those recovered from the spot of the methyl esters exhibited the response of the ECD, which was similar to that observed with the unfractionated fatty acid methyl esters. The lack of the response of the ECD for compounds in the “D” spot appears to be due to the fact that they cannot be eluted from the column.On the basis of the analytical results, it can be postulated that the “D” spot contains compounds formed by a chain termination addition reaction of free radicals derived from CCl₄ (probably trichloromethyl free radicals) to fatty acid free radicals containing conjugated dienes. On the other hand, the spot of the methyl esters appears to contain also, together with unmodified fatty acids, the fatty acids in which a simple addition of CCl₄ free radicals to double bonds has occurred.     

The polyphyletic origin of the "Cheilostomata" (Bryszoa)

The relative appearance of the parietal muscles in the development of the zooids has been studied in several ctenostomatous and “cheilostomatous” species. A comparison of the different on-togenetical sequences demonstrated that a “cheilostomatous” type of organization of the zooids with a great probability has been achieved in minimum three times independentl and originated from different ctenostomatous sub-grous: the Membranidea from plesiomorgic victorelloids (ancestors of Bulbella with not yet developed peristomial tube), the Inoviceiata (Aetea) from advanced forms of victorelloids with reduced primary parietal muscles (perhaps stcies related to Pottsiella), and Penetrantia from arachnidioid or vesicularioid ancestors (?). Therefore, the classical orders ^α“Ctenostomata” and “Cheilostomata” represent only “stage groups” but no monohyletic systematical units. Because of the new concept and interpretation I propose a new name for the united group: Cteno-Cheilostomata, supra-ord. nov.     

Properties of biologically active messenger RNA from human placenta. Cell-free synthesis of two immunoreactive forms of placental lactogen.

In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.     

Water binding by glycogen molecules

Low temperature NMR spectra have been obtained of the water bound to glycogen. These data have allowed the evaluation of the amount of water bound and the energy and entropy associated with this bonding. High molecular weight glycogen (approx. 1·10⁹) exhibits water binding properties analogous to those previously found for other glycoproteins. Low molecular weight glycogen (approx. 1 · 10⁷), however, shows anomalous binding characteistics, with large amounts of associated “non-freezing” water. These findings are discussed in terms of previously proposed molecular architecture.     

A study of dextran hydration in dilute,aqueous solution by the proton magnetic relaxation method

The times of proton magnetic relaxation in dilute (<1%), aqueous solutions of dextrans, having a molecular weight range of 17 x 10³?500 x 10³, are highly sensitive to the temperature—time prehistory of the samples investigated. Reliable results have been obtained only after preliminary heating of the solutions at 100° for 30 min. On the basis of the model of “two states of water in a solution”, the dependence of the degree of hydration of a dextran on its molecular weight has been obtained. In the molecular weight range 17 x 10³?110 x 10³, only a fraction of the D-glucose residues are hydrated, the degree of hydration increasing with the molecular weight. The data obtained are considered to be a consequence of intersegmentary interaction in a dextran macromolecule.     

The preparation of high molecular weight divalent "antigens" of defined size

A simple, rapid procedure for the synthesis of divalent “antigens” that consist of high molecular weight polyethylene oxide chains bearing dinitrophenyl groups at their ends and for their fractionation to near size-homogeneity is described.