Low Molecular Weight Degradation Products Research Articles

Carboxymethyl cellulase from Bacillus sp.: Isolation, macromolecular organization, and cellular location.

A Bacillus sp. strain isolated from termite (Odontotermes obesus) mount soils was found to produce extracellular carboxymethyl cellulase (CMCase). For further characterization the enzyme was isolated and purified from the culture supernatant. It revealed a molecular weight around 250kDa and a tetrameric structure, with a molecular weight of the subunits around 62kDa. Only one type of subunit was found. The enzyme was active over a wide range of conditions (30°C to 65°C; pH 3.0 to pH 11.0). Highest enzyme activity was measured at 50°C and a pH of 6.0. The enzyme was found to be not glycosylated. Glucose and cellobiose, the low molecular weight degradation products produced by the enzyme, did not induce or repress enzyme synthesis. A polyclonal antiserum was raised against the purified enzyme and applied for studies on the cellular location of the enzyme prior to secretion into the culture medium. The enzyme was shown to be located within the cytoplasm and at the cell periphery.

Fibrin and Fibrinogen Degradation Products With an Intact D-Domain C-Terminal γ Chain Inhibit an Early Step in Accessory Cell-Dependent Lymphocyte Mitogenesis

Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.

Fibrin and fibrinogen degradation products with an intact D-domain C- terminal gamma chain inhibit an early step in accessory cell-dependent lymphocyte mitogenesis

Although the low molecular weight degradation products of fibrinogen (FgDP) and fibrin (FbDP) are known to inhibit lymphocyte blastogenesis, the effect of purified macro-molecular FgDP and FbDP (molecular weight, 90 to 200 Kd) is unclear. We have examined the effect of these latter FgDP and FbDP and find that products that contain the D domain inhibit lymphocyte proliferation in response to T-cell mitogens, allogeneic mononuclear leukocytes, and anti-CD3 in vitro. Plasmic digestion of D1 in the absence of calcium with removal of the C-terminal end of the gamma chain or disruption of the gamma-gamma C-terminal cross-link site of D-dimer (DD) by puffadder venom (PAV-D) abrogates their inhibitory potential. Prior incubation of monocytes with DD or D1 inhibits subsequent lymphocyte transformation. Binding studies with radiolabeled DD and PAV-D confirm that monocytes interact only with DD. This specific binding may be competitively inhibited by monoclonal antibodies to CD11b/CD18 or by peptide analogues of the C-terminal gamma chain of fibrinogen that mimic the adhesion recognition site of integrins. We postulate that DD and D1 bind to CD11b/CD18 on adherent monocytes and modulate lymphocyte activation. These products are typically present in the plasma of patients with disseminated intravascular coagulation with sepsis and could therefore influence inflammatory processes in vivo.

GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer, Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47°C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassic® blue, Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.

GPR-phoresis, a novel approach to determining fibrin monomer and other macromolecular derivatives of fibrinogen and fibrin in blood

An electrophoretic method for determining (i) cross-linked fibrin-complexes, (ii) fibrin-monomer, (iii) fibrinogen-dimers, (iv) normal fibrinogen and (v) degradation products in plasma, has been devised. The technique is based on differences in their migration characteristics in the presence and absence of Gly-Pro-Arg (GPR), a specific inhibitor of fibrin aggregation. In buffer containing 2.5 mM GPR, fibrin monomer and fibrinogen co-migrate anodally, but, unlike fibrinogen which does not depend on GPR for solubility, the fibrin monomers precipitate when they traverse a boundary between buffer containing and buffer lacking GPR. By limiting the GPR to a 2 cm zone of buffer under the conditions employed, the precipitation of fibrin monomer occurs in a sharp band 4 mm anodally to the sample application point. Cross-linked fibrin complexes have slower mobility than fibrin monomer and precipitate in a broad band behind the monomer. Dimeric fibrinogen, like fibrinogen itself but unlike the fibrin complexes, is not constrained to migration within the GPR boundary and passes through it, but behind the band for normal fibrinogen due to sieving by the gel. Fibrinogen and all but low molecular weight degradation products can be specifically precipitated within electrophoregrams by heat denaturation at 47 degrees C. After washing unrelated protein away, the fibrin(ogen) derivatives can be measured by staining with Coomassie blue. Since the method does not depend on immunoprobing for specific staining, it provides an inexpensive and rapid means for differential assessment of the prevalence of the fibrinogen derivatives in disease states and in models of disease, regardless of animal species.

Environment-adaptable polymers

Scott-Gilead low density polyethylene (LDPE) containing pro-oxidant metal complexes has been subjected to ageing in a weatherometer ( i.e. photo-oxidation). Samples were analyzed using Fourier-transform infrared spectroscopy and differential scanning calorimetry to monitor changes in the carbonyl index and in the crystallinity during photo-oxidation. Volatile low molecular weight degradation products were detected using head-space gas chromatography (hs-GC) and changes in tensile strength were monitored in an Instron tester. Other methods for accelerating biodegradation are discussed.

Anomalies in reduction-mediated technetium-99m labelling of monoclonal antibodies

A reduction-mediated technetium-99m labelling method has been evaluated with a range of tumour-specific monoclonal antibodies. Antibodies reduced with 2-mercaptoethanol (2-ME) had free sulphydryl groups, but their number was much higher than could be accounted for by only limited intra-chain reduction of disulphide bonds. Reduced antibody could be labelled efficiently with 99mTc using an methylene diphosphonate (MDP) bone-scanning kit, although this seemed to depend on the presence of residual 2-ME in the preparations. With a carcinoembryonic antigen (CEA)-specific antibody, immunoreactivity of labelled antibody was confirmed, and after injection into nude with CEA-producing xenografts there was localisation into the tumours. Sephacryl S300 gel filtration showed the radiolabel eluting at a single discrete peak at the expected 150 kDa. However, examination of all of the labelled antibodies by polyacrylamide gel electrophoresis (PAGE) and autoradiography showed the presence of a large number of radiolabelled low molecular weight degradation products of the antibodies. These degradation products seemed to be formed in previously reduced antibodies during processing for PAGE, indicating some fragility of the reduced antibody.

Renal uptake and degradation of trapped-label calcitonin

In order to quantitate the role of the kidneys in the clearance and degradation of calcitonin, a trapped-label procedure was used to label human calcitonin. In contrast to conventional [ 125I]calcitonin, the trapped-label preparation allows quantitative measurements of the extent of uptake as well as of degradation in vivo because the final degradation products do not leave the cells. Trapped-label calcitonin activated adenylate cyclase of bone cells and kidney, as did the native hormone. Ten minutes after intravenous injection into rats, 16% of a trace dose was found in the kidneys. Renal recovery increased to 20% after one hour; in addition, 14% of the injected dose was found in the urine. Eighty per cent of the radioactivity in the urine was in high-molecular weight material. After 90 min, the sum of the accumulated radioactivities in the kidneys and the urine reached 40% of the dose. More than 80% of the radioactivity was sedimentable by centrifuging in a density gradient, indicating that intact calcitonin, as well as the degradation products in the cells, were enclosed within membrane-bound vesicles. Two minutes after injection of trapped-label calcitonin, the peak of radioactivity was found in light gradient fractions associated with cell membrane marker enzymes. Between 5 and 15 min, the peak migrated from light fractions to heavy fractions containing lysosomal marker enzymes. After just 2.5 min, 61% of the renal radioactivity was in low-molecular weight degradation products, as determined by gel filtration. The kinetics of renal degradation of calcitonin indicate that substantial amounts of endocytosed calcitonin is degraded before the hormone reaches the lysosomes.

Plasma clearance of chicken and human insulin-like growth factor-I and their association with circulating binding proteins in chickens

We have investigated the clearance of 125I-labelled chicken and recombinant human insulin-like growth factor-I (IGF-I) from the circulation of chickens as well as the role that IGF-binding proteins play in this process. Analysis of plasma samples by high-performance liquid chromatography (HPLC) neutral gel permeation on a TSK G3000SW column indicated that the i.v. injected radioactivity was rapidly partitioned between at least three pools. Most of the radioactivity occurred in a complex with binding protein(s), while smaller amounts of radioactivity chromatographed in the free IGF-I peak or appeared as low molecular weight degradation products. The labelled chicken and human IGF-I were rapidly cleared during the first 90 min. The calculated half-life for total labelled IGF-I during this period was 54 min for the chicken tracer and 33 min for the human tracer. The clearance was monitored for 10 h during which the human tracer continued to be cleared more rapidly than the chicken tracer. The proportion of radioactivity appearing as low molecular weight degradation products increased with time. Acid gel permeation and reverse-phase HPLC of the binding protein-associated radioactivity demonstrated that the labelled IGF-I bound was intact IGF-I. Sephadex G-200 gel permeation chromatography of chicken plasma samples at pH 7 x 4 showed that the binding protein complex labelled in vivo with chicken IGF-I tracer had a molecular mass of 55 kDa. Furthermore, the tracer associated with the binding protein coeluted with the major peak of endogenous IGF-I, suggesting that the tracer was bound to the physiologically relevant binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Disposition of recombinant human interleukin-2 (S-6820). (II): Studies using 125I-labeled S-6820 in rats.

The distribution, excretion and metabolism of recombinant human interleukin-2 (S-6820) were studied using 125I-labeled compound (125I-S-6820)1. At 5min after intravenous injection of 125I-S-6820 to male and female rats, high radioactivity was observed in the kidney. Radioactivities in the other organs were lower than the serum level. Results obtained by whole-body autoradiography showed that high concentrations of radioactivity were found in the cortex renis.2. At 5min after intravenous injection of 125I-S-6820 to 20-th day pregnant rats, no radioactivities were detected in the amniotic fluid and fetus.3. Within 24hr after intravenous injection of 125I-S-6820, 78% and 1 % of administered radioactivity were excreted in the urine and feces, respectively. However, 98% of excreted radioactivity in the urine was not precipitated with trichloroacetic acid.4. In the kidney after intravenous injection of 125I-S-6820, a low molecular weight degradation products of 125I-S-6820 were observed as revealed by gel filtration radio-chromatography. In addition, micro-autoradiogram of cortex renis after intravenous injection of 125I-S-6820 showed that S-6820 was likely to be ultrafiltrated by the glomerulus and absorbed by proximal tubules. S-6820 appeared to be degraded in the kidney.

Insulin processing in primary endosomes is not responsible for insulin resistance observed in parametrial adipocytes from lactating rats

The fate of [125I insulin and the insulin receptor after internalization was characterized in parametrial adipocytes from virgin rats. Parallel experiments were carried out on parametrial adipocytes from 2-4-day lactating rats, which are insulin resistant. Similar results were obtained in adipocytes from either group of animals. Insulin caused 10% of the plasma membrane insulin receptor to be translocated to a compartment resistant to extracellular trypsin. The intracellularly located insulin receptor rapidly recycled to the plasma membrane at 37 degrees C. An endosomal compartment involved in both the endocytosis and subsequent recycling of [125I]insulin and the insulin receptor to the plasma membrane was identified on sucrose density floatation gradients. [125I]Insulin internalized at 37 degrees C accumulated in a fraction of modal density 1.12 g/ml. Crosslinking experiments revealed the presence of intact [125I]insulin-insulin receptor complexes in endosomes. After a pulse with [125I]insulin, 55-60% of the 125I radioactivity recovered in the endosome compartment was intact [125I]insulin. The remainder was composed of low molecular weight degradation products. Endosomal 125I radioactivity was rapidly retroendocytosed to the medium with a mean half-life of 6 min. These results suggest: (1) [125I]insulin and the insulin receptor are internalized by parametrial adipocytes into an early endosomal compartment (primary endosomes), from which the receptor, intact [125I]insulin, and [125I]tyrosine are returned to the cell surface; and (2) the damping of the insulin signal observed in parametrial adipocytes from lactating rats is not expressed at the level of altered endocytotic processing of [125I]insulin and the insulin receptor.

Elimination of Native and Carbohydrate-Modified Tissue Plasminogen Activator in Rabbits

The elimination of native and carbohydrate-modified tissue type plasminogen activator (t-PA) after an i.v. bolus injection was studied in rabbits. t-PA with a low content of mannose (man-t-PA) was obtained by treatment with alpha-mannosidase, which cleaves terminal mannose from the carbohydrate side chains of the molecule. About 50% of the total mannose was removed from the native t-PA. Clearance of t-PA in rabbits was followed by measurements of both the fibrinolytic activity of the activators and the radioactivity of the iodine-labelled compounds. A biphasic mode of elimination of the fibrinolytic activity as well as the radioactivity was found with both the native and the carbohydrate modified t-PA. The initial half-life (t 1/2 alpha) was the same, about 1.5 minutes, for both compounds, irrespective of method of analysis. The late half-life (t 1/2 beta) was also the same, about 15 minutes, for both compounds. However, the beta-elimination of native t-PA could only be determined accurately from radioactivity data in the dosages tested, i.e. up to 2 mg of t-PA. No dose dependency in the elimination pattern was observed. In gel filtration experiments, with plasma from the rabbits, it was demonstrated that in addition to fibrinolytically active t-PA and man-t-PA, both fibrinolytically inactive high molecular weight complexes and low molecular weight degradation products of the activators were present in plasma after injection of the compounds. The second phase of elimination (beta) was much more pronounced after mannosidase treatment of the t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal uptake and degradation of trapped-label insulin.

In order to study the kinetics of insulin degradation in the kidneys and liver, insulin was labelled by a trapped-label procedure and injected into rats. In contrast to conventional 125I-insulin, the trapped-label preparation allows quantitative measurements of the extent of degradation in vivo because the final degradation products do not leave the cells. One hour after injection, the amount of radioactivity in the kidneys from a trace dose of trapped-label insulin was 10 times higher that from conventionally labelled insulin; over 80% of the increase was due to low molecular weight degradation products which were retained in the kidneys. The amount of acid-precipitable radioactivity in the blood was the same for both labelled preparations, indicating that their rates of clearance were similar. In the kidney, we detected no degradation products of molecular weight intermediate between intact insulin and the end products of proteolysis. After 2 h, 33% of the injected dose remained in the kidneys and only 13% in the liver. Over 80% of the renal radioactivity was sedimentable in an isotonic density gradient, indicating that intact insulin, as well as degradation products in the cells, were enclosed within membrane-bound vesicles.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

An enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 81% for free tPA and tPA in complex with PAI-1, PAI-2, alpha 2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about 100%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasma, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Aminoterminal Propeptide of Type III Procollagen is Cleared from the Circulation by Receptor-Mediated Endocytosis in Liver Endothelial Cells

Intravenously administered 125I -labelled bovine aminoterminal propeptide of typeIII procollagen (125I -BPIIINP) had a half-life in blood of about 2 minutes. Low molecular weight degradation products appeared in the circulation about 5 minutes after injection. BPIIINP coupled to 125I -labelled tyramine cellobiose (125I -TC-BPIIINP) was administered intravenously to determine the cellular site of uptake. TC is non-degradable and is therefore accumulated intralysosomally. With this ligand I could show that PIIINP is taken up mainly by the liver endothelial cells (LEC), with very low uptake in other types of liver cells and at extrahepatic sites. Studies on binding and endocytosis of labelled PIIINP in cultures of purified populations of liver cells can be summarized as follows: 1) Uptake and degradation were observed mainly in LEC; 2) PIIINP associated with Kupffer and parenchymal cells, but degradation was very low; 3) Serum was not required for binding of PIIINP to LEC; 4) Binding was specific, that is, other ligands, such as collagen type III, hyaluronan, chondroitin sulfate, formaldehyde-treated albumin, and mannose, that are recognized by distinct receptors on LEC, did not compete with PIIINP for binding; 5) BPIIINP, TC-BPIIINP, and rat PIIINP (RPIIINP) were recognized with the same specificity by LEC; 6) BPIIINP bound to LEC with high affinity (dissociation constant = 1 nM), and about 4.2 × 105, 3.2 × 105, and 1.6 × 105 molecules of BPIIINP, TC-PIIINP, and R-PIIINP, respectively were bound per cell; 7) PIIINP could not be degraded by conditioned medium from cultured Kupffer cells; 8) Leupeptin, which is a strong inhibitor of lysosomal collagenolysis, only weakly inhibited degradation of PIIINP; 9) Binding and endocytosis of PIIINP was not Ca++-dependent; 10) Agents that inhibit the endocytic machinery inhibited uptake and degradation of PIIINP. In conclusion, the present results suggest that PIIINP is rapidly eliminated from the circulation by receptor-mediated endocytosis in LEC.

Post-translational regulation of Lcr plasmid-mediated peptides in pesticinogenic Yersinia pestis

The low calcium response of wild type Yersinia pestis, the causative agent of bubonic plague, and of enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica is known to be mediated by a shared Lcr plasmid of about 70 kb. At 37 °C in Ca 2+-deficient medium, this element promotes restriction of growth with concomitant production of virulence functions including the common V antigen and a set of yersiniae outer membrane peptides termed YOPs (Lcr +). The latter are expressed by the enteropathogenic species but not by wild type Y. pestis which possesses a unique 10 kb Pst plasmid associated with pesticinogeny (Pst +). We show in this report that, after pulse with 35S-methionine, peptides with molecular weights corresponding to YOPs of 78, 47, 45, 44, 36, and 26 kDa are synthesized during the low calcium response by both Lcr +, Pst + and Lcr +, Pst − cells of Y. pestis. Although stable in the latter, radioactivity in YOPs of wild type was rapidly chased into lower molecular weight degradation products. At least four soluble peptides, including V, were also labeled during starvation for Ca 2+; these structures were stable in both Lcr +, Pst + and Lcr +, Pst − yersiniae. These findings suggest that a product encoded by the Pst plasmid of Y. pestis is required for post-translational regulation of outer membrane but not soluble peptides mediated by a second unrelated Lcr plasmid.

Structure and proteolysis of the growth hormone receptor on rat hepatocytes.

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.

Permeability of renal capillaries. I. Preparation of neutral and charged protein probes.

This paper describes the preparation of charged and uncharged protein molecular probes for study of the permselectivity of renal capillaries. Horse heart myoglobin was used as a neutral myoglobin. Since it contained several fractions with different isoelectric points, it was purified by fast protein liquid chromatography (FPLC). To obtain a negatively charged myoglobin, the original horse heart myoglobin was treated with cyanate, resulting in net charge of -5.7 +/- 0.3 at physiological pH (mean +/- SEM). The charge was determined from the Donnan potential which develops over a semipermeable membrane separating the inside solution in which the protein was dissolved from a surrounding bath of equal ionic strength. Sperm whale myoglobin was similarly purified by FPLC and used as a positively (+1.7 +/- 0.2) charged isomer. Horseradish peroxidase (HRP) was purified by means of gel and ion-exchange chromatography and found to be neutral at physiological pH. Negatively charged (-14.0 +/- 0.5) HRP was obtained by succinylation. Two isomers of lactate dehyrogenase (LDH) were used, namely the slightly positive (+2) LDH-M4 and the strongly negative (-19) LDH-H4. These isomers, which occur naturally, did not require further purification. The Stokes-Einstein radii, as measured by gel chromatography, of inulin, myoglobin, HRP and LDH were 11, 17.5, 32 and 46 A, respectively. The chemical modifications did not alter the Stokes-Einstein radii. In biological studies on rat kidneys samples of both plasma and renal hilar lymph were found to contain radioactive low molecular weight degradation products in addition to the intact proteins. This necessitated separation of all individual samples on small Sephadex columns prior to analysis.

The effect of insulin concentration on retroendocytosis in isolated rat adipocytes.

After internalization by isolated rat adipocytes, insulin can be degraded or released intact from the cell, a process termed retroendocytosis. To determine whether the amount of ligand entering the cell could modulate its ultimate intracellular disposition, adipocytes were incubated with 0.4-25 ng/ml radiolabeled insulin for 20 min at 37 C to reach steady state binding and internalization. After this, surface bound insulin was removed by acid extraction and the cells were reincubated in insulin-free 37 C buffer. The fractional rate of release of internalized cell associated radioactivity was similar at all insulin concentrations. However, insulin enhanced the appearance of trichloroacetic acid (TCA)-precipitable material in a dose-dependent manner reaching a maximum at an insulin concentration of 10 ng/ml. At 0.4 ng/ml insulin, 18 +/- 2% of the released radioactivity was TCA precipitable whereas 36 +/- 3% was precipitable at 25 ng/ml. Sephadex G-50 gel chromatography and reverse phase HPLC analysis of the reincubation medium confirmed TCA-precipitable material was intact insulin. To further investigate the dual pathways of intracellular insulin processing, adipocytes were incubated with 0.4 and 25 ng/ml insulin for 20 min, acid extracted to remove surface receptor insulin, and solubilized. Sephadex G-50 and HPLC analysis revealed that proportionately less insulin intermediates and low molecular weight degradation products are found in cells incubated at the higher insulin concentrations. In conclusion, as adipocytes internalize more insulin, less is converted into insulin intermediates and low molecular weight degradation products and more is diverted to retroendocytosis.

Soluble, crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers : 2. Effect of molecular weight on blood clearance and body distribution in the rat after intravenous administration. Distribution of unfractionated copolymer after intraperitoneal, subcutaneous or oral administration

Preparation and fractionation of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers containing oligopeptide side-chains crosslinked with dityrosylhexamethylenediamine to a level below the gel point has been described previously [1]. Fractions of mean molecular weight ( M ̄ w) from 34,000 to > 400,000 were radiolabelled with 125I and used to study the effect of molecular weight on the fate of polymer in vivo. The blood clearance and body distribution (60 min) of each molecular weight fraction was measured after intravenous administration to rats. Blood clearance was clearly related to molecular weight. In addition the body distribution of unfractionated HPMA copolymer ( M ̄ w = 190,000) was monitored at various times after intraperitoneal, subcutaneous or oral administration to rats. Copolymer administered intraperitoneally progressively moved out of the peritoneal cavity into the bloodstream, and after 24 h most radioactivity was recovered in the urine. Sepharose 4B/6B column chromatography of urine samples showed that most radioactivity represented low molecular weight polymer but low molecular weight degradation products were also present. After subcutaneous administration polymer persisted at the injection site, gradually moving away over 24 h. Material entering the body was recovered principally from the bloodstream, eventually being excreted in the urine and faeces. Oral administration led to rapid transfer of radioactivity along the gastrointestinal tract, after 24 h most radioactivity being recovered in the urine and faeces. Sepharose 4B/6B chromatography of radioactivity recovered from the caecum showed that extensive degradation of copolymer had taken place. Incubation of 126I-labelled HPMA copolymers in rat plasma and a mixture of rat liver lysosomal enzymes, followed by analysis with Sepharose 4B/6B or Sephadex G-15 chromatography, showed that the copolymers used in this study were relatively stable in plasma but degradable by lysosomal enzymes.