High Molecular Weight Peptides Research Articles

Reserpine changes the dynamic state of enkephalin stores in rat striatum and adrenal medulla by different mechanisms

The administration of reserpine increases enkephalin content in rat striatum and adrenal medulla. In order to investigate the mechanisms operative in this increase, we have studied in vivo the dynamic state of enkephalin stores by determining the content of proenkephalin mRNA, enkephalin precursors, and enkephalins in rats receiving reserpine. We measured proenkephalin mRNA by using a cDNA probe for human proenkephalin mRNA which hybridizes to the same species of mRNA either in the brain or in adrenal medulla. (Met5)-Enkephalin-Arg6-Phe7, as well as the high and low molecular weight forms of the enkephalins separated by Sephadex G-75 column chromatography, were measured by radioimmunoassay. Reserpine (2 mg/kg, i.p., repeated daily for two consecutive days) led 3 to 5 days later to an increase in the striatal content of proenkephalin mRNA as well as high and low molecular weight peptides containing enkephalin. The same treatment produced, in adrenal medulla, a shift from higher molecular weight to lower molecular weight enkephalin-containing peptides, an increase of enkephalin peptides, and a decrease of proenkephalin mRNA content. The results in striatum suggest that reserpine increases enkephalin synthesis by removing a tonic dopaminergic inhibition: those in adrenal medulla indicate that reserpine causes an accumulation of enkephalins by blocking the release and/or increasing the processing which may trigger a feedback-regulatory mechanism leading to a decrease in proenkephalin mRNA content.

Rat platelets activate high molecular weight atrial natriuretic peptides in vitro.

Evidence suggests that the more biologically active low molecular weight forms (less than 10,000) of rat atrial natriuretic peptides are proteolytically derived from a less active precursor of higher molecular weight. Conversion and activation could occur within the myocyte as well as during circulation. The present study found that in vitro rat blood and platelets were capable of converting the high molecular weight atrial natriuretic peptides (greater than 10,000) to low molecular weight atrial natriuretic peptides within minutes and that enhanced biological activity attended the conversion. Rat high molecular weight peptides were partially purified by gel filtration, lyophilized, and reconstituted in Krebs-Ringer bicarbonate buffer. One milliliter of fresh rat blood was incubated with the high molecular weight peptides at 37 degrees C for 2 minutes. After centrifugation, the supernatant was fractionated on Sephadex G-75. Natriuretic activity was determined by bioassay in anesthetized rats. In contrast to the results following incubation of high molecular weight peptides in Krebs-Ringer bicarbonate buffer alone, which showed that 95% of the natriuretic activity remained in the high molecular weight peptide region, the natriuretic activity of the blood-treated high molecular weight peptides eluted almost exclusively in the low molecular weight peptide region, which indicates conversion. Blood was separated into plasma, erythrocytes, lymphocytes, and platelets. Conversion of high to low molecular weight peptides occurred only after incubation with platelets. Compared with control high molecular weight peptides, rat platelet-treated high molecular weight peptides had significantly greater activity in relaxing histamine-contracted rabbit aortic smooth muscle (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecytokinin (CCK) gene-related peptides: distribution and characterization of immunoreactive pro-CCK and an amino-terminal pro-CCK fragment in rat brain

An antiserum specific for the amino terminus of pro-CCKK detects 3 major peptides in rat brain with molecular weithts of 13, 000, 8000 and 2700 daltons. These peptides are abundant both in CCK terminal-field regions and regions rich in CCK perikarya. The two high molecular weight peptides probably represent CCK precursor molecules. The smaller peptide is probably the amino-terminal fragment of pro-CCK and may be stored and released along with CCK-8, and have a potential role in synaptic transmission.

Identification of the peptides of the crystals of [formula omitted] involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B. thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (<44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry−) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.

Routine Fast Atom Bombardment Mass Spectral analysis of high molecular weight peptides — Atrial gland peptides from [formula omitted

Three peptides isolated from the atrial glands of Aplysia californica were analysed by Fast Atom Bombardment Mass Spectrometry. Survey scans over the mass range 1650 to 7500 at 500 resolution were used to locate signals for the protonated molecular ion and two subunits which result from cleavage of a single disulfide bond. A more accurate mass determination was made by accumulating scans over a narrow mass range. The amounts of sample used for each measurement ranged between 10 and 30 pmoles. Measured mass values are within 0.5 amu of calculated average molecular weights. Results illustrate the utility of the technique for accurate molecular weight determinations on limited quantities of high molecular weight peptides.

Composition of Undigested Peptide Fragments from Gluten with Regard to Bioavailability of Essential Amino Acids

ABSTRACTOur purpose was to isolate and characterize the high molecular weight peptides resulting from the in vitro digestion of wheat gluten in a system simulating actual in vivo conditions, and to determine to what extent essential amino acids were included in these peptides. Large peptides were separated from the smaller products by CuSephadex G‐25, TLE and TLC, treated with dansyl chloride for end group determination, and redansylated to identify the remaining amino acids. Fifteen peptides, hexapeptides or larger, were isolated and were found to contain about 16.1% of the threonine, 15.2% of the methionine and 12.3% of the lysine originally present in wheat gluten.

Different patterns of molecular forms of somatostatin are released by the rat median eminence and hypothalamus

The molecular forms of somatostatin (SOM) released from hypothalamic slices and from the isolated median eminence (ME) in vitro were compared by high-performance liquid chromatography and radioimmunoassay. SOM-14 was the predominant form of the peptide released from hypothalamic slices, although small amounts of SOM-28 were detected. Perifusates of ME tissue contained a larger proportion of SOM-28 and higher molecular weight peptides were present; depolarization increased the rates of release of all molecular forms of SOM. These results suggest that the capacity to release SOM-28 and high-molecular-weight forms of SOM may be a specialized function of nerve terminals in the ME.

Substantivity to hair and skin of 125l‐labelled collagen hydrolysates under application simulating conditions

Synopsis Substantivity of various (125)I-labelled collagen hydrolysates to virgin or bleached and dyed European hair or human callus was tested under conditions which simulated actual application of such hydrolysates in shampoos or foam baths. Factors which most strongly influence protein substantivity were identified. The molecular properties of the hydrolysate are the most important factor. The hydrolysate which adsorbed best had the lowest overall isoelectric point, the highest pH in solution and the largest content of high molecular weight peptides. The extent of adsorption was also a function of concentration of protein, of the duration of application and of the nature of the components of the formulation. Thus, the combination of shampoo and hydrolysate can be adjusted so that a desired substantivity is attained.

The opioid octapeptide Met 5-enkephalin-Arg 6-Gly 7-Leu 8: Characterization and distribution in rat spinal cord

The regional quantitation, immunohistochemical localization and molecular heterogeneity of Met 5-enkephalin-Arg 6-Gly 7-Leu 8 were examined in rat spinal cord with a specific radioimmunoassay. A rostrocaudal gradient in Met 5-enkephalin-Arg 6-Gly 7-Leu 8 content was observed; the highest levels occurred in sacral cord. Dorsal cord content was higher than that of ventral cord at all spinal segments. Immunohistochemical staining supported and refined the latter observation: a dense network of perikarya and fibers was found in Laminae I and II of the dorsal horn. Cell bodies were frequently observed in lamina IV. Additional terminals were seen around the central canal and in the ventral gray matter, often outlining perikarya of motor neurons. Total Met 5-enkephalin-Arg 6-Gly 7-Leu 8 immunoreactivity could be fractionated into two main components using gel filtration chromatography. Nearly half of the total immunoreactivity eluted as a high molecular weight peptide; the other half which co-eluted with Met 5-enkephalin-Arg 6-Gly 7-Leu 8 was further identified to be authentic Met 5-enkephalin-Arg 6-Gly 7-Leu 8 on reverse phase high pressure liquid chromatography. The present data, in conjunction with our previous study of Met 5-enkephaline and Met 5-enkephalin-Arg 6-Phe 7 indicates that all opioid peptides derived from preproenkephalin A are present in spinal cord and most likely are stored in the same neurons. Immunohistochemical localization of Met 5-enkephalin-Arg 6-Gly 7-Leu 8 in dorsal and ventral cord suggest a role for this peptide in both sensory and motor integration.

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.

PROTEIN INSOLUBILIZATION IN FROZEN GREENLAND HALIBUT (REINHARDTIUS HIPPOGLOSSOIDES)

Frozen storage of minced Greenland halibut (Reinhardtius hippoglossoides) at -10°C resulted in a rapid loss in salt solubility of “my ofibrillar proteins” (approximately 50% in 15 days) and in a gradual loss in water solubility of “sarcoplasmic proteins” (approximately 40% in 120 days). The water and salt inextractable protein from frozen mince (R) was completely soluble in 4% sodium dodecylsulfate (SDS) when a disulfide bond reducing agent such as mercaptoethanol (ME) was present. Other reagents, including urea and Triton X 100, were less effective in solubilizing the protein from mince after frozen storage. Evidence presented supports the thesis that disulfide bond formation contributes to the observed loss in protein extractability during frozen storage of mince. Addition of various thiol reagents to mince prior to freezing was effective in minimizing protein insolubilization. An estimated 50% of the reduced cysteine associated with protein is oxidized in mince after long time storage at -10°C. However, the kinetics of disulfide bond formation do not parallel the time course of protein insolubilization; accordingly, the possibility that disulfide bond formation is a secondary event cannot be excluded. Other lines of evidence indicate that additional covalent or strong bond interactions contribute to the formation of(R). Solubilization and boiling of(R) in SDS/ME does not dissociate high molecular weight aggregates (500–1000 Kdaltons) although these aggregates are disrupted by sodium borohydride reduction. In addition, the degree of hydrolysis of proteins in frozen mince by certain proteolytic enzymes is lower then that of fresh mince despite the complete solubilization of the mince protein by these proteolytic enzymes. The mince protein from frozen fish contains substantially more fluorescence than that of fresh fish. Fluorescence is associated with the high molecular weight peptide fraction after pepsin catalyzed hydrolysis. The amino acid composition of mince proteins after HCl or formic acid/HCl hydrolysis did not change appreciably as a result of frozen storage. The data indicate that covalent bond formation by sulfhydryl residues and other borohydride and acid labile linkages contribute to the loss in solubility of the protein in Greenland halibut mince during frozen storage at -10°C.

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin.

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolyic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 μg/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.

Immunocytochemical localization of atrial natriuretic factor in the heart and salivary glands

Antibodies produced in the mouse by repeated intraperitoneal injections of partly purified atrial natriuretic factor (low molecular weight peptide (LMWP) and high molecular weight peptide (HMWP)) have been used to localize these factors by immunohistochemistry (immunofluorescence and immunoperoxidase method) and by immunocytochemistry (protein A-gold technique) in the heart of rats and of a variety of animal species including man and in the rat salivary glands. Immunofluorescence and the immunoperoxidase method gave identical results; in the rat, atrial cardiocytes gave a positive reaction at both nuclear poles while ventricular cardiocytes were consistently negative. The cardiocytes of the right atrial appendage were more intensely reactive than those localized in the left appendage. A decreasing gradient of intensity was observed from the subpericardial to the subendocardial cardiocytes. The cardiocytes of the interatrial septum were only lightly granulated. Sodium deficiency and thirst (deprivation of drinking water for 5 days) produced, as already shown at the ultrastructural level, a marked increase in the reactivity of all cardiocytes from both atria with the same gradient of intensity as in control animals. Cross-reactivity of intragranular peptides with the rat antibodies allowed visualization of specific granules in a variety of animal species (mouse, guinea pig, rabbit, rat, dog) and in human atrial appendages. No reaction could be elicited in the frog atrium and ventricle although, in this species, specific granules have been shown to be present by electron microscopy in all cardiac chambers. With the protein A-gold technique, at the ultrastructural level, single labeling (use of one antibody on one face of a fine section) or double labeling (use of two antibodies on the two faces of a fine section) showed that the two peptides are localized simultaneously in all three types (A, B and D) of specific granules. In the rat salivary glands, immunofluorescence and the immunoperoxidase method showed reactivity exclusively in the acinar cells. The reaction was most intense in the acinar cells of the parotid gland. In the sublingual gland, only the serous cells, sometimes forming abortive "demi-lunes", were reactive. In the submaxillary gland, the reaction was weaker and distributed seemingly haphazardly in the gland. The most constantly reactive cells were localized near the capsule while many cells did not contain visible reaction product.

Comparative investigations of partial amino-acid sequences of prolamines and glutelins from cereals

The main peptide fractions obtained from prolamins and glutelins of wheat, rye, barley, and corn by chymotryptic hydrolysis, gel filtration and cation exchange chromatography [1, 2] were further separated by anion exchange chromatography. The amino-acid compositions of the peptides obtained from wheat, rye and barley prolamins are closely related. Typical compositions are Glx greater than 44, Pro greater than 28, and Phe greater than 7 mol-%. The peptide fractions from corn prolamine are different (Glx greater than 20, Leu greater than 20, Ala greater than 12, Pro greater than 10 mol-%). The peptide fractions from wheat, rye and barley glutelins are less similar. Typical compositions are Glx greater than 30, Pro greater than 15, and Gly greater than 8 mol-%. Most of the fractions from corn glutelin contain many amino-acids with Glx, Leu, and Ala predominating. Wheat differs significantly from the other cereals because of the unique composition of some high molecular weight peptide fractions from glutelin, which are rich in Glx (greater than 49) and Gly (greater than 19 mol-%).

Proteolytic activation of a bioactive cardiac peptide by in vitro trypsin cleavage.

Mammalian cardiac atria possess several unidentified biologically active peptides. Fractionation of rat atrial extracts by gel filtration chromatography revealed two major fractions [apparent molecular weights of 20,000-30,000 (peak I) and less than 10,000 (peak II)], both of which were potent natriuretic agents (eliciting a 25-fold increase in sodium excretion) and smooth muscle relaxants. Vigorous treatment with trypsin (100 units/ml at 37 degrees C for 15 min) of both fractions abolished all biological activity. Further purification of the lower molecular weight fraction (peak II) by ion-exchange chromatography indicated two subfractions that possessed potent natriuretic activity and that preferentially relaxed either intestinal (designated peak IIA) or vascular (peak IIB) smooth muscle assay tissues. The similarity of the biological effect of the high (peak I) and low (peak II) molecular weight peptides led us to test the possibility of precursor-product relationship. Mild proteolytic treatment of the high molecular weight peptide with trypsin (1 unit/ml at room temperature) markedly enhanced the smooth muscle relaxant activity. Subsequent analysis of the trypsin (1 unit/ml)-treated high molecular weight peptide (peak I) by gel filtration and ion-exchange chromatography revealed that the peptide now resembled the low molecular weight peptides (peaks IIA and IIB) present in the original atrial extract. These data suggest that the cardiac atria contain a relatively inactive (smooth muscle relaxant) high molecular weight peptide and suggest that biologically active low molecular weight peptides can subsequently be generated by proteolytic cleavage.

Pepsin cleavage of band 3 produces its membrane-crossing domains

After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid) or with N- ethylmaleimide , probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N- ethylmaleimide . Because the location of the N- ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.

“Joining peptide” of pro-opiomelanocortin. I. Radioimmunoassay and extraction of related peptides from pituitary glands

In order to immunoassay the specific region of bovine pituitary pro-opiomelanocortin (POMC) between ACTH and γ-MSH, referred to as “joining peptide,” antisera were prepared against the synthetic amidated decapeptide Val-Ala-Val-Gly-Glu-Gly-Pro-Gly-Pro-Arg-NH 2. The non-amidated peptide represents residues −23 to −14 of bovine POMC [25]. An NH 2-terminal tyrosine analog of the decapeptide was used as the radioligand. Under optimal conditions, immunoassays with selected antisera exhibited a sensitivity (50% displacement of the radioligand) toward the decapeptide in the range of 31–55 pg. Immunoreactivity found in extracts of fresh or lyophilized bovine pituitary glands displaced the iodinated Tyr-decapeptide in the RIA in a parallel manner. The amount of imunoreactive (ir)-material was dependent upon the state of preservation of the tissue, the method of extraction, and the particular antiserum used. Extractable immunoreactivity was separated into low (Mr 1,500) and high (Mr 17,000) molecular weight peptides using gel chromatography (G-75). Additional ir-material appeared in the void volume ( Mr>22,500 ). Thus, these antisera have the capacity to interact not only with a region of the joining peptide but also with its larger, and apparent precursor forms. The immunoassay developed should be valuable in understanding the disposition and processing in this specific region of POMC.

Met 5]-enkephalin-like peptides of the adrenal medulla: Release by nerve stimulation and functional implications

In adrenal chromaffin cells, various molecular forms of polypeptides cross-reacting with [met 5]-enkephalin antisera are stored in granules together with catecholamines and soluble proteins. Splanchnic nerve stimulation increases the release of enkephalin-like peptides from the adrenal gland into the adrenal vein. The release of these peptides appears to be mediated by a cholinergic nicotinic receptor. Fractionation of plasma extracts on Bio-gel P-2 shows the presence of only low molecular weight peptides in the resting condition. The low molecular weight fraction contains mainly [met 5]-enkephalin and [leu 5]-enkephalin. When the splanchnic nerve is stimulated high and low molecular weight peptides are released and the amount of low molecular weight peptides in plasma is increased. The content of enkephalin-like peptides in adrenal venous plasma was similar in control and reserpinized dogs. Splanchnic nerve stimulation increased the peptide content but not the epinephrine content of plasma in reserpinized dogs. This also caused a fall of arterial blood pressure which could be prevented by pretreatment with naloxone. A decrease in blood pressure was also directly elicited by the injection of [met 5]-enkephalin-[arg 6-phe 7]. The duration of the hypotensive effect of this peptide was prolonged by prior injection of captopril.

A high-molecular weight peptide hydrolase in erythrocytes

1. 1. A peptide hydrolase has been partially purified from the soluble fraction of erythrocyte lysates. 2. 2. The enzyme has a molecular weight of approximately 600,000 and hydrolyses the chymotrypsin substrate glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin (pH optimum 7.0) and the trypsin substrate CBZ-Gly-Gly-Arg-2-naphthylamide. The two activities could not be separated by the purification procedure used. 3. 3. The activity towards glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin in rat reticulocytes was four times that in mature erythrocytes. 4. 4. Activity was abolished by 10 μM p-hydroxymercuriphenylsulphonic acid.